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Sample records for preventing protein aggregation

  1. Targeting Protein Aggregation for the Treatment of Degenerative Diseases

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    Eisele, Yvonne S.; Monteiro, Cecilia; Fearns, Colleen; Encalada, Sandra E.; Wiseman, R. Luke; Powers, Evan T.; Kelly, Jeffery W.

    2015-01-01

    The aggregation of specific proteins is hypothesized to underlie several degenerative diseases, collectively called amyloid disorders. However, the mechanistic connection between the process of protein aggregation and tissue degeneration is not yet fully understood. Here, we review current and emerging strategies to ameliorate aggregation-associated degenerative disorders, with a focus on disease-modifying strategies that prevent the formation of and/or eliminate protein aggregates. Persuasive pharmacologic and genetic evidence now support protein aggregation as the cause of post-mitotic tissue dysfunction or loss. However, a more detailed understanding of the factors that trigger and sustain aggregate formation, as well as the structure-activity relationships underlying proteotoxicity are needed to develop future disease-modifying therapies. PMID:26338154

  2. The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

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    Bani Kumar Pathak

    Full Text Available BACKGROUND: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains. RESULTS: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein. CONCLUSION: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

  3. Preventing disulfide bond formation weakens non-covalent forces among lysozyme aggregates.

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    Vijay Kumar Ravi

    Full Text Available Nonnative disulfide bonds have been observed among protein aggregates in several diseases like amyotrophic lateral sclerosis, cataract and so on. The molecular mechanism by which formation of such bonds promotes protein aggregation is poorly understood. Here in this work we employ previously well characterized aggregation of hen eggwhite lysozyme (HEWL at alkaline pH to dissect the molecular role of nonnative disulfide bonds on growth of HEWL aggregates. We employed time-resolved fluorescence anisotropy, atomic force microscopy and single-molecule force spectroscopy to quantify the size, morphology and non-covalent interaction forces among the aggregates, respectively. These measurements were performed under conditions when disulfide bond formation was allowed (control and alternatively when it was prevented by alkylation of free thiols using iodoacetamide. Blocking disulfide bond formation affected growth but not growth kinetics of aggregates which were ∼50% reduced in volume, flatter in vertical dimension and non-fibrillar in comparison to control. Interestingly, single-molecule force spectroscopy data revealed that preventing disulfide bond formation weakened the non-covalent interaction forces among monomers in the aggregate by at least ten fold, thereby stalling their growth and yielding smaller aggregates in comparison to control. We conclude that while constrained protein chain dynamics in correctly disulfide bonded amyloidogenic proteins may protect them from venturing into partial folded conformations that can trigger entry into aggregation pathways, aberrant disulfide bonds in non-amyloidogenic proteins (like HEWL on the other hand, may strengthen non-covalent intermolecular forces among monomers and promote their aggregation.

  4. Endoplasmic reticulum proteins SDF2 and SDF2L1 act as components of the BiP chaperone cycle to prevent protein aggregation.

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    Fujimori, Tsutomu; Suno, Ryoji; Iemura, Shun-Ichiro; Natsume, Tohru; Wada, Ikuo; Hosokawa, Nobuko

    2017-08-01

    The folding of newly synthesized proteins in the endoplasmic reticulum (ER) is assisted by ER-resident chaperone proteins. BiP (immunoglobulin heavy-chain-binding protein), a member of the HSP70 family, plays a central role in protein quality control. The chaperone function of BiP is regulated by its intrinsic ATPase activity, which is stimulated by ER-resident proteins of the HSP40/DnaJ family, including ERdj3. Here, we report that two closely related proteins, SDF2 and SDF2L1, regulate the BiP chaperone cycle. Both are ER-resident, but SDF2 is constitutively expressed, whereas SDF2L1 expression is induced by ER stress. Both luminal proteins formed a stable complex with ERdj3 and potently inhibited the aggregation of different types of misfolded ER cargo. These proteins associated with non-native proteins, thus promoting the BiP-substrate interaction cycle. A dominant-negative ERdj3 mutant that inhibits the interaction between ERdj3 and BiP prevented the dissociation of misfolded cargo from the ERdj3-SDF2L1 complex. Our findings indicate that SDF2 and SDF2L1 associate with ERdj3 and act as components in the BiP chaperone cycle to prevent the aggregation of misfolded proteins, partly explaining the broad folding capabilities of the ER under various physiological conditions. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  5. Proteins aggregation and human diseases

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    Hu, Chin-Kun

    2015-01-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease. (paper)

  6. Proteins aggregation and human diseases

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    Hu, Chin-Kun

    2015-04-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease.

  7. Disaggregases, molecular chaperones that resolubilize protein aggregates

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    David Z. Mokry

    2015-08-01

    Full Text Available The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of protein aggregates, an insoluble association of polypeptides that harm cell physiology, either by themselves or in the process of formation. Several biological processes have evolved to prevent and eliminate the existence of non-functional and amyloidogenic aggregates, as they are associated with several human pathologies. Molecular chaperones and heat shock proteins are specialized in controlling the quality of the proteins in the cell, specifically by aiding proper folding, and dissolution and clearance of already formed protein aggregates. The latter is a function of disaggregases, mainly represented by the ClpB/Hsp104 subfamily of molecular chaperones, that are ubiquitous in all organisms but, surprisingly, have no orthologs in the cytosol of metazoan cells. This review aims to describe the characteristics of disaggregases and to discuss the function of yeast Hsp104, a disaggregase that is also involved in prion propagation and inheritance.

  8. Effect of protein-surfactant interactions on aggregation of β-lactoglobulin.

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    Hansted, Jon G; Wejse, Peter L; Bertelsen, Hans; Otzen, Daniel E

    2011-05-01

    The milk protein β-lactoglobulin (βLG) dominates the properties of whey aggregates in food products. Here we use spectroscopic and calorimetric techniques to elucidate how anionic, cationic and non-ionic surfactants interact with bovine βLG and modulate its heat-induced aggregation. Alkyl trimethyl ammonium chlorides (xTAC) strongly promote aggregation, while sodium alkyl sulfates (SxS) and alkyl maltopyranosides (xM) reduce aggregation. Sodium dodecyl sulfate (SDS) binds to non-aggregated βLG in several steps, but reduction of aggregation was associated with the first binding step, which occurs far below the critical micelle concentration. In contrast, micellar concentrations of xMs are required to reduce aggregation. The ranking order for reduction of aggregation (normalized to their tendency to self-associate) was C10-C12>C8>C14 for SxS and C8>C10>C12>C14>C16 for xM. xTAC promote aggregation in the same ranking order as xM reduce it. We conclude that SxS reduce aggregation by stabilizing the protein's ligand-bound state (the melting temperature t(m) increases by up to 10°C) and altering its charge potential. xM monomers also stabilize the protein's ligand-bound state (increasing t(m) up to 6°C) but in the absence of charged head groups this is not sufficient by itself to prevent aggregation. Although micelles of both anionic and non-ionic surfactants destabilize βLG, they also solubilize unfolded protein monomers, leaving them unavailable for protein-protein association and thus inhibiting aggregation. Cationic surfactants promote aggregation by a combination of destabilization and charge neutralization. The food compatible surfactant sodium dodecanoate also inhibited aggregation well below the cmc, suggesting that surfactants may be a practical way to modulate whey protein properties. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Effect of the addition of CMC on the aggregation behaviour of proteins

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    Yu, H.; Sabato, S.F.; D' Aprano, G.; Lacroix, M. E-mail: monique.lacroix@inrs-iaf.uquebec.ca

    2004-10-01

    The effect of carboxymethylcellulose (CMC) on the aggregation of formulation based on calcium caseinate, commercial whey protein (WPC), and a 1:1 mixture of soy protein isolate (SPI) and whey protein isolate (WPI) was investigated. Protein aggregation could be observed upon addition of CMC, as demonstrated by size-exclusion chromatography. This aggregation behaviour was enhanced by means of physical treatments, such as heating at 90 deg. C for 30 min or gamma-irradiation at 32 kGy. A synergy resulted from the combination of CMC to gamma-irradiation in Caseinate/CMC and SPI/WPI/CMC formulations. Furthermore, CMC prevented precipitation in irradiated protein solutions for a period of more than 3 months at 4 deg. C.

  10. Effect of the addition of CMC on the aggregation behaviour of proteins

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    Yu, H.; Sabato, S.F.; D'Aprano, G.; Lacroix, M.

    2004-01-01

    The effect of carboxymethylcellulose (CMC) on the aggregation of formulation based on calcium caseinate, commercial whey protein (WPC), and a 1:1 mixture of soy protein isolate (SPI) and whey protein isolate (WPI) was investigated. Protein aggregation could be observed upon addition of CMC, as demonstrated by size-exclusion chromatography. This aggregation behaviour was enhanced by means of physical treatments, such as heating at 90 deg. C for 30 min or gamma-irradiation at 32 kGy. A synergy resulted from the combination of CMC to gamma-irradiation in Caseinate/CMC and SPI/WPI/CMC formulations. Furthermore, CMC prevented precipitation in irradiated protein solutions for a period of more than 3 months at 4 deg. C

  11. CPAD, Curated Protein Aggregation Database: A Repository of Manually Curated Experimental Data on Protein and Peptide Aggregation.

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    Thangakani, A Mary; Nagarajan, R; Kumar, Sandeep; Sakthivel, R; Velmurugan, D; Gromiha, M Michael

    2016-01-01

    Accurate distinction between peptide sequences that can form amyloid-fibrils or amorphous β-aggregates, identification of potential aggregation prone regions in proteins, and prediction of change in aggregation rate of a protein upon mutation(s) are critical to research on protein misfolding diseases, such as Alzheimer's and Parkinson's, as well as biotechnological production of protein based therapeutics. We have developed a Curated Protein Aggregation Database (CPAD), which has collected results from experimental studies performed by scientific community aimed at understanding protein/peptide aggregation. CPAD contains more than 2300 experimentally observed aggregation rates upon mutations in known amyloidogenic proteins. Each entry includes numerical values for the following parameters: change in rate of aggregation as measured by fluorescence intensity or turbidity, name and source of the protein, Uniprot and Protein Data Bank codes, single point as well as multiple mutations, and literature citation. The data in CPAD has been supplemented with five different types of additional information: (i) Amyloid fibril forming hexa-peptides, (ii) Amorphous β-aggregating hexa-peptides, (iii) Amyloid fibril forming peptides of different lengths, (iv) Amyloid fibril forming hexa-peptides whose crystal structures are available in the Protein Data Bank (PDB) and (v) Experimentally validated aggregation prone regions found in amyloidogenic proteins. Furthermore, CPAD is linked to other related databases and resources, such as Uniprot, Protein Data Bank, PUBMED, GAP, TANGO, WALTZ etc. We have set up a web interface with different search and display options so that users have the ability to get the data in multiple ways. CPAD is freely available at http://www.iitm.ac.in/bioinfo/CPAD/. The potential applications of CPAD have also been discussed.

  12. Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

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    Wang Hongmin; Monteiro, Mervyn J.

    2007-01-01

    Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases

  13. Exploring new biological functions of amyloids: bacteria cell agglutination mediated by host protein aggregation.

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    Marc Torrent

    Full Text Available Antimicrobial proteins and peptides (AMPs are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.

  14. PE859, a novel tau aggregation inhibitor, reduces aggregated tau and prevents onset and progression of neural dysfunction in vivo.

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    Michiaki Okuda

    Full Text Available In tauopathies, a neural microtubule-associated protein tau (MAPT is abnormally aggregated and forms neurofibrillary tangle. Therefore, inhibition of the tau aggregation is one of the key approaches for the treatment of these diseases. Here, we have identified a novel tau aggregation inhibitor, PE859. An oral administration of PE859 resulted in the significant reduction of sarkosyl-insoluble aggregated tau along with the prevention of onset and progression of the motor dysfunction in JNPL3 P301L-mutated human tau transgenic mice. These results suggest that PE859 is useful for the treatment of tauopathies.

  15. Protein carbonylation, protein aggregation and neuronal cell death in a murine model of multiple sclerosis

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    Dasgupta, Anushka

    /spatial relationship between carbonylation, protein aggregation and cellular apoptosis. Furthermore, the effectiveness of the carbonyl scavenger hydralazine, histidine hydrazide and methoxylamine at preventing cell death identifies protein carbonyls as the toxic species. Experiments using well-characterized apoptosis inhibitors place protein carbonylation downstream of the mitochondrial transition pore opening and upstream of caspase activation. These in vitro studies demonstrate for the first time a causal relationship between carbonylation, protein aggregation and apoptosis of neurons undergoing oxidative damage. This relationship was further strengthened with the experiments carried out in chapter 4, which show that inhibition of protein aggregation with congo red (CR) or 2-hydroxypropyl beta-cyclodextrin (HPCD) significantly reduced neuronal cell death without affecting the levels of oxidized proteins. Interestingly, large, juxta-nuclear aggregates are not formed upon GSH depletion, suggesting that the small protein aggregates are the cytotoxic species. Together, our data suggest that protein carbonylation causes protein aggregation to mediate neuronal apoptosis in vitro and that a similar mechanism might be contributing to neuronal/glial apoptosis in EAE. These studies provide the basis for testing protein carbonylation scavengers and protein aggregation inhibitors for the treatment of inflammatory demyelinating disorders.

  16. Cellular Handling of Protein Aggregates by Disaggregation Machines.

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    Mogk, Axel; Bukau, Bernd; Kampinga, Harm H

    2018-01-18

    Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key.

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    Utpal Das

    Full Text Available BACKGROUND: Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. METHODOLOGY: We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Abeta(1-42 peptide is modified. Changes in the hydrodynamic volume of Abeta(1-42 in the presence of arginine measured by size exclusion chromatography show that arginine binds to Abeta(1-42. Arginine increases the solubility of Abeta(1-42 peptide in aqueous medium. It decreases the aggregation of Abeta(1-42 as observed by atomic force microscopy. CONCLUSIONS: Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation.

  18. Understanding curcumin-induced modulation of protein aggregation.

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    Ahmad, Basir; Borana, Mohanish S; Chaudhary, Ankur P

    2017-07-01

    Curcumin, a diarylheptanoid compound, found in spice turmeric is known to alter the aggregation of proteins and reduce the toxicity of the aggregates. This review looks at the molecular basis of modulating protein aggregation and toxicity of the aggregates. Foremost, we identify the interaction of curcumin and its derivatives with proteins/peptides and the effect of their interaction on the conformational stability and unfolding/folding pathway(s). The unfolding/folding processes generate partially folded/unfolded intermediate, which serve as aggregation precursor state. Secondly, we discuss the effect of curcumin binding on the kinetics parameters of the aggregation process, which give information about the mechanism of the aggregation inhibition. We describe, in addition, that curcumin can accelerate/promote fibril formation by binding to oligomeric intermediate(s) accumulated in the aggregation pathway. Finally, we discuss the correlation of curcumin-induced monomeric and/or oligomeric precursor states with aggregate structure and toxicity. On the basis of these discussions, we propose a model describing curcumin-induced inhibition/promotion of formation of amyloid-like fibrils. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: A study of DNAJB6 chaperone

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    Rasha Mohamed Hussein

    2015-07-01

    Full Text Available Alzheimer’s disease is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation-prone peptides containing large poly glutamines (associated with CAG repeat diseases both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of HEK293 cells with Aβ-GFP fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly Q peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells.

  20. Aggregation of natively folded proteins: a theoretical approach

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    Trovato, Antonio; Maritan, Amos; Seno, Flavio

    2007-01-01

    The reliable identification of β-aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimer's and Parkinson's diseases, as well as other pathological conditions associated with protein deposition. While the list of aggregation related diseases is growing, it has also been shown that many proteins that are normally well behaved can be induced to aggregate in vitro. This fact suggests the existence of a unified framework that could explain both folding and aggregation. By assuming this universal behaviour, we have recently introduced an algorithm (PASTA: prediction of amyloid structure aggregation), which is based on a sequence-specific energy function derived from the propensity of two residue types to be found paired in neighbouring strands within β-sheets in globular proteins. The algorithm is able to predict the most aggregation-prone portions of several proteins initially unfolded, in excellent agreement with experimental results. Here, we apply the method to a set of proteins which are known to aggregate, but which are natively folded. The quality of the prediction is again very high, corroborating the hypothesis that the amyloid structure is stabilized by the same physico-chemical determinants as those operating in folded proteins

  1. Protein aggregate turbidity: Simulation of turbidity profiles for mixed-aggregation reactions.

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    Hall, Damien; Zhao, Ran; Dehlsen, Ian; Bloomfield, Nathaniel; Williams, Steven R; Arisaka, Fumio; Goto, Yuji; Carver, John A

    2016-04-01

    Due to their colloidal nature, all protein aggregates scatter light in the visible wavelength region when formed in aqueous solution. This phenomenon makes solution turbidity, a quantity proportional to the relative loss in forward intensity of scattered light, a convenient method for monitoring protein aggregation in biochemical assays. Although turbidity is often taken to be a linear descriptor of the progress of aggregation reactions, this assumption is usually made without performing the necessary checks to provide it with a firm underlying basis. In this article, we outline utilitarian methods for simulating the turbidity generated by homogeneous and mixed-protein aggregation reactions containing fibrous, amorphous, and crystalline structures. The approach is based on a combination of Rayleigh-Gans-Debye theory and approximate forms of the Mie scattering equations. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  2. Protein aggregation and misfolding: good or evil?

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    Pastore, Annalisa; Temussi, Pierandrea

    2012-06-01

    Protein aggregation and misfolding have important implications in an increasing number of fields ranging from medicine to biology to nanotechnology and material science. The interest in understanding this field has accordingly increased steadily over the last two decades. During this time the number of publications that have been dedicated to protein aggregation has increased exponentially, tackling the problem from several different and sometime contradictory perspectives. This review is meant to summarize some of the highlights that come from these studies and introduce this topical issue on the subject. The factors that make a protein aggregate and the cellular strategies that defend from aggregation are discussed together with the perspectives that the accumulated knowledge may open.

  3. Protein aggregation and misfolding: good or evil?

    International Nuclear Information System (INIS)

    Pastore, Annalisa; Temussi, Pierandrea

    2012-01-01

    Protein aggregation and misfolding have important implications in an increasing number of fields ranging from medicine to biology to nanotechnology and material science. The interest in understanding this field has accordingly increased steadily over the last two decades. During this time the number of publications that have been dedicated to protein aggregation has increased exponentially, tackling the problem from several different and sometime contradictory perspectives. This review is meant to summarize some of the highlights that come from these studies and introduce this topical issue on the subject. The factors that make a protein aggregate and the cellular strategies that defend from aggregation are discussed together with the perspectives that the accumulated knowledge may open. (topical review)

  4. The Mechanisms of Aberrant Protein Aggregation

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    Cohen, Samuel; Vendruscolo, Michele; Dobson, Chris; Knowles, Tuomas

    2012-02-01

    We discuss the development of a kinetic theory for understanding the aberrant loss of solubility of proteins. The failure to maintain protein solubility results often in the assembly of organized linear structures, commonly known as amyloid fibrils, the formation of which is associated with over 50 clinical disorders including Alzheimer's and Parkinson's diseases. A true microscopic understanding of the mechanisms that drive these aggregation processes has proved difficult to achieve. To address this challenge, we apply the methodologies of chemical kinetics to the biomolecular self-assembly pathways related to protein aggregation. We discuss the relevant master equation and analytical approaches to studying it. In particular, we derive the underlying rate laws in closed-form using a self-consistent solution scheme; the solutions that we obtain reveal scaling behaviors that are very generally present in systems of growing linear aggregates, and, moreover, provide a general route through which to relate experimental measurements to mechanistic information. We conclude by outlining a study of the aggregation of the Alzheimer's amyloid-beta peptide. The study identifies the dominant microscopic mechanism of aggregation and reveals previously unidentified therapeutic strategies.

  5. The proteome of neurofilament-containing protein aggregates in blood

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    Rocco Adiutori

    2018-07-01

    Full Text Available Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf, the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP, for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS. Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.

  6. Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes.

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    Castillo, Virginia; Graña-Montes, Ricardo; Sabate, Raimon; Ventura, Salvador

    2011-06-01

    In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of β-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Widespread protein aggregation as an inherent part of aging in C. elegans.

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    Della C David

    Full Text Available Aberrant protein aggregation is a hallmark of many age-related diseases, yet little is known about whether proteins aggregate with age in a non-disease setting. Using a systematic proteomics approach, we identified several hundred proteins that become more insoluble with age in the multicellular organism Caenorhabditis elegans. These proteins are predicted to be significantly enriched in beta-sheets, which promote disease protein aggregation. Strikingly, these insoluble proteins are highly over-represented in aggregates found in human neurodegeneration. We examined several of these proteins in vivo and confirmed their propensity to aggregate with age. Different proteins aggregated in different tissues and cellular compartments. Protein insolubility and aggregation were significantly delayed or even halted by reduced insulin/IGF-1-signaling, which also slows aging. We found a significant overlap between proteins that become insoluble and proteins that influence lifespan and/or polyglutamine-repeat aggregation. Moreover, overexpressing one aggregating protein enhanced polyglutamine-repeat pathology. Together our findings indicate that widespread protein insolubility and aggregation is an inherent part of aging and that it may influence both lifespan and neurodegenerative disease.

  8. Plasmodium falciparum Hep1 Is Required to Prevent the Self Aggregation of PfHsp70-3.

    Directory of Open Access Journals (Sweden)

    David O Nyakundi

    Full Text Available The majority of mitochondrial proteins are encoded in the nucleus and need to be imported from the cytosol into the mitochondria, and molecular chaperones play a key role in the efficient translocation and proper folding of these proteins in the matrix. One such molecular chaperone is the eukaryotic mitochondrial heat shock protein 70 (Hsp70; however, it is prone to self-aggregation and requires the presence of an essential zinc-finger protein, Hsp70-escort protein 1 (Hep1, to maintain its structure and function. PfHsp70-3, the only Hsp70 predicted to localize in the mitochondria of P. falciparum, may also rely on a Hep1 orthologue to prevent self-aggregation. In this study, we identified a putative Hep1 orthologue in P. falciparum and co-expression of PfHsp70-3 and PfHep1 enhanced the solubility of PfHsp70-3. PfHep1 suppressed the thermally induced aggregation of PfHsp70-3 but not the aggregation of malate dehydrogenase or citrate synthase, thus showing specificity for PfHsp70-3. Zinc ions were indeed essential for maintaining the function of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Soluble and functional PfHsp70-3, acquired by co-expression with PfHep-1, will facilitate the biochemical characterisation of this particular Hsp70 protein and its evaluation as a drug target for the treatment of malaria.

  9. Protein Colloidal Aggregation Project

    Science.gov (United States)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  10. Protein Folding and Aggregation into Amyloid: The Interference by Natural Phenolic Compounds

    Directory of Open Access Journals (Sweden)

    Massimo Stefani

    2013-06-01

    Full Text Available Amyloid aggregation is a hallmark of several degenerative diseases affecting the brain or peripheral tissues, whose intermediates (oligomers, protofibrils and final mature fibrils display different toxicity. Consequently, compounds counteracting amyloid aggregation have been investigated for their ability (i to stabilize toxic amyloid precursors; (ii to prevent the growth of toxic oligomers or speed that of fibrils; (iii to inhibit fibril growth and deposition; (iv to disassemble preformed fibrils; and (v to favor amyloid clearance. Natural phenols, a wide panel of plant molecules, are one of the most actively investigated categories of potential amyloid inhibitors. They are considered responsible for the beneficial effects of several traditional diets being present in green tea, extra virgin olive oil, red wine, spices, berries and aromatic herbs. Accordingly, it has been proposed that some natural phenols could be exploited to prevent and to treat amyloid diseases, and recent studies have provided significant information on their ability to inhibit peptide/protein aggregation in various ways and to stimulate cell defenses, leading to identify shared or specific mechanisms. In the first part of this review, we will overview the significance and mechanisms of amyloid aggregation and aggregate toxicity; then, we will summarize the recent achievements on protection against amyloid diseases by many natural phenols.

  11. Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates.

    Directory of Open Access Journals (Sweden)

    Maggie P Wear

    Full Text Available Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12 cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID domains (≥ 100 amino acids and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.

  12. Distinct role of hydration water in protein misfolding and aggregation revealed by fluctuating thermodynamics analysis.

    Science.gov (United States)

    Chong, Song-Ho; Ham, Sihyun

    2015-04-21

    Protein aggregation in aqueous cellular environments is linked to diverse human diseases. Protein aggregation proceeds through a multistep process initiated by conformational transitions, called protein misfolding, of monomer species toward aggregation-prone structures. Various forms of aggregate species are generated through the association of misfolded monomers including soluble oligomers and amyloid fibrils. Elucidating the molecular mechanisms and driving forces involved in the misfolding and subsequent association has been a central issue for understanding and preventing protein aggregation diseases such as Alzheimer's, Parkinson's, and type II diabetes. In this Account, we provide a thermodynamic perspective of the misfolding and aggregation of the amyloid-beta (Aβ) protein implicated in Alzheimer's disease through the application of fluctuating thermodynamics. This approach "dissects" the conventional thermodynamic characterization of the end states into the one of the fluctuating processes connecting them, and enables one to analyze variations in the thermodynamic functions that occur during the course of protein conformational changes. The central quantity in this approach is the solvent-averaged effective energy, f = Eu + Gsolv, comprising the protein potential energy (Eu) and the solvation free energy (Gsolv), whose time variation reflects the protein dynamics on the free energy landscape. Protein configurational entropy is quantified by the magnitude of fluctuations in f. We find that misfolding of the Aβ monomer when released from a membrane environment to an aqueous phase is driven by favorable changes in protein potential energy and configurational entropy, but it is also accompanied by an unfavorable increase in solvation free energy. The subsequent dimerization of the misfolded Aβ monomers occurs in two steps. The first step, where two widely separated monomers come into contact distance, is driven by water-mediated attraction, that is, by a

  13. N-acetyl-L-cysteine prevents stress-induced desmin aggregation in cellular models of desminopathy.

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    Bertrand-David Segard

    Full Text Available Mutations within the human desmin gene are responsible for a subcategory of myofibrillar myopathies called desminopathies. However, a single inherited mutation can produce different phenotypes within a family, suggesting that environmental factors influence disease states. Although several mouse models have been used to investigate organ-specific desminopathies, a more general mechanistic perspective is required to advance our knowledge toward patient treatment. To improve our understanding of disease pathology, we have developed cellular models to observe desmin behaviour in early stages of disease pathology, e.g., upon formation of cytoplasmic desmin aggregates, within an isogenic background. We cloned the wildtype and three mutant desmin cDNAs using a Tet-On Advanced® expression system in C2C12 cells. Mutations were selected based on positioning within desmin and capacity to form aggregates in transient experiments, as follows: DesS46Y (head domain; low aggregation, DesD399Y (central rod domain; high aggregation, and DesS460I (tail domain; moderate aggregation. Introduction of these proteins into a C2C12 background permitted us to compare between desmin variants as well as to determine the role of external stress on aggregation. Three different types of stress, likely encountered during muscle activity, were introduced to the cell models-thermal (heat shock, redox-associated (H2O2 and cadmium chloride, and mechanical (stretching stresses-after which aggregation was measured. Cells containing variant DesD399Y were more sensitive to stress, leading to marked cytoplasmic perinuclear aggregations. We then evaluated the capacity of biochemical compounds to prevent this aggregation, applying dexamethasone (an inducer of heat shock proteins, fisetin or N-acetyl-L-cysteine (antioxidants before stress induction. Interestingly, N-acetyl-L-cysteine pre-treatment prevented DesD399Y aggregation during most stress. N-acetyl-L-cysteine has recently been

  14. Protein aggregates as depots for the release of biologically active compounds.

    Science.gov (United States)

    Artemova, Natalya V; Kasakov, Alexei S; Bumagina, Zoya M; Lyutova, Elena M; Gurvits, Bella Ya

    2008-12-12

    Protein misfolding and aggregation is one of the most serious problems in cell biology, molecular medicine, and biotechnology. Misfolded proteins interact with each other or with other proteins in non-productive or damaging ways. However, a new paradigm arises that protein aggregation may be exploited by nature to perform specific functions in different biological contexts. From this consideration, acceleration of stress-induced protein aggregation triggered by any factor resulting in the formation of soluble aggregates may have paradoxical positive consequences. Here, we suggest that amorphous aggregates can act as a source for the release of biologically active proteins after removal of stress conditions. To address this concept, we investigated the kinetics of thermal aggregation in vitro of yeast alcohol dehydrogenase (ADH) as a model substrate in the presence of two amphiphilic peptides: Arg-Phe or Ala-Phe-Lys. Using dynamic light scattering (DLS) and turbidimetry, we have demonstrated that under mild stress conditions the concentration-dependent acceleration of ADH aggregation by these peptides results in formation of large but soluble complexes of proteins prone to refolding.

  15. Protein aggregation studied by forward light scattering and light transmission analysis

    Science.gov (United States)

    Penzkofer, A.; Shirdel, J.; Zirak, P.; Breitkreuz, H.; Wolf, E.

    2007-12-01

    The aggregation of the circadian blue-light photo-receptor cryptochrome from Drosophila melanogaster (dCry) is studied by transmission and forward light scattering measurement in the protein transparent wavelength region. The light scattering in forward direction is caused by Rayleigh scattering which is proportional to the degree of aggregation. The light transmission through the samples in the transparent region is reduced by Mie light scattering in all directions. It depends on the degree of aggregation and the monomer volume fill factor of the aggregates (less total scattering with decreasing monomer volume fill factor of protein globule) allowing a distinction between tightly packed protein aggregation (monomer volume fill factor 1) and loosely packed protein aggregation (monomer volume fill factor less than 1). An increase in aggregation with temperature, concentration, and blue-light exposure is observed. At a temperature of 4 °C and a protein concentration of less than 0.135 mM no dCry aggregation was observed, while at 24 °C and 0.327 mM gelation occurred (loosely packed aggregates occupying the whole solution volume).

  16. Heat-shock protein dysregulation is associated with functional and pathological TDP-43 aggregation

    Science.gov (United States)

    Chang, Hsiang-Yu; Hou, Shin-Chen; Way, Tzong-Der; Wong, Chi-Huey; Wang, I.-Fan

    2013-11-01

    Conformational disorders are involved in various neurodegenerative diseases. Reactive oxygen species (ROS) are the major contributors to neurodegenerative disease; however, ROS that affect the structural changes in misfolded disease proteins have yet to be well characterized. Here we demonstrate that the intrinsic propensity of TDP-43 to aggregate drives the assembly of TDP-43-positive stress granules and soluble toxic TDP-43 oligomers in response to a ROS insult via a disulfide crosslinking-independent mechanism. Notably, ROS-induced TDP-43 protein assembly correlates with the dynamics of certain TDP-43-associated chaperones. The heat-shock protein (HSP)-90 inhibitor 17-AAG prevents ROS-induced TDP-43 aggregation, alters the type of TDP-43 multimers and reduces the severity of pathological TDP-43 inclusions. In summary, our study suggests that a common mechanism could be involved in the pathogenesis of conformational diseases that result from HSP dysregulation.

  17. Protein aggregation in bacteria: the thin boundary between functionality and toxicity.

    Science.gov (United States)

    Bednarska, Natalia G; Schymkowitz, Joost; Rousseau, Frederic; Van Eldere, Johan

    2013-09-01

    Misfolding and aggregation of proteins have a negative impact on all living organisms. In recent years, aggregation has been studied in detail due to its involvement in neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, and type II diabetes--all associated with accumulation of amyloid fibrils. This research highlighted the central importance of protein homeostasis, or proteostasis for short, defined as the cellular state in which the proteome is both stable and functional. It implicates an equilibrium between synthesis, folding, trafficking, aggregation, disaggregation and degradation. In accordance with the eukaryotic systems, it has been documented that protein aggregation also reduces fitness of bacterial cells, but although our understanding of the cellular protein quality control systems is perhaps most detailed in bacteria, the use of bacterial proteostasis as a drug target remains little explored. Here we describe protein aggregation as a normal physiological process and its role in bacterial virulence and we shed light on how bacteria defend themselves against the toxic threat of aggregates. We review the impact of aggregates on bacterial viability and look at the ways that bacteria use to maintain a balance between aggregation and functionality. The proteostasis in bacteria can be interrupted via overexpression of proteins, certain antibiotics such as aminoglycosides, as well as antimicrobial peptides--all leading to loss of cell viability. Therefore intracellular protein aggregation and disruption of proteostatic balance in bacteria open up another strategy that should be explored towards the discovery of new antimicrobials.

  18. Cellular strategies to cope with protein aggregation

    NARCIS (Netherlands)

    Scior, Annika; Juenemann, Katrin; Kirstein, Janine

    2016-01-01

    Nature has evolved several mechanisms to detoxify intracellular protein aggregates that arise upon proteotoxic challenges. These include the controlled deposition of misfolded proteins at distinct cellular sites, the protein disaggregation and refolding by molecular chaperones and/or degradation of

  19. DENDRIMER CONJUGATES FOR SELECTIVE OF PROTEIN AGGREGATES

    DEFF Research Database (Denmark)

    2004-01-01

    Dendrimer conjugates are presented, which are formed between a dendrimer and a protein solubilising substance. Such dendrimer conjugates are effective in the treatment of protein aggregate-related diseases (e.g. prion-related diseases). The protein solubilising substance and the dendrimer together...

  20. Prevention of H-Aggregates Formation in Cy5 Labeled Macromolecules

    Directory of Open Access Journals (Sweden)

    Jing Kang

    2010-01-01

    Full Text Available H-aggregates of the cyanine dye Cy5 are formed during covalent linkage to the cationic macromolecule Poly(allylamine (PAH. The nonfluorescent H-aggregates strongly restrict the usage of the dye for analytical purposes and prevent a quantitative determination of the labeled macromolecules. The behavior of the H-aggregates has been studied by investigation of the absorption and fluorescence spectra of the dye polymer in dependence on solvent, label degree and additional sulfonate groups. H-aggregate formation is caused by an inhomogeneous distribution of the Cy5 molecules on the polymer chain. The H-aggregates can be destroyed by conformational changes of the PAH induced by interactions with polyanions or in organic solvents. It has been found that the polymer labeling process in high content of organic solvents can prevent the formation of H-aggregates. The results offer a better understanding and improvement of the use of the Cy5 dye for labeling purposes in fluorescence detection of macromolecules.

  1. In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL.

    Science.gov (United States)

    Kumar, Vipul; Punetha, Ankita; Sundar, Durai; Chaudhuri, Tapan K

    2012-01-01

    Molecular chaperones appear to have been evolved to facilitate protein folding in the cell through entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES. They bind newly synthesized or non-native polypeptides through hydrophobic interactions and prevent their aggregation. Some proteins do not interact with GroEL, hence even though they are aggregation prone, cannot be assisted by GroEL for their folding. In this study, we have attempted to engineer these non-substrate proteins to convert them as the substrate for GroEL, without compromising on their function. We have used a computational biology approach to generate mutants of the selected proteins by selectively mutating residues in the hydrophobic patch, similar to GroES mobile loop region that are responsible for interaction with GroEL, and compared with the wild counterparts for calculation of their instability and aggregation propensities. The energies of the newly designed mutants were computed through molecular dynamics simulations. We observed increased aggregation propensity of some of the mutants formed after replacing charged amino acid residues with hydrophobic ones in the well defined hydrophobic patch, raising the possibility of their binding ability to GroEL. The newly generated mutants may provide potential substrates for Chaperonin GroEL, which can be experimentally generated and tested for their tendency of aggregation, interactions with GroEL and the possibility of chaperone-assisted folding to produce functional proteins.

  2. Resolving the paradox for protein aggregation diseases: a common mechanism for aggregated proteins to initially attack membranes without needing aggregates [v1; ref status: indexed, http://f1000r.es/221

    Directory of Open Access Journals (Sweden)

    Haina Qin

    2013-10-01

    Full Text Available Paradoxically, aggregation of specific proteins is characteristic of many human diseases and aging, yet aggregates have been found to be unnecessary for initiating pathogenesis. Here we determined the NMR topology and dynamics of a helical mutant in a membrane environment transformed from the 125-residue cytosolic all-β MSP by the ALS-causing P56S mutation. Unexpectedly, despite its low hydrophobicity, the P56S major sperm protein (MSP domain becomes largely embedded in the membrane environment with high backbone rigidity. Furthermore it is composed of five helices with amphiphilicity comparable to those of the partly-soluble membrane toxin mellitin and α-synuclein causing Parkinson's disease. Consequently, the mechanism underlying this chameleon transformation becomes clear: by disrupting the specific tertiary interaction network stabilizing the native all-β MSP fold to release previously-locked amphiphilic segments, the P56S mutation acts to convert the classic MSP fold into a membrane-active protein that is fundamentally indistinguishable from mellitin and α-synuclein which are disordered in aqueous solution but spontaneously partition into membrane interfaces driven by hydrogen-bond energetics gained from forming α-helix in the membrane environments. As segments with high amphiphilicity exist in all proteins, our study successfully resolves the paradox by deciphering that the proteins with a higher tendency to aggregate have a stronger potential to partition into membranes through the same mechanism as α-synuclein to initially attack membranes to trigger pathogenesis without needing aggregates. This might represent the common first step for various kinds of aggregated proteins to trigger familiar, sporadic and aging diseases. Therefore the homeostasis of aggregated proteins in vivo is the central factor responsible for a variety of human diseases including aging. The number and degree of the membrane attacks by aggregated proteins may

  3. Aspirin-Mediated Acetylation Protects Against Multiple Neurodegenerative Pathologies by Impeding Protein Aggregation.

    Science.gov (United States)

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Kakraba, Samuel; Alla, Ramani; Mehta, Jawahar L; Shmookler Reis, Robert J

    2017-12-10

    Many progressive neurological disorders, including Alzheimer's disease (AD), Huntington's disease, and Parkinson's disease (PD), are characterized by accumulation of insoluble protein aggregates. In prospective trials, the cyclooxygenase inhibitor aspirin (acetylsalicylic acid) reduced the risk of AD and PD, as well as cardiovascular events and many late-onset cancers. Considering the role played by protein hyperphosphorylation in aggregation and neurodegenerative diseases, and aspirin's known ability to donate acetyl groups, we asked whether aspirin might reduce both phosphorylation and aggregation by acetylating protein targets. Aspirin was substantially more effective than salicylate in reducing or delaying aggregation in human neuroblastoma cells grown in vitro, and in Caenorhabditis elegans models of human neurodegenerative diseases in vivo. Aspirin acetylates many proteins, while reducing phosphorylation, suggesting that acetylation may oppose phosphorylation. Surprisingly, acetylated proteins were largely excluded from compact aggregates. Molecular-dynamic simulations indicate that acetylation of amyloid peptide energetically disfavors its association into dimers and octamers, and oligomers that do form are less compact and stable than those comprising unacetylated peptides. Hyperphosphorylation predisposes certain proteins to aggregate (e.g., tau, α-synuclein, and transactive response DNA-binding protein 43 [TDP-43]), and it is a critical pathogenic marker in both cardiovascular and neurodegenerative diseases. We present novel evidence that acetylated proteins are underrepresented in protein aggregates, and that aggregation varies inversely with acetylation propensity after diverse genetic and pharmacologic interventions. These results are consistent with the hypothesis that aspirin inhibits protein aggregation and the ensuing toxicity of aggregates through its acetyl-donating activity. This mechanism may contribute to the neuro-protective, cardio

  4. Strain-dependent profile of misfolded prion protein aggregates.

    Science.gov (United States)

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V; Soto, Claudio

    2016-02-15

    Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration.

  5. Role of foam drainage in producing protein aggregates in foam fractionation.

    Science.gov (United States)

    Li, Rui; Zhang, Yuran; Chang, Yunkang; Wu, Zhaoliang; Wang, Yanji; Chen, Xiang'e; Wang, Tao

    2017-10-01

    It is essential to obtain a clear understanding of the foam-induced protein aggregation to reduce the loss of protein functionality in foam fractionation. The major effort of this work is to explore the roles of foam drainage in protein aggregation in the entire process of foam fractionation with bovine serum albumin (BSA) as a model protein. The results show that enhancing foam drainage increased the desorption of BSA molecules from the gas-liquid interface and the local concentration of desorbed molecules in foam. Therefore, it intensified the aggregation of BSA in foam fractionation. Simultaneously, it also accelerated the flow of BSA aggregates from rising foam into the residual solution along with the drained liquid. Because enhancing foam drainage increased the relative content of BSA molecules adsorbed at the gas-liquid interface, it also intensified the aggregation of BSA during both the defoaming process and the storage of the foamate. Furthermore, enhancing foam drainage more readily resulted in the formation of insoluble BSA aggregates. The results are highly important for a better understanding of foam-induced protein aggregation in foam fractionation. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Role of Prion Protein Aggregation in Neurotoxicity

    Directory of Open Access Journals (Sweden)

    Tullio Florio

    2012-07-01

    Full Text Available In several neurodegenerative diseases, such as Parkinson, Alzheimer’s, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP, the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126 and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.

  7. Protein corona between nanoparticles and bacterial proteins in activated sludge: Characterization and effect on nanoparticle aggregation.

    Science.gov (United States)

    Zhang, Peng; Xu, Xiao-Yan; Chen, You-Peng; Xiao, Meng-Qian; Feng, Bo; Tian, Kai-Xun; Chen, Yue-Hui; Dai, You-Zhi

    2018-02-01

    In this work, the protein coronas of activated sludge proteins on TiO 2 nanoparticles (TNPs) and ZnO nanoparticles (ZNPs) were characterized. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and their effects of on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of protein coronas on the aggregations of TNPs and ZNPs were evaluated. Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than to ZNPs. The aggregation percentages of ZNPs at 35, 53, and 106 mg/L of proteins were 13%, 14%, and 18%, respectively, whereas those of TNPs were 21%, 30%, 41%, respectively. The proteins contributed to ZNPs aggregation by dissolved Zn ion-bridging, whereas the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Uncoupling of Protein Aggregation and Neurodegeneration in a Mouse Amyotrophic Lateral Sclerosis Model.

    Science.gov (United States)

    Lee, Joo-Yong; Kawaguchi, Yoshiharu; Li, Ming; Kapur, Meghan; Choi, Su Jin; Kim, Hak-June; Park, Song-Yi; Zhu, Haining; Yao, Tso-Pang

    2015-01-01

    Aberrant accumulation of protein aggregates is a pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Although a buildup of protein aggregates frequently leads to cell death, whether it is the key pathogenic factor in driving neurodegenerative disease remains controversial. HDAC6, a cytosolic ubiquitin-binding deacetylase, has emerged as an important regulator of ubiquitin-dependent quality control autophagy, a lysosome-dependent degradative system responsible for the disposal of misfolded protein aggregates and damaged organelles. Here, we show that in cell models HDAC6 plays a protective role against multiple disease-associated and aggregation-prone cytosolic proteins by facilitating their degradation. We further show that HDAC6 is required for efficient localization of lysosomes to protein aggregates, indicating that lysosome targeting to autophagic substrates is regulated. Supporting a critical role of HDAC6 in protein aggregate disposal in vivo, genetic ablation of HDAC6 in a transgenic SOD1G93A mouse, a model of ALS, leads to dramatic accumulation of ubiquitinated SOD1G93A protein aggregates. Surprisingly, despite a robust buildup of SOD1G93A aggregates, deletion of HDAC6 only moderately modified the motor phenotypes. These findings indicate that SOD1G93A aggregation is not the only determining factor to drive neurodegeneration in ALS, and that HDAC6 likely modulates neurodegeneration through additional mechanisms beyond protein aggregate clearance. © 2015 S. Karger AG, Basel.

  9. Heat-Induced Soluble Protein Aggregates from Mixed Pea Globulins and β-Lactoglobulin.

    Science.gov (United States)

    Chihi, Mohamed-Lazhar; Mession, Jean-luc; Sok, Nicolas; Saurel, Rémi

    2016-04-06

    The present work investigates the formation of protein aggregates (85 °C, 60 min incubation) upon heat treatment of β-lactoglobulin (βlg)-pea globulins (Glob) mixtures at pH 7.2 and 5 mM NaCl from laboratory-prepared protein isolates. Various βlg/Glob weight ratios were applied, for a total protein concentration of 2 wt % in admixture. Different analytical methods were used to determine the aggregation behavior of "mixed" aggregates, that is, surface hydrophobicity and also sulfhydryl content, protein interactions by means of SDS-PAGE electrophoresis, and molecule size distribution by DLS and gel filtration. The production of "mixed" thermal aggregates would involve both the formation of new disulfide bonds and noncovalent interactions between the denatured βlg and Glob subunits. The majority of "mixed" soluble aggregates displayed higher molecular weight and smaller diameter than those for Glob heated in isolation. The development of pea-whey protein "mixed" aggregates may help to design new ingredients for the control of innovative food textures.

  10. Aggregation propensity of critical regions of the protein Tau

    Science.gov (United States)

    Muthee, Micaiah; Ahmed, Azka; Larini, Luca

    The Alzheimer's disease is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, which eventually leads to the ability to not able to carry out the simplest tasks. The Alzheimer's disease is characterized by the formation of protein aggregates both within and outside of the brain's cells, the neurons. Within the neurons, the aggregation of the protein tau leads to the destruction of the microtubules in the axon of the neuron. Tau belongs to a group of proteins referred to as Microtubule-Associated Proteins. It is extremely flexible and is classified as an intrinsically unstructured protein due to its low propensity to form secondary structure. Tau promotes tubulin assembly into microtubules thereby stabilizing the cytoskeleton of the axon of the neurons. The microtubule binding region of tau consists of 4 pseudo-repeats. In this study, we will focus on the aggregation propensity of two fragments. In this study we will focus on the PHF43 fragment that contains the third pseudo-repeat and has been shown experimentally to aggregate readily. Another fragment that contains the second pseudo-repeat will be considered as well. Mutations in this region are associated with various form of dementia and for this reason we will consider the mutant P301L.

  11. Small surfactant-like peptides can drive soluble proteins into active aggregates

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2012-01-01

    Full Text Available Abstract Background Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF and a short beta structure peptide ELK16 (LELELKLKLELELKLK have a similar property. Results In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6 were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used, Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same, Bacillus pumilus xylosidase (XynB, and green fluorescent protein (GFP, and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core. Conclusions This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in

  12. Al cation induces aggregation of serum proteins.

    Science.gov (United States)

    Chanphai, P; Kreplak, L; Tajmir-Riahi, H A

    2017-07-15

    Al cation is known to induce protein fibrillation and causes several neurodegenerative disorders. We report the spectroscopic, thermodynamic analysis and AFM imaging for the Al cation binding process with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. Hydrophobicity played a major role in Al-protein interactions with more hydrophobic b-LG forming stronger Al-protein complexes. Thermodynamic parameters ΔS, ΔH and ΔG showed Al-protein bindings occur via hydrophobic and H-bonding contacts for b-LG, while van der Waals and H-bonding interactions prevail in HSA and BSA adducts. AFM clearly indicated that aluminum cations are able to force BSA and b-LG into larger or more robust aggregates than HSA, with HSA 4±0.2 (SE, n=801) proteins per aggregate, for BSA 17±2 (SE, n=148), and for b-LG 12±3 (SE, n=151). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced major alterations of protein conformations with the order of perturbations b-LG>BSA>HSA. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Age- and Hypertension-Associated Protein Aggregates in Mouse Heart Have Similar Proteomic Profiles.

    Science.gov (United States)

    Ayyadevara, Srinivas; Mercanti, Federico; Wang, Xianwei; Mackintosh, Samuel G; Tackett, Alan J; Prayaga, Sastry V S; Romeo, Francesco; Shmookler Reis, Robert J; Mehta, Jawahar L

    2016-05-01

    Neurodegenerative diseases are largely defined by protein aggregates in affected tissues. Aggregates contain some shared components as well as proteins thought to be specific for each disease. Aggregation has not previously been reported in the normal, aging heart or the hypertensive heart. Detergent-insoluble protein aggregates were isolated from mouse heart and characterized on 2-dimensional gels. Their levels increased markedly and significantly with aging and after sustained angiotensin II-induced hypertension. Of the aggregate components identified by high-resolution proteomics, half changed in abundance with age (392/787) or with sustained hypertension (459/824), whereas 30% (273/901) changed concordantly in both, each Phypertensive hearts, we posited that aging of fibroblasts may contribute to the aggregates observed in cardiac tissue. Indeed, as cardiac myofibroblasts "senesced" (approached their replicative limit) in vitro, they accrued aggregates with many of the same constituent proteins observed in vivo during natural aging or sustained hypertension. In summary, we have shown for the first time that compact (detergent-insoluble) protein aggregates accumulate during natural aging, chronic hypertension, and in vitro myofibroblast senescence, sharing many common proteins. Thus, aggregates that arise from disparate causes (aging, hypertension, and replicative senescence) may have common underlying mechanisms of accrual. © 2016 American Heart Association, Inc.

  14. Localization of aggregating proteins in bacteria depends on the rate of addition

    Directory of Open Access Journals (Sweden)

    Karl eScheu

    2014-08-01

    Full Text Available Many proteins are observed to localize to specific subcellular regions within bacteria. Recent experiments have shown that proteins that have self-interactions that lead them to aggregate tend to localize to the poles. Theoretical modeling of the localization of aggregating protein within bacterial cell geometries shows that aggregates can spontaneously localize to the pole due to nucleoid occlusion. The resulting polar localization, whether it be to a single pole or to both was shown to depend on the rate of protein addition. Motivated by these predictions we selected a set of genes from E. coli, whose protein products have been reported to localize when tagged with GFP, and explored the dynamics of their localization. We induced protein expression from each gene at different rates and found that in all cases unipolar patterning is favored at low rates of expression whereas bipolar is favored at higher rates of expression. Our findings are consistent with the predictions of the model, suggesting that localization may be due to aggregation plus nucleoid occlusion. When we expressed GFP by itself under the same conditions, no localization was observed. These experiments highlight the potential importance of protein aggregation, nucleoid occlusion and rate of protein expression in driving polar localization of functional proteins in bacteria.

  15. Uncovering methods for the prevention of protein aggregation and improvement of product quality in a transient expression system.

    Science.gov (United States)

    Estes, Bram; Hsu, Yueh-Rong; Tam, Lei-Ting; Sheng, Jackie; Stevens, Jennitte; Haldankar, Raj

    2015-01-01

    Mammalian expression systems are used routinely for the production of recombinant proteins as therapeutic molecules as well as research tools. Transient expression has become increasingly popular in recent years due to its rapid timeline and improvements in expression level. While improvements to transient expression systems have focused mainly on the level of protein expression, the aspect of protein quality has received little attention. The removal of undesirable products, such as aggregation, depends primarily on purification, requiring additional cumbersome steps, which can lead to a lower product yield and longer timelines. In this study, we show that reducing the level of transcription by transfecting at a lower gene dose improves the quality of secreted molecules prone to aggregation. For gene dosing to have this effect, it is critical for the carrier DNA to be an empty vector containing the same elements as the gene containing plasmid. This approach can be used in combination with a temperature shift to hypothermic conditions during production to enhance the effect. The observed improvements not only minimized aggregation levels, but also generated products with overall superior quality, including more homogeneous signal peptide cleavage and N-linked glycosylation profiles. These techniques have produced a similar improvement in product quality with a variety of other molecules, suggesting that this may be a general approach to enhance product quality from transient expression systems. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

  16. Protein aggregation in food models: effect of γ-irradiation and lipid oxidation

    International Nuclear Information System (INIS)

    Delincee, H.; Paul, P.

    1981-01-01

    Myoglobin and serum albumin have been irradiated in aqueous solution in the presence of varying amounts of carbohydrates and lipids, and the yield of protein aggregates has been determined by gel filtration. With myoglobin the formation of aggregates evolving from the reaction with oxidizing lipids was observed, which was not found for serum albumin. The production of protein-lipid complexes, in which lipid material was occluded in the high-molecular aggregates by physical forces was demonstrated. Gel filtration and gel electrophoresis, both in the presence of SDS, and thin-layer isoelectric focusing revealed distinct structural differenes between the protein aggregates induced by irradiation and the aggregates formed by interaction with oxidizing lipids

  17. Hsp72 (HSPA1A Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment.

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    Paola C Rosas

    Full Text Available Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A, significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity.

  18. Computational study of aggregation mechanism in human lysozyme[D67H].

    Directory of Open Access Journals (Sweden)

    Dharmeshkumar Patel

    Full Text Available Aggregation of proteins is an undesired phenomena that affects both human health and bioengineered products such as therapeutic proteins. Finding preventative measures could be facilitated by a molecular-level understanding of dimer formation, which is the first step in aggregation. Here we present a molecular dynamics (MD study of dimer formation propensity in human lysozyme and its D67H variant. Because the latter protein aggregates while the former does not, they offer an ideal system for testing the feasibility of the proposed MD approach which comprises three stages: i partially unfolded conformers involved in dimer formation are generated via high-temperature MD simulations, ii potential dimer structures are searched using docking and refined with MD, iii free energy calculations are performed to find the most stable dimer structure. Our results provide a detailed explanation for how a single mutation (D67H turns human lysozyme from non-aggregating to an aggregating protein. Conversely, the proposed method can be used to identify the residues causing aggregation in a protein, which can be mutated to prevent it.

  19. Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

    Directory of Open Access Journals (Sweden)

    Pilar Eraso

    Full Text Available The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.

  20. Dynamics of proteins aggregation. II. Dynamic scaling in confined media

    Science.gov (United States)

    Zheng, Size; Shing, Katherine S.; Sahimi, Muhammad

    2018-03-01

    In this paper, the second in a series devoted to molecular modeling of protein aggregation, a mesoscale model of proteins together with extensive discontinuous molecular dynamics simulation is used to study the phenomenon in a confined medium. The medium, as a model of a crowded cellular environment, is represented by a spherical cavity, as well as cylindrical tubes with two aspect ratios. The aggregation process leads to the formation of β sheets and eventually fibrils, whose deposition on biological tissues is believed to be a major factor contributing to many neuro-degenerative diseases, such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis diseases. Several important properties of the aggregation process, including dynamic evolution of the total number of the aggregates, the mean aggregate size, and the number of peptides that contribute to the formation of the β sheets, have been computed. We show, similar to the unconfined media studied in Paper I [S. Zheng et al., J. Chem. Phys. 145, 134306 (2016)], that the computed properties follow dynamic scaling, characterized by power laws. The existence of such dynamic scaling in unconfined media was recently confirmed by experiments. The exponents that characterize the power-law dependence on time of the properties of the aggregation process in spherical cavities are shown to agree with those in unbounded fluids at the same protein density, while the exponents for aggregation in the cylindrical tubes exhibit sensitivity to the geometry of the system. The effects of the number of amino acids in the protein, as well as the size of the confined media, have also been studied. Similarities and differences between aggregation in confined and unconfined media are described, including the possibility of no fibril formation, if confinement is severe.

  1. Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

    Science.gov (United States)

    Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

    2016-02-01

    Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation. © 2015 Wiley Periodicals, Inc.

  2. Fundamentals of unfolding, refolding and aggregation of food proteins

    NARCIS (Netherlands)

    Broersen, K.

    2005-01-01

    Protein functionality in food products strongly relies on the fact that proteins can undergo intermolecular interactions, called aggregation. It was found that very subtle dynamics inherent to the protein of interest can have consequences for the functional properties of proteins. The aim of this

  3. Aggregation of egg white proteins with pulsed electric fields and thermal processes.

    Science.gov (United States)

    Wu, Li; Zhao, Wei; Yang, Ruijin; Yan, Wenxu; Sun, Qianyan

    2016-08-01

    Pulsed electric field (PEF) processing is progressing towards application for liquid egg to ensure microbial safety. However, it usually causes protein aggregation, and the mechanism is still unclear. In this study, egg white protein was applied to investigate the changes in protein structure and mechanism of aggregates formation and a comparison was made with thermal treatment. Soluble protein content decreased with the increase of turbidity after both treatments. Fluorescence intensity and free sulfhydryl content were increased after being treated at 70 °C for 4 min. Less-remarkable changes of hydrophobicity were observed after PEF treatments (30 kV cm(-1) , 800 µs). Soluble and insoluble aggregates were observed by thermal treatment, and disulfide bonds were the main binding forces. The main components of insoluble aggregates formed by thermal treatment were ovotransferrin (30.58%), lysozyme (18.47%) and ovalbumin (14.20%). While only insoluble aggregates were detected during PEF processes, which consists of ovotransferrin (11.86%), lysozyme (21.11%) and ovalbumin (31.07%). Electrostatic interaction played a very important role in the aggregates formation. PEF had a minor impact on the structure of egg white protein. PEF had insignificant influence on heat-sensitive protein, indicating that PEF has potential in processing food with high biological activity and heat sensitive properties. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  4. Effects of Polymer Hydrophobicity on Protein Structure and Aggregation Kinetics in Crowded Milieu.

    Science.gov (United States)

    Breydo, Leonid; Sales, Amanda E; Frege, Telma; Howell, Mark C; Zaslavsky, Boris Y; Uversky, Vladimir N

    2015-05-19

    We examined the effects of water-soluble polymers of various degrees of hydrophobicity on the folding and aggregation of proteins. The polymers we chose were polyethylene glycol (PEG) and UCON (1:1 copolymer of ethylene glycol and propylene glycol). The presence of additional methyl groups in UCON makes it more hydrophobic than PEG. Our earlier analysis revealed that similarly sized PEG and UCON produced different changes in the solvent properties of water in their solutions and induced morphologically different α-synuclein aggregates [Ferreira, L. A., et al. (2015) Role of solvent properties of aqueous media in macromolecular crowding effects. J. Biomol. Struct. Dyn., in press]. To improve our understanding of molecular mechanisms defining behavior of proteins in a crowded environment, we tested the effects of these polymers on secondary and tertiary structure and aromatic residue solvent accessibility of 10 proteins [five folded proteins, two hybrid proteins; i.e., protein containing ordered and disordered domains, and three intrinsically disordered proteins (IDPs)] and on the aggregation kinetics of insulin and α-synuclein. We found that effects of both polymers on secondary and tertiary structures of folded and hybrid proteins were rather limited with slight unfolding observed in some cases. Solvent accessibility of aromatic residues was significantly increased for the majority of the studied proteins in the presence of UCON but not PEG. PEG also accelerated the aggregation of protein into amyloid fibrils, whereas UCON promoted aggregation to amyloid oligomers instead. These results indicate that even a relatively small change in polymer structure leads to a significant change in the effect of this polymer on protein folding and aggregation. This is an indication that protein folding and especially aggregation are highly sensitive to the presence of other macromolecules, and an excluded volume effect is insufficient to describe their effect.

  5. Acid-Induced Cold Gelation of Globular Proteins: Effects of Protein Aggregate Characteristics and Disulfide Bonding on Rheological Properties

    NARCIS (Netherlands)

    Alting, A.C.; Weijers, M.; Hoog, E.H.A. de; Pijpekamp, A.M. van de; Cohen Stuart, M.A.; Hamer, R.J.; Kruif, C.G. de; Visschers, R.W.

    2004-01-01

    The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked

  6. Protein aggregation in aqueous casein solution

    International Nuclear Information System (INIS)

    Yousri, R.M.

    1980-01-01

    From the vast amount of research efforts dealing with various aspects of radiation effects on foods and food components (11, 18, 5, 12, 19, 8, 9, 6, 13, 15, 17, 20), it is apparent up to now that much remains to be studied in depth, much may have to be added or corrected about radiation-induced physico-chemical changes in foods. A great many reactions that take place when foodstuffs are subjected to ionizing radiation are still not fully understood. The better understanding of some of the radiation-induced changes in pure proteins as such or in mixture with other food constituents could yield much data which could be meaningfully extrapolated to intact foods and consequently could help to improve the assessment of the wholesomeness of irradiated foods. It was the purpose of our investigations to elucidate some of the changes in the chemical structure of a pure protein (casein), irradiated as such or which added carbohydrate and/or lipid. The effect of subsequent storage of the irradiated solutions has been also examined. The formation of protein aggregates was studied by gel filtration technique. The application of thin-layer gel filtration, its speed and adaptability to very small samples facilitated the measurements of the extent of aggregation which occurred in protein molecules after irradiation. (orig.) [de

  7. Sulfur dioxide induced aggregation of wine thaumatin-like proteins: Role of disulfide bonds.

    Science.gov (United States)

    Chagas, Ricardo; Laia, César A T; Ferreira, Ricardo B; Ferreira, Luísa M

    2018-09-01

    Aggregation of heat unstable wine proteins is responsible for the economically and technologically detrimental problem called wine protein haze. This is caused by the aggregation of thermally unfolded proteins that can precipitate in bottled wine. To study the influence of SO 2 in this phenomenon, wine proteins were isolated and thaumatins were identified has the most prone to aggregate in the presence of this compound. Isolated wine thaumatins aggregation was followed by dynamic light scattering (DLS), circular dichroism (CD), fluorescence spectroscopy and size exclusion chromatography (SEC). Our experimental results demonstrate that protein thermal unfolding after exposure of the protein to 70 °C does not present differences whether SO 2 is present or not. Conversely, when the protein solution is cooled to 15 °C (after heat stress) significant analytical changes can be observed between samples with and without SO 2 . A remarkable change of circular dichroism spectra in the region 220-230 nm is observed (which can be related to S-S torsion angles), as well as an increase in tryptophan fluorescence intensity (absence of fluorescence quenching by S-S bonds). Formation of covalently-linked dimeric and tetrameric protein species were also detected by SEC. The ability to dissolve the aggregates with 8 M urea seems to indicate that hydrophobic interactions are prevalent in the formed aggregates. Also, the reduction of these aggregates with tris (2-carboxyethyl) phosphine (TCEP) to only monomeric species reveals the presence of intermolecular S-S bonds. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Dynamics of protein aggregation and oligomer formation governed by secondary nucleation

    Energy Technology Data Exchange (ETDEWEB)

    Michaels, Thomas C. T., E-mail: tctm3@cam.ac.uk; Lazell, Hamish W.; Arosio, Paolo; Knowles, Tuomas P. J., E-mail: tpjk2@cam.ac.uk [Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW (United Kingdom)

    2015-08-07

    The formation of aggregates in many protein systems can be significantly accelerated by secondary nucleation, a process where existing assemblies catalyse the nucleation of new species. In particular, secondary nucleation has emerged as a central process controlling the proliferation of many filamentous protein structures, including molecular species related to diseases such as sickle cell anemia and a range of neurodegenerative conditions. Increasing evidence suggests that the physical size of protein filaments plays a key role in determining their potential for deleterious interactions with living cells, with smaller aggregates of misfolded proteins, oligomers, being particularly toxic. It is thus crucial to progress towards an understanding of the factors that control the sizes of protein aggregates. However, the influence of secondary nucleation on the time evolution of aggregate size distributions has been challenging to quantify. This difficulty originates in large part from the fact that secondary nucleation couples the dynamics of species distant in size space. Here, we approach this problem by presenting an analytical treatment of the master equation describing the growth kinetics of linear protein structures proliferating through secondary nucleation and provide closed-form expressions for the temporal evolution of the resulting aggregate size distribution. We show how the availability of analytical solutions for the full filament distribution allows us to identify the key physical parameters that control the sizes of growing protein filaments. Furthermore, we use these results to probe the dynamics of the populations of small oligomeric species as they are formed through secondary nucleation and discuss the implications of our work for understanding the factors that promote or curtail the production of these species with a potentially high deleterious biological activity.

  9. The missing piece in the puzzle: Prediction of aggregation via the protein-protein interaction parameter A∗2.

    Science.gov (United States)

    Koepf, Ellen; Schroeder, Rudolf; Brezesinski, Gerald; Friess, Wolfgang

    2018-07-01

    The tendency of protein pharmaceuticals to form aggregates is a major challenge during formulation development, as aggregation affects quality and safety of the product. In particular, the formation of large native-like particles in the context of liquid-air interfacial stress is a well-known but not fully understood problem. Focusing on the two most fundamental criteria of protein formulation affecting protein-protein interaction, the impact of pH and ionic strength on the interaction parameter A ∗ 2 and its link to aggregation upon mechanical stress was investigated. A ∗ 2 of two monoclonal antibodies (mABs) and a polyclonal IgG was determined using dynamic light scattering and was correlated to the number of particles formed upon shaking in vials analyzed by visual inspection, turbidity analysis, light obscuration and micro-flow imaging. A good correlation between aggregation induced by interfacial stress and formulation pH was given. It could be shown that A ∗ 2 was highest for mAB 1 and lowest for IgG, what was in good accordance with the number of particles formed. Shaking of IgG resulted in overall higher numbers of particles compared to the two mABs. A ∗ 2 decreased and particle numbers increased with increasing pH. Different to pH, ionic strength only slightly affected A ∗ 2 . Nevertheless, at high ionic (100 mM) strength the samples exhibited more pronounced particle formation, particularly of large particles >25 µm, which was most pronounced at high pH. Protein solutions were identified to form continuous films with an inhomogeneous protein distribution at the liquid-air interface. These areas of agglomerated, native-like protein material can be transferred into the bulk solution by compression-decompression of the interface. Whether or not those clusters lead to the appearance of large protein aggregates or fall apart depends on the attractive or repulsive forces between protein molecules. Thus, protein aggregation due to interfacial

  10. Effect of electrostatics on aggregation of prion protein Sup35 peptide

    International Nuclear Information System (INIS)

    Portillo, Alexander M; Krasnoslobodtsev, Alexey V; Lyubchenko, Yuri L

    2012-01-01

    Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. The GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI = 5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0), suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 h, whereas the lag time decreases ∼5 times when the ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 h under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. The ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics to dissociation of transient peptide dimers. (paper)

  11. Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity

    Science.gov (United States)

    Cheng, Pin-Nan; Liu, Cong; Zhao, Minglei; Eisenberg, David; Nowick, James S.

    2012-11-01

    The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloid-β peptide associated with Alzheimer's disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

  12. Effect of dynamic high pressure homogenization on the aggregation state of soy protein.

    Science.gov (United States)

    Keerati-U-Rai, Maneephan; Corredig, Milena

    2009-05-13

    Although soy proteins are often employed as functional ingredients in oil-water emulsions, very little is known about the aggregation state of the proteins in solution and whether any changes occur to soy protein dispersions during homogenization. The effect of dynamic high pressure homogenization on the aggregation state of the proteins was investigated using microdifferential scanning calorimetry and high performance size exclusion chromatography coupled with multiangle laser light scattering. Soy protein isolates as well as glycinin and beta-conglycinin fractions were prepared from defatted soy flakes and redispersed in 50 mM sodium phosphate buffer at pH 7.4. The dispersions were then subjected to homogenization at two different pressures, 26 and 65 MPa. The results demonstrated that dynamic high pressure homogenization causes changes in the supramolecular structure of the soy proteins. Both beta-conglycinin and glycinin samples had an increased temperature of denaturation after homogenization. The chromatographic elution profile showed a reduction in the aggregate concentration with homogenization pressure for beta-conglycinin and an increase in the size of the soluble aggregates for glycinin and soy protein isolate.

  13. Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect.

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Coquel

    2013-04-01

    Full Text Available Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian. Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of "soft" intracellular structuring (based on

  14. Curcumin inhibits aggregation of alpha-synuclein.

    Science.gov (United States)

    Pandey, Neeraj; Strider, Jeffrey; Nolan, William C; Yan, Sherry X; Galvin, James E

    2008-04-01

    Aggregation of amyloid-beta protein (Abeta) is a key pathogenic event in Alzheimer's disease (AD). Curcumin, a constituent of the Indian spice Turmeric is structurally similar to Congo Red and has been demonstrated to bind Abeta amyloid and prevent further oligomerization of Abeta monomers onto growing amyloid beta-sheets. Reasoning that oligomerization kinetics and mechanism of amyloid formation are similar in Parkinson's disease (PD) and AD, we investigated the effect of curcumin on alpha-synuclein (AS) protein aggregation. In vitro model of AS aggregation was developed by treatment of purified AS protein (wild-type) with 1 mM Fe3+ (Fenton reaction). It was observed that the addition of curcumin inhibited aggregation in a dose-dependent manner and increased AS solubility. The aggregation-inhibiting effect of curcumin was next investigated in cell culture utilizing catecholaminergic SH-SY5Y cell line. A model system was developed in which the red fluorescent protein (DsRed2) was fused with A53T mutant of AS and its aggregation examined under different concentrations of curcumin. To estimate aggregation in an unbiased manner, a protocol was developed in which the images were captured automatically through a high-throughput cell-based screening microscope. The obtained images were processed automatically for aggregates within a defined dimension of 1-6 microm. Greater than 32% decrease in mutant alpha-synuclein aggregation was observed within 48 h subsequent to curcumin addition. Our data suggest that curcumin inhibits AS oligomerization into higher molecular weight aggregates and therefore should be further explored as a potential therapeutic compound for PD and related disorders.

  15. Aggregation in concentrated protein solutions: Insights from rheology, neutron scattering and molecular simulations

    Science.gov (United States)

    Castellanos, Maria Monica

    Aggregation of therapeutic proteins is currently one of the major challenges in the bio-pharmaceutical industry, because aggregates could induce immunogenic responses and compromise the quality of the product. Current scientific efforts, both in industry and academia, are focused on developing rational approaches to screen different drug candidates and predict their stability under different conditions. Moreover, aggregation is promoted in highly concentrated protein solutions, which are typically required for subcutaneous injection. In order to gain further understanding about the mechanisms that lead to aggregation, an approach that combined rheology, neutron scattering, and molecular simulations was undertaken. Two model systems were studied in this work: Bovine Serum Albumin in surfactant-free Phosphate Buffered Saline at pH = 7.4 at concentrations from 11 mg/mL up to ˜519 mg/mL, and a monoclonal antibody in 20 mM Histidine/Histidine Hydrochloride at pH = 6.0 with 60 mg/mL trehalose and 0.2 mg/mL polysorbate-80 at concentrations from 53 mg/mL up to ˜220 mg/mL. The antibody used here has three mutations in the CH2 domain, which result in lower stability upon incubation at 40 °C with respect to the wild-type protein, based on size-exclusion chromatography assays. This temperature is below 49 °C, where unfolding of the least stable, CH2 domain occurs, according to differential scanning calorimetry. This dissertation focuses on identifying the role of aggregation on the viscosity of protein solutions. The protein solutions of this work show an increase in the low shear viscosity in the absence of surfactants, because proteins adsorb at the air/water interface forming a viscoelastic film that affects the measured rheology. Stable surfactant-laden protein solutions behave as simple Newtonian fluids. However, the surfactant-laden antibody solution also shows an increase in the low shear viscosity from bulk aggregation, after prolonged incubation at 40 °C. Small

  16. State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation.

    Science.gov (United States)

    Kitamura, Akira; Kinjo, Masataka

    2018-03-23

    Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and Huntington's disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In the current review, we present an overview of the principles and application of several methodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N&B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases.

  17. Protein aggregates and novel presenilin gene variants in idiopathic dilated cardiomyopathy.

    Science.gov (United States)

    Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E; del Monte, Federica

    2010-03-16

    Heart failure is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as idiopathic dilated cardiomyopathy (iDCM), the origin of heart failure is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of beta-amyloid impair cell function and lead to cell death. We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients, pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca(2+) homeostasis. Additionally, we have identified 2 new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 coimmunoprecipitates with SERCA2a. On the basis of these findings, we propose that 2 mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca(2+) handling and a direct effect of PSEN1 sequence variants on excitation-contraction coupling protein function.

  18. Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system.

    Science.gov (United States)

    Wu, Li; Zhao, Wei; Yang, Ruijin; Yan, Wenxu

    2015-05-15

    The aggregation of multi-proteins is of great interest in food processing and a good understanding of the formation of aggregates during PEF processing is needed for the application of the process to pasteurize protein-based foods. The aggregates formation of a multi-protein system (containing ovalbumin, ovotransferrin and lysozyme) was studied through turbidity, size exclusion chromatography and SDS-PAGE patterns for interaction studies and binding forces. Results from size exclusion chromatography indicated that there was no soluble aggregates formed during PEF processing. The existence of lysozyme was important to form insoluble aggregates in the chosen ovalbumin solution. The results of SDS-PAGE patterns indicated that lysozyme was prone to precipitate, and was relatively the higher component of aggregates. Citric acid could be effective in inhibiting lysozyme from interacting with other proteins during PEF processing. Blocking the free sulphydryl by N-ethylmaleimide (NEM) did not affect aggregation inhibition. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Enzyme-induced aggregation of whey proteins with Bacillus licheniformis protease

    NARCIS (Netherlands)

    Creusot, N.P.

    2006-01-01

    Whey proteins are commonly used as ingredient in food. In relation with the gelation properties of whey proteins, this thesis deals with understanding the mechanism of peptide-induced aggregation of whey protein hydrolysates made with Bacillus licheniformis protease (BLP). The results show that BLP

  20. Aggregation of the protein TRIOBP-1 and its potential relevance to schizophrenia.

    Directory of Open Access Journals (Sweden)

    Nicholas J Bradshaw

    Full Text Available We have previously proposed that specific proteins may form insoluble aggregates as a response to an illness-specific proteostatic dysbalance in a subset of brains from individuals with mental illness, as is the case for other chronic brain conditions. So far, established risk factors DISC1 and dysbindin were seen to specifically aggregate in a subset of such patients, as was a novel schizophrenia-related protein, CRMP1, identified through a condition-specific epitope discovery approach. In this process, antibodies are raised against the pooled insoluble protein fractions (aggregomes of post mortem brain samples from schizophrenia patients, followed by epitope identification and confirmation using additional techniques. Pursuing this epitope discovery paradigm further, we reveal TRIO binding protein (TRIOBP to be a major substrate of a monoclonal antibody with a high specificity to brain aggregomes from patients with chronic mental illness. TRIOBP is a gene previously associated with deafness which encodes for several distinct protein species, each involved in actin cytoskeletal dynamics. The 3' splice variant TRIOBP-1 is found to be the antibody substrate and has a high aggregation propensity when over-expressed in neuroblastoma cells, while the major 5' splice variant, TRIOBP-4, does not. Endogenous TRIOBP-1 can also spontaneously aggregate, doing so to a greater extent in cell cultures which are post-mitotic, consistent with aggregated TRIOBP-1 being able to accumulate in the differentiated neurons of the brain. Finally, upon expression in Neuroscreen-1 cells, aggregated TRIOBP-1 affects cell morphology, indicating that TRIOBP-1 aggregates may directly affect cell development, as opposed to simply being a by-product of other processes involved in major mental illness. While further experiments in clinical samples are required to clarify their relevance to chronic mental illness in the general population, TRIOBP-1 aggregates are thus

  1. The effect of transient conditions on synovial fluid protein aggregation lubrication.

    Science.gov (United States)

    Myant, Connor William; Cann, Philippa

    2014-06-01

    Little is known about the prevailing lubrication mechanisms in artificial articular joints and the way in which these mechanisms determine implant performance. The authors propose that interfacial film formation is determined by rheological changes local to the contact and is driven by aggregation of synovial fluid proteins within the contact inlet region. A direct relationship between contact film thickness and size of the protein aggregation within the inlet region has been observed. In this paper the latest experimental observations of the protein aggregation mechanism are presented for conditions which more closely mimic joint kinematics and loading. Lubricant films were measured for a series of bovine calf serum solutions for CoCrMo femoral component sliding against a glass disc. An optical interferometric apparatus was employed to study the effects of transient motion on lubricant film formation. Central film thickness was measured as a function of time for a series of transient entrainment conditions; start-up motion, steady-state and non-steady-state uni-directional sliding, and bi-directional sliding. The size of the inlet aggregations was found to be dependent upon the type of transient condition. Thick protective protein films were observed to build up within the main contact region for all uni-directional tests. In contrast the inlet aggregation was not observed for bi-directional tests. Contact film thickness and wear was found to be directly proportional to the presence of the inlet protein phase. The inlet phase and contact films were found to be fragile when disrupted by surface scratches or subjected to reversal of the sliding direction. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Congo red and protein aggregation in neurodegenerative diseases.

    Science.gov (United States)

    Frid, Petrea; Anisimov, Sergey V; Popovic, Natalija

    2007-01-01

    Congo red is a commonly used histological dye for amyloid detection. The specificity of this staining results from Congo red's affinity for binding to fibril proteins enriched in beta-sheet conformation. Unexpectedly, recent investigations indicate that the dye also possesses the capacity to interfere with processes of protein misfolding and aggregation, stabilizing native protein monomers or partially folded intermediates, while reducing concentration of more toxic protein oligomers. Inhibitory effects of Congo red upon amyloid toxicity may also range from blockade of channel formation and interference with glycosaminoglycans binding or immune functions, to the modulation of gene expression. Particularly, Congo red exhibits ameliorative effect in models of neurodegenerative disorders, such as Alzheimer's, Parkinson's, Huntington's and prion diseases. Another interesting application of Congo red analogues is the development of imaging probes. Based on their small molecular size and penetrability through blood-brain barrier, Congo red congeners can be used for both antemortem and in vivo visualization and quantification of brain amyloids. Therefore, understanding mechanisms involved in dye-amyloidal fibril binding and inhibition of aggregation will provide instructive guides for the design of future compounds, potentially useful for monitoring and treating neurodegenerative diseases.

  3. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders.

    Science.gov (United States)

    Caughey, Byron; Lansbury, Peter T

    2003-01-01

    Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates.

  4. Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

    Science.gov (United States)

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-03-20

    The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  5. Influence of Pea Protein Aggregates on the Structure and Stability of Pea Protein/Soybean Polysaccharide Complex Emulsions

    Directory of Open Access Journals (Sweden)

    Baoru Yin

    2015-03-01

    Full Text Available The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS, and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  6. Kinetics of fibrilar aggregation of food proteins

    NARCIS (Netherlands)

    Arnaudov, L.N.

    2005-01-01

    In this thesis we study the kinetics of fibrilar aggregation of two model proteins widely used in the food industry -b-lactoglobulin (b-lg) and hen

  7. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

    Science.gov (United States)

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

  8. CELLS OVEREXPRESSING HSP27 SHOW ACCELERATED RECOVERY FROM HEAT-INDUCED NUCLEAR-PROTEIN AGGREGATION

    NARCIS (Netherlands)

    KAMPINGA, HH; BRUNSTING, JF; STEGE, GJJ; KONINGS, AWT; LANDRY, J

    1994-01-01

    Protein denaturation/aggregation upon cell exposure to heat shock is a likely cause of cell death. in the nucleus, protein aggregation has often been correlated to inhibition of nuclear located processes and heat-induced cell killing. in Chinese hamster 023 cells made thermotolerant by a prior

  9. Protein Aggregates and Novel Presenilin Gene Variants in Idiopathic Dilated Cardiomyopathy

    Science.gov (United States)

    Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M.; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E.; Monte, Federica del

    2010-01-01

    Background Heart failure (HF) is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as Idiopathic Dilated Cardiomyopathy (iDCM), the origin of HF is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca2+ homeostasis. Additionally, we have identified two new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 co-immunoprecipitates with SERCA2a. Conclusions Based on these findings we propose that two mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca2+ handling and a direct effect of PSEN1 sequence variants on EC-coupling protein function. PMID:20194882

  10. Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

    Science.gov (United States)

    Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

    2012-01-01

    The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

  11. Entrapment of Aβ1-40 peptide in unstructured aggregates

    International Nuclear Information System (INIS)

    Corsale, C; Carrotta, R; Mangione, M R; Vilasi, S; Provenzano, A; Bulone, D; San Biagio, P L; Cavallaro, G

    2012-01-01

    Recognizing the complexity of the fibrillogenesis process provides a solid ground for the development of therapeutic strategies aimed at preventing or inhibiting protein-protein aggregation. Under this perspective, it is meaningful to identify the possible aggregation pathways and their relative products. We found that Aβ-peptide dissolved in a pH 7.4 solution at small peptide concentration and low ionic strength forms globular aggregates without typical amyloid β-conformation. ThT binding kinetics was used to monitor aggregate formation. Circular dichroism spectroscopy, AFM imaging, static and dynamic light scattering were used for structural and morphological characterization of the aggregates. They appear stable or at least metastable with respect to fiber growth, therefore appearing as an incidental product in the pathway of fibrillogenesis. (paper)

  12. The effect of carbohydrates and lipids on the radiation-induced aggregation of proteins

    International Nuclear Information System (INIS)

    Delincee, H.; Jakubick, V.

    1977-01-01

    Myoglobin, ovalbumin and serum albumin have been irradiated in aqueous solution in the presence of varying amounts of carbohydrates and lipids, simulating a model food. Gel chromatography revealed the induction of protein aggregates, the formation of which depended strongly on protein concentration. The addition of carbohydrates (trehalose, starch) greatly reduced the amount of radiation-induced aggregates, whereas the addition of lipids (sunflower oil) had practically no effect on aggregate formation. However, if both carbohydrates and lipids were added, the decrease in aggregation caused by the carbohydrate addition was counteracted by the addition of the lipid; as increasing amounts of lipid were added, the effect of carbohydrate addition became smaller. (author)

  13. Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques

    Directory of Open Access Journals (Sweden)

    Akira Kitamura

    2015-03-01

    Full Text Available Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS and Huntington’s disease (HD. A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET, fluorescence correlation spectroscopy (FCS, and bimolecular fluorescence complementation (BiFC have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes, and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.

  14. Protein aggregation and degradation during iodine labeling and its consequences for protein adsorption to biomaterials

    DEFF Research Database (Denmark)

    Holmberg, Maria; Jensen, Karin Bagger Stibius; Ndoni, Sokol

    2007-01-01

    Protein adsorption on modified and unmodified polymer surfaces investigated through radiolabeling experiments showed a tendency for higher than expected albumin and immunoglobulin G (IgG) adsorption. Possible enhanced protein aggregation and degradation caused by the iodine labeling method used w...

  15. Structure based descriptors for the estimation of colloidal interactions and protein aggregation propensities.

    Directory of Open Access Journals (Sweden)

    Michael Brunsteiner

    Full Text Available The control of protein aggregation is an important requirement in the development of bio-pharmaceutical formulations. Here a simple protein model is proposed that was used in molecular dynamics simulations to obtain a quantitative assessment of the relative contributions of proteins' net-charges, dipole-moments, and the size of hydrophobic or charged surface patches to their colloidal interactions. The results demonstrate that the strength of these interactions correlate with net-charge and dipole moment. Variation of both these descriptors within ranges typical for globular proteins have a comparable effect. By comparison no clear trends can be observed upon varying the size of hydrophobic or charged patches while keeping the other parameters constant. The results are discussed in the context of experimental literature data on protein aggregation. They provide a clear guide line for the development of improved algorithms for the prediction of aggregation propensities.

  16. Robustness of the Process of Nucleoid Exclusion of Protein Aggregates in Escherichia coli

    Science.gov (United States)

    Neeli-Venkata, Ramakanth; Martikainen, Antti; Gupta, Abhishekh; Gonçalves, Nadia; Fonseca, Jose

    2016-01-01

    ABSTRACT Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. Combined with cell divisions, this generates heterogeneous aggregate distributions in subsequent cell generations. We studied the robustness of this process with differing medium richness and antibiotics stress, which affect nucleoid size, using multimodal, time-lapse microscopy of live cells expressing both a fluorescently tagged chaperone (IbpA), which identifies in vivo the location of aggregates, and HupA-mCherry, a fluorescent variant of a nucleoid-associated protein. We find that the relative sizes of the nucleoid's major and minor axes change widely, in a positively correlated fashion, with medium richness and antibiotic stress. The aggregate's distribution along the major cell axis also changes between conditions and in agreement with the nucleoid exclusion phenomenon. Consequently, the fraction of aggregates at the midcell region prior to cell division differs between conditions, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, from the location of the peak of anisotropy in the aggregate displacement distribution, the nucleoid relative size, and the spatiotemporal aggregate distribution, we find that the exclusion of detectable aggregates from midcell is most pronounced in cells with mid-sized nucleoids, which are most common under optimal conditions. We conclude that the aggregate management mechanisms of E. coli are significantly robust but are not immune to stresses due to the tangible effect that these have on nucleoid size. IMPORTANCE Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. From live single-cell microscopy studies of the robustness of this process to various stresses known to affect nucleoid size, we find that nucleoid size and aggregate preferential locations change concordantly between conditions. Also, the degree of influence of the nucleoid

  17. Chlorin photosensitizers sterically designed to prevent self-aggregation.

    Science.gov (United States)

    Uchoa, Adjaci F; de Oliveira, Kleber T; Baptista, Mauricio S; Bortoluzzi, Adailton J; Iamamoto, Yassuko; Serra, Osvaldo A

    2011-11-04

    The synthesis and photophysical evaluation of new chlorin derivatives are described. The Diels-Alder reaction between protoporphyrin IX dimethyl ester and substituted maleimides furnishes endo-adducts that completely prevent the self-aggregation of the chlorins. Fluorescence, resonant light scattering (RLS) and (1)H NMR experiments, as well as X-ray crystallographic have demonstrated that the configurational arrangement of the synthesized chlorins prevent π-stacking interactions between macrocycles, thus indicating that it is a nonaggregating photosensitizer with high singlet oxygen (Φ(Δ)) and fluorescence (Φ(f)) quantum yields. Our results show that this type of synthetic strategy may provide the lead to a new generation of PDT photosensitizers.

  18. Cytoplasmic Dynein Is Required for the Spatial Organization of Protein Aggregates in Filamentous Fungi

    Directory of Open Access Journals (Sweden)

    Martin J. Egan

    2015-04-01

    Full Text Available Eukaryotes have evolved multiple strategies for maintaining cellular protein homeostasis. One such mechanism involves neutralization of deleterious protein aggregates via their defined spatial segregation. Here, using the molecular disaggregase Hsp104 as a marker for protein aggregation, we describe the spatial and temporal dynamics of protein aggregates in the filamentous fungus Aspergillus nidulans. Filamentous fungi, such as A. nidulans, are a diverse group of species of major health and economic importance and also serve as model systems for studying highly polarized eukaryotic cells. We find that microtubules promote the formation of Hsp104-positive aggregates, which coalesce into discrete subcellular structures in a process dependent on the microtubule-based motor cytoplasmic dynein. Finally, we find that impaired clearance of these inclusions negatively impacts retrograde trafficking of endosomes, a conventional dynein cargo, indicating that microtubule-based transport can be overwhelmed by chronic cellular stress.

  19. Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Erik Kvam

    2009-05-01

    Full Text Available Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to

  20. Natural Biomolecules and Protein Aggregation: Emerging Strategies against Amyloidogenesis

    Directory of Open Access Journals (Sweden)

    Antonella Sgarbossa

    2012-12-01

    Full Text Available Biomolecular self-assembly is a fundamental process in all organisms. As primary components of the life molecular machinery, proteins have a vast array of resources available to them for self-assembly in a functional structure. Protein self-assembly, however, can also occur in an aberrant way, giving rise to non-native aggregated structures responsible for severe, progressive human diseases that have a serious social impact. Different neurodegenerative disorders, like Huntington’s, Alzheimer’s, and spongiform encephalopathy diseases, have in common the presence of insoluble protein aggregates, generally termed “amyloid,” that share several physicochemical features: a fibrillar morphology, a predominantly beta-sheet secondary structure, birefringence upon staining with the dye Congo red, insolubility in common solvents and detergents, and protease resistance. Conformational constrains, hydrophobic and stacking interactions can play a key role in the fibrillogenesis process and protein–protein and peptide–peptide interactions—resulting in self-assembly phenomena of peptides yielding fibrils—that can be modulated and influenced by natural biomolecules. Small organic molecules, which possess both hydrophilic and hydrophobic moieties able to bind to peptide/protein molecules through hydrogen bonds and hydrophobic and aromatic interactions, are potential candidates against amyloidogenesis. In this review some significant case examples will be critically discussed.

  1. Myofibrillar protein oxidation affects filament charges, aggregation and water-holding

    NARCIS (Netherlands)

    Bao, Yulong; Boeren, Sjef; Ertbjerg, Per

    2018-01-01

    Hypochlorous acid (HClO) is a strong oxidant that is able to mediate protein oxidation. In order to study the effect of oxidation on charges, aggregation and water-holding of myofibrillar proteins, extracted myofibrils were oxidized by incubation with different concentrations of HClO (0, 1, 5,

  2. Two-Step Amyloid Aggregation: Sequential Lag Phase Intermediates

    Science.gov (United States)

    Castello, Fabio; Paredes, Jose M.; Ruedas-Rama, Maria J.; Martin, Miguel; Roldan, Mar; Casares, Salvador; Orte, Angel

    2017-01-01

    The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation.

  3. Self-assembly of caseinomacropeptide as a potential key mechanism in the formation of visible storage induced aggregates in acidic whey protein isolate dispersions

    DEFF Research Database (Denmark)

    Villumsen, Nanna Stengaard; Jensen, Hanne Bak; Thu Le, Thao Thi

    2015-01-01

    Visible aggregates formed during storage in acidic whey protein isolate (WPI) dispersions represent a challenge to the beverage industry. Batch-to-batch variations are observed that prevents consistent quality and shelf-life prediction. Heat-treatment of WPI dispersions at 120°C for 20s instead...

  4. Identification of CLN6 as a molecular entity of endoplasmic reticulum-driven anti-aggregate activity.

    Science.gov (United States)

    Yamashita, Arisa; Hiraki, Yuri; Yamazaki, Tetsuo

    2017-06-10

    αB-crystallin (αBC) is a small heat shock protein. Mutations in the αBC gene are linked to α-crystallinopathy, a hereditary myopathy histologically characterized by intracellular accumulation of protein aggregates. The disease-causing R120G αBC mutant, harboring an arginine-to-glycine replacement at position 120, is an aggregate-prone protein. We previously showed that the R120G mutant's aggregation in HeLa cells was prevented by enforced expression of αBC on the endoplasmic reticulum (ER). To elucidate the molecular nature of the preventive effect on the R120G mutant, we isolated proteins binding to ER-anchored αBC (TMαBC). The ER transmembrane CLN6 protein was identified as a TMαBC's binder. CLN6 knockdown in HeLa cells attenuated TMαBC's anti-aggregate activity against the R120G mutant. Conversely, CLN6 overexpression enhanced the activity, indicating that CLN6 operates as a downstream effector of TMαBC. CLN6 physically interacted with the R120G mutant, and repressed its aggregation in HeLa cells even when TMαBC was not co-expressed. Furthermore, CLN6's antagonizing effect on the R120G mutant was compromised upon treatment with a lysosomal inhibitor, suggesting CLN6 requires the intact autophagy-lysosome system to prevent the R120G mutant from aggregating. We hence conclude that CLN6 is not only a molecular entity of the anti-aggregate activity conferred by the ER manipulation using TMαBC, but also serves as a potential target of therapeutic interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

    Energy Technology Data Exchange (ETDEWEB)

    Takanashi, Keisuke; Yamaguchi, Atsushi, E-mail: atsyama@restaff.chiba-u.jp

    2014-09-26

    Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

  6. Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold

    Science.gov (United States)

    Mazzer, Alice R.; Perraud, Xavier; Halley, Jennifer; O’Hara, John; Bracewell, Daniel G.

    2015-01-01

    Protein A chromatography is a near-ubiquitous method of mAb capture in bioprocesses. The use of low pH buffer for elution from protein A is known to contribute to product aggregation. Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly. This paper presents a well-defined method for investigating this problem. An IgG4 was incubated in elution buffer after protein A chromatography (typical of the viral inactivation hold) and the quantity of monomer in neutralised samples was determined by size exclusion chromatography; elution buffers of different pH values predetermined to induce aggregation of the IgG4 were used. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH. Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested. PMID:26346187

  7. Role of pH-induced structural change in protein aggregation in foam fractionation of bovine serum albumin

    Directory of Open Access Journals (Sweden)

    Rui Li

    2016-03-01

    Full Text Available For reducing protein aggregation in foam fractionation, the role of pH-induced structural change in the interface-induced protein aggregation was analyzed using bovine serum albumin (BSA as a model protein. The results show that the decrease in pH from 7.0 to 3.0 gradually unfolded the BSA structure to increase the molecular size and the relative content of β-sheet and thus reduced the stability of BSA in the aqueous solution. At the isoelectric point (pH 4.7, BSA suffered the lowest level in protein aggregation induced by the gas–liquid interface. In the pH range from 7.0 to 4.7, most BSA aggregates were formed in the defoaming process while in the pH range from 4.7 to 3.0, the BSA aggregates were formed at the gas–liquid interface due to the unfolded BSA structure and they further aggregated to form insoluble ones in the desorption process.

  8. Heavy metals and metalloids as a cause for protein misfolding and aggregation.

    Science.gov (United States)

    Tamás, Markus J; Sharma, Sandeep K; Ibstedt, Sebastian; Jacobson, Therese; Christen, Philipp

    2014-02-25

    While the toxicity of metals and metalloids, like arsenic, cadmium, mercury, lead and chromium, is undisputed, the underlying molecular mechanisms are not entirely clear. General consensus holds that proteins are the prime targets; heavy metals interfere with the physiological activity of specific, particularly susceptible proteins, either by forming a complex with functional side chain groups or by displacing essential metal ions in metalloproteins. Recent studies have revealed an additional mode of metal action targeted at proteins in a non-native state; certain heavy metals and metalloids have been found to inhibit the in vitro refolding of chemically denatured proteins, to interfere with protein folding in vivo and to cause aggregation of nascent proteins in living cells. Apparently, unfolded proteins with motile backbone and side chains are considerably more prone to engage in stable, pluridentate metal complexes than native proteins with their well-defined 3D structure. By interfering with the folding process, heavy metal ions and metalloids profoundly affect protein homeostasis and cell viability. This review describes how heavy metals impede protein folding and promote protein aggregation, how cells regulate quality control systems to protect themselves from metal toxicity and how metals might contribute to protein misfolding disorders.

  9. Thermodiffusion as a close-to-interface effect that matters in non-isothermal (dis)orderly protein aggregations

    Energy Technology Data Exchange (ETDEWEB)

    Gadomski, A., E-mail: agad@utp.edu.pl; Kruszewska, N., E-mail: nkruszewska@utp.edu.pl

    2014-08-01

    The goal of this discussion letter is to argue how and why an inherent nanoscale thermodiffusion (Soret-type) effect can be relevant in (dis)orderly protein aggregation. We propose a model in which the aggregation of proteins, in the presence of temperature gradient, is described in terms of Smoluchowski dynamics in the phase space of nuclei sizes. The Soret coefficient of the aggregation is proportional to the variations of the aggregation free energy over temperature. The free energy is related to the (interface) boundary condition of the system. When boundary condition is of equilibrium Gibbs–Thomson type, with a well-stated surface tension of the nucleus, to the system can be assigned a negative Soret effect. On the contrary, when a non-equilibrium perturbing (salting-out) term enters the boundary condition, a positive Soret effect may manifest. A zero-value Soret regime is expected to occur in between, yielding very soft (“fragile”) non-Kossel protein-type crystals. - Highlights: • Comprehension for non-isothermal formation of (dis)orderly protein aggregation. • Classification of temperature-sensitive morphologies in colloid-type aggregation. • Morphologies split into near-equilibrium and nonequilibrium structural outcomes. • Classification on mesoscopic nonequilibrium thermodynamics near local equilibrium.

  10. p53 Aggregates penetrate cells and induce the co-aggregation of intracellular p53.

    Directory of Open Access Journals (Sweden)

    Karolyn J Forget

    Full Text Available Prion diseases are unique pathologies in which the infectious particles are prions, a protein aggregate. The prion protein has many particular features, such as spontaneous aggregation, conformation transmission to other native PrP proteins and transmission from an individual to another. Protein aggregation is now frequently associated to many human diseases, for example Alzheimer's disease, Parkinson's disease or type 2 diabetes. A few proteins associated to these conformational diseases are part of a new category of proteins, called prionoids: proteins that share some, but not all, of the characteristics associated with prions. The p53 protein, a transcription factor that plays a major role in cancer, has recently been suggested to be a possible prionoid. The protein has been shown to accumulate in multiple cancer cell types, and its aggregation has also been reproduced in vitro by many independent groups. These observations suggest a role for p53 aggregates in cancer development. This study aims to test the «prion-like» features of p53. Our results show in vitro aggregation of the full length and N-terminally truncated protein (p53C, and penetration of these aggregates into cells. According to our findings, the aggregates enter cells using macropinocytosis, a non-specific pathway of entry. Lastly, we also show that once internalized by the cell, p53C aggregates can co-aggregate with endogenous p53 protein. Together, these findings suggest prion-like characteristics for p53 protein, based on the fact that p53 can spontaneously aggregate, these aggregates can penetrate cells and co-aggregate with cellular p53.

  11. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition.

    Science.gov (United States)

    Moll, Lorna; Ben-Gedalya, Tziona; Reuveni, Hadas; Cohen, Ehud

    2016-04-01

    The discovery that the alteration of aging by reducing the activity of the insulin/IGF-1 signaling (IIS) cascade protects nematodes and mice from neurodegeneration-linked, toxic protein aggregation (proteotoxicity) raises the prospect that IIS inhibitors bear therapeutic potential to counter neurodegenerative diseases. Recently, we reported that NT219, a highly efficient IGF-1 signaling inhibitor, protects model worms from the aggregation of amyloid β peptide and polyglutamine peptides that are linked to the manifestation of Alzheimer's and Huntington's diseases, respectively. Here, we employed cultured cell systems to investigate whether NT219 promotes protein homeostasis (proteostasis) in mammalian cells and to explore its underlying mechanisms. We found that NT219 enhances the aggregation of misfolded prion protein and promotes its deposition in quality control compartments known as "aggresomes." NT219 also elevates the levels of certain molecular chaperones but, surprisingly, reduces proteasome activity and impairs autophagy. Our findings show that IGF-1 signaling inhibitors in general and NT219 in particular can promote proteostasis in mammalian cells by hyperaggregating hazardous proteins, thereby bearing the potential to postpone the onset and slow the progression of neurodegenerative illnesses in the elderly.-Moll, L., Ben-Gedalya, T., Reuveni, H., Cohen, E. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition. © FASEB.

  12. TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

    OpenAIRE

    Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

    2015-01-01

    In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP....

  13. Characterisation of heat-induced protein aggregation in whey protein isolate and the influence of aggregation on the availability of amino groups as measured by the ortho-phthaldialdehyde (OPA) and trinitrobenzenesulfonic acid (TNBS) methods.

    Science.gov (United States)

    Mulcahy, Eve M; Fargier-Lagrange, Maéva; Mulvihill, Daniel M; O'Mahony, James A

    2017-08-15

    Whey protein isolate (WPI) solutions, with different levels of aggregated protein, were prepared by heating (5% protein, pH 7, 90°C for 30min) WPI solutions with either 20mM added NaCl (WPI+NaCl), 5mM N-ethylmaleimide (WPI+NEM) or 20mM added NaCl and 5mM NEM (WPI+NaCl+NEM). Gel electrophoresis demonstrated that the heated WPI and WPI+NaCl solutions had higher levels of aggregated protein, due to more covalent interactions between proteins, than the heated WPI+NEM and WPI+NaCl+NEM solutions. There were marked differences in the levels of amino groups between all heated WPI solutions when measured by the OPA and TNBS methods, with lower levels being measured by the TNBS method than by the OPA method. These results demonstrate that the measurement of available amino groups by the OPA method is less impacted than by the TNBS method after heat-induced structural changes, arising from disulfide or sulfhydryl-disulfide bond-mediated aggregation of whey protein molecules. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Disulphide bond formation in food protein aggregation and gelation

    NARCIS (Netherlands)

    Visschers, R.W.; Jongh, de H.H.J.

    2005-01-01

    In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified.

  15. Intermolecular crosslinks mediate aggregation of phospholipid vesicles by pulmonary surfactant-associated protein SAP-35

    International Nuclear Information System (INIS)

    Ross, G.R.; Sawyer, J.; Whitsett, J.

    1987-01-01

    Pulmonary surfactant-associated protein, Mr=35,000 (SAP-35) is known to bind phospholipids and is hypothesized to function in the organization of surfactant lipid membranes. SAP-35 has been observed to accelerate the calcium-induced aggregation of phospholipid vesicles. In order to define the molecular domains of SAP-35 which function in phospholipid aggregation, they have measured the light scattering properties (400nm) of purified canine SAP-35-phospholipid vesicle suspensions. Accelerated aggregation of unilamellar vesicles, requires SAP-35 and at least 2mM free calcium. The initial rate of A 400 change is proportional to the amount of native SAP-35 added over lipid:protein molar ratios ranging from 100:1 to 5000:1. Removal of the SAP-35 collagen-like domain and a specific cysteine residue involved in intermolecular disulfide bonding by bacterial collagenase digestion destroys the protein's lipid aggregation activity. Pre-incubation of SAP-35 with dithiothreitol (DTT) under nondenaturing conditions also results in a time-dependent loss of aggregation activity. Sucrose density gradient floatation of SAP-35 with 14 C dipalmitoyl phosphatidycholine labelled vesicles in the absence or presence of DTT suggests retention of SAP-35 lipid binding capacity. These data demonstrate the importance of SAP-35 triple helix and disulfide crosslinking integrity for the aggregation of unilamellar phospholipid vesicles

  16. The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions

    International Nuclear Information System (INIS)

    Mezzenga, Raffaele; Fischer, Peter

    2013-01-01

    The aggregation of proteins is of fundamental relevance in a number of daily phenomena, as important and diverse as blood coagulation, medical diseases, or cooking an egg in the kitchen. Colloidal food systems, in particular, are examples that have great significance for protein aggregation, not only for their importance and implications, which touches on everyday life, but also because they allow the limits of the colloidal science analogy to be tested in a much broader window of conditions, such as pH, ionic strength, concentration and temperature. Thus, studying the aggregation and self-assembly of proteins in foods challenges our understanding of these complex systems from both the molecular and statistical physics perspectives. Last but not least, food offers a unique playground to study the aggregation of proteins in three, two and one dimensions, that is to say, in the bulk, at air/water and oil/water interfaces and in protein fibrillation phenomena. In this review we will tackle this very ambitious task in order to discuss the current understanding of protein aggregation in the framework of foods, which is possibly one of the broadest contexts, yet is of tremendous daily relevance. (review article)

  17. The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions.

    Science.gov (United States)

    Mezzenga, Raffaele; Fischer, Peter

    2013-04-01

    The aggregation of proteins is of fundamental relevance in a number of daily phenomena, as important and diverse as blood coagulation, medical diseases, or cooking an egg in the kitchen. Colloidal food systems, in particular, are examples that have great significance for protein aggregation, not only for their importance and implications, which touches on everyday life, but also because they allow the limits of the colloidal science analogy to be tested in a much broader window of conditions, such as pH, ionic strength, concentration and temperature. Thus, studying the aggregation and self-assembly of proteins in foods challenges our understanding of these complex systems from both the molecular and statistical physics perspectives. Last but not least, food offers a unique playground to study the aggregation of proteins in three, two and one dimensions, that is to say, in the bulk, at air/water and oil/water interfaces and in protein fibrillation phenomena. In this review we will tackle this very ambitious task in order to discuss the current understanding of protein aggregation in the framework of foods, which is possibly one of the broadest contexts, yet is of tremendous daily relevance.

  18. The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions

    Science.gov (United States)

    Mezzenga, Raffaele; Fischer, Peter

    2013-04-01

    The aggregation of proteins is of fundamental relevance in a number of daily phenomena, as important and diverse as blood coagulation, medical diseases, or cooking an egg in the kitchen. Colloidal food systems, in particular, are examples that have great significance for protein aggregation, not only for their importance and implications, which touches on everyday life, but also because they allow the limits of the colloidal science analogy to be tested in a much broader window of conditions, such as pH, ionic strength, concentration and temperature. Thus, studying the aggregation and self-assembly of proteins in foods challenges our understanding of these complex systems from both the molecular and statistical physics perspectives. Last but not least, food offers a unique playground to study the aggregation of proteins in three, two and one dimensions, that is to say, in the bulk, at air/water and oil/water interfaces and in protein fibrillation phenomena. In this review we will tackle this very ambitious task in order to discuss the current understanding of protein aggregation in the framework of foods, which is possibly one of the broadest contexts, yet is of tremendous daily relevance.

  19. Surface properties of heat-induced soluble soy protein aggregates of different molecular masses.

    Science.gov (United States)

    Guo, Fengxian; Xiong, Youling L; Qin, Fang; Jian, Huajun; Huang, Xiaolin; Chen, Jie

    2015-02-01

    Suspensions (2% and 5%, w/v) of soy protein isolate (SPI) were heated at 80, 90, or 100 °C for different time periods to produce soluble aggregates of different molecular sizes to investigate the relationship between particle size and surface properties (emulsions and foams). Soluble aggregates generated in these model systems were characterized by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heat treatment increased surface hydrophobicity, induced SPI aggregation via hydrophobic interaction and disulfide bonds, and formed soluble aggregates of different sizes. Heating of 5% SPI always promoted large-size aggregate (LA; >1000 kDa) formation irrespective of temperature, whereas the aggregate size distribution in 2% SPI was temperature dependent: the LA fraction progressively rose with temperature (80→90→100 °C), corresponding to the attenuation of medium-size aggregates (MA; 670 to 1000 kDa) initially abundant at 80 °C. Heated SPI with abundant LA (>50%) promoted foam stability. LA also exhibited excellent emulsifying activity and stabilized emulsions by promoting the formation of small oil droplets covered with a thick interfacial protein layer. However, despite a similar influence on emulsion stability, MA enhanced foaming capacity but were less capable of stabilizing emulsions than LA. The functionality variation between heated SPI samples is clearly related to the distribution of aggregates that differ in molecular size and surface activity. The findings may encourage further research to develop functional SPI aggregates for various commercial applications. © 2015 Institute of Food Technologists®

  20. Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States); Bernstein, Sanford I., E-mail: sbernst@sciences.sdsu.edu [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States)

    2010-05-28

    UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, {alpha}-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.

  1. Behavior of Heat-Denatured Whey: Buttermilk Protein Aggregates during the Yogurt-Making Process and Their Influence on Set-Type Yogurt Properties

    Directory of Open Access Journals (Sweden)

    Maxime Saffon

    2013-09-01

    Full Text Available The objective of this study was to assess the impact of using heat-denatured whey:buttermilk protein aggregate in acid-set type yogurt production. Whey and buttermilk (25:75 protein concentrate was adjusted to pH 4.6, heated at 90 °C for 5 min, homogenized and freeze-dried. Set-type yogurts were prepared from skim milk standardized to 15% (w/v total solids and 4.2% (w/v protein using different levels of powdered skim milk or freeze-dried protein aggregate. The use of the protein aggregate significantly modified yogurt texture, but did not affect the water-holding capacity of the gel. Confocal laser-scanning microscope images showed the presence of large particles in milk enriched with protein aggregate, which directly affected the homogeneity of the clusters within the protein matrix. Thiol groups were freed during heating of the protein aggregate suspended in water, suggesting that the aggregates could interact with milk proteins during heating.

  2. CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

    Directory of Open Access Journals (Sweden)

    Ashley A. Zurawel

    2016-09-01

    Full Text Available Proteins containing polyglutamine (polyQ regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD. To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms.

  3. Hemin as a generic and potent protein misfolding inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yanqin [School of Chemistry and Physics, The University of Adelaide, Adelaide, SA 5005 (Australia); Carver, John A. [Discipline of Pharmacology, The University of Adelaide, Adelaide, SA 5005 (Australia); Ho, Lam H.; Elias, Abigail K. [School of Chemistry and Physics, The University of Adelaide, Adelaide, SA 5005 (Australia); Musgrave, Ian F. [Research School of Chemistry, The Australian National University, Canberra, ACT 0200 (Australia); Pukala, Tara L., E-mail: tara.pukala@adelaide.edu.au [School of Chemistry and Physics, The University of Adelaide, Adelaide, SA 5005 (Australia)

    2014-11-14

    Highlights: • Hemin prevents Aβ42, α-synuclein and RCM-κ-casein forming amyloid fibrils. • Hemin inhibits the β-sheet structure formation of Aβ42. • Hemin reduces the cell toxicity caused by fibrillar Aβ42. • Hemin dissociates partially formed Aβ42 fibrils. • Hemin prevents amorphous aggregation by ADH, catalase and γs-crystallin. - Abstract: Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.

  4. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.

    Science.gov (United States)

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.

  5. Towards control of aggregational behaviour of alpha-lactalbumin at acidic pH.

    Science.gov (United States)

    Pedersen, Jane B; Fojan, Peter; Sorensen, John; Petersen, Steffen B

    2006-07-01

    alpha-Lactalbumin (alpha-La) undergoes considerable structural changes upon loss of bound Ca2+ at acidic pH, leaving alpha-La in a molten globule structure. Using fluorescence the present work provides more insight into the structural transition of alpha-La at acidic pH leading to protein aggregation, most likely caused by a combination of hydrophobic and electrostatic interactions. The rate of aggregation is determined by the protein concentration and temperature applied. Availability of Ca2+ stabilises the protein, and thus prevent aggregation at pH values as low as pH 2.9. In contrast, presence of Cu2+ induces a destabilisation of the protein, which can be explained by a binding to the Zn2+ binding site in alpha-La, possibly resulting in structural alterations of the protein. In general, presence of anions destabilize alpha-La at pH values below pI, with SO4(2-) exhibiting the strongest effect on the protein stability, thus correlating well with the Hofmeister series. At more acidic pH values far from pI, alpha-La becomes more stable towards ion induced aggregation, since higher ion activity is required to efficiently screen the charges on the protein surface. The results presented in this paper provide detailed knowledge on the external parameters leading to aggregation of alpha-La at acidic pH, thus permitting rational design of the aggregation process.

  6. Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases.

    Science.gov (United States)

    Alam, Parvez; Chaturvedi, Sumit Kumar; Siddiqi, Mohammad Khursheed; Rajpoot, Ravi Kant; Ajmal, Mohd Rehan; Zaman, Masihuz; Khan, Rizwan Hasan

    2016-05-27

    Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

  7. Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease.

    Science.gov (United States)

    Ruiz-Riquelme, Alejandro; Sánchez-Iglesias, Sofía; Rábano, Alberto; Guillén-Navarro, Encarna; Domingo-Jiménez, Rosario; Ramos, Adriana; Rosa, Isaac; Senra, Ana; Nilsson, Peter; García, Ángel; Araújo-Vilar, David; Requena, Jesús R

    2015-11-01

    Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Aggregation of polyQ proteins is increased upon yeast aging and affected by Sir2 and Hsf1: novel quantitative biochemical and microscopic assays.

    Directory of Open Access Journals (Sweden)

    Aviv Cohen

    Full Text Available Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.

  9. Revealing Linear Aggregates of Light Harvesting Antenna Proteins in Photosynthetic Membranes

    OpenAIRE

    He, Yufan; Zeng, Xiaohua; Mukherjee, Saptarshi; Rajapaksha, Suneth; Kaplan, Samuel; Lu, H. Peter

    2010-01-01

    How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under...

  10. The effect of oil type on network formation by protein aggregates into oleogels

    NARCIS (Netherlands)

    Vries, de Auke; Lopez Gomez, Yuly; Linden, van der Erik; Scholten, Elke

    2017-01-01

    The aim of this study was to assess the effect of oil type on the network formation of heat-set protein aggregates in liquid oil. The gelling properties of such aggregates to structure oil into so-called ‘oleogels’ are related to both the particle-particle and particle-solvent interactions. To

  11. Sensitive Electrochemical Detection of Native and Aggregated x-Synuclein Protein Involved in Parkinson's Disease

    NARCIS (Netherlands)

    Masarik, Michal; Stobiecka, Agata; Kizek, René; Jelen, Frantisek; Pechan, Zdenk; Hoyer, Wolfgang; Subramaniam, Vinod; Palecek, Emil

    2004-01-01

    The aggregation of α-synuclein, a 14 kDa protein, is involved in several human neurodegenerative disorders, including Parkinson's disease. We studied native and in vitro aggregated α-synuclein by circular dichroism (CD), atomic force microscopy (AFM) and electrochemical methods. We used constant

  12. Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

    Science.gov (United States)

    Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

    2018-04-01

    Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

  13. A dynamic study of protein secretion and aggregation in the secretory pathway.

    Science.gov (United States)

    Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Sitia, Roberto; Anelli, Tiziana

    2014-01-01

    Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

  14. A dynamic study of protein secretion and aggregation in the secretory pathway.

    Directory of Open Access Journals (Sweden)

    Maria Francesca Mossuto

    Full Text Available Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

  15. Lysosomal membrane permeability stimulates protein aggregate formation in neurons of a lysosomal disease.

    Science.gov (United States)

    Micsenyi, Matthew C; Sikora, Jakub; Stephney, Gloria; Dobrenis, Kostantin; Walkley, Steven U

    2013-06-26

    Protein aggregates are a common pathological feature of neurodegenerative diseases and several lysosomal diseases, but it is currently unclear what aggregates represent for pathogenesis. Here we report the accumulation of intraneuronal aggregates containing the macroautophagy adapter proteins p62 and NBR1 in the neurodegenerative lysosomal disease late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 disease is caused by a deficiency in the lysosomal enzyme tripeptidyl peptidase I, which results in aberrant lysosomal storage of catabolites, including the subunit c of mitochondrial ATP synthase (SCMAS). In an effort to define the role of aggregates in CLN2, we evaluated p62 and NBR1 accumulation in the CNS of Cln2(-/-) mice. Although increases in p62 and NBR1 often suggest compromised degradative mechanisms, we found normal ubiquitin-proteasome system function and only modest inefficiency in macroautophagy late in disease. Importantly, we identified that SCMAS colocalizes with p62 in extra-lysosomal aggregates in Cln2(-/-) neurons in vivo. This finding is consistent with SCMAS being released from lysosomes, an event known as lysosomal membrane permeability (LMP). We predicted that LMP and storage release from lysosomes results in the sequestration of this material as cytosolic aggregates by p62 and NBR1. Notably, LMP induction in primary neuronal cultures generates p62-positive aggregates and promotes p62 localization to lysosomal membranes, supporting our in vivo findings. We conclude that LMP is a previously unrecognized pathogenic event in CLN2 disease that stimulates cytosolic aggregate formation. Furthermore, we offer a novel role for p62 in response to LMP that may be relevant for other diseases exhibiting p62 accumulation.

  16. Aggregated forms of bull seminal plasma proteins and their heparin-binding activity

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Ryšlavá, H.; Liberda, J.; Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 69, - (2004), s. 616-630 ISSN 0010-0765 R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : bull seminal plasma proteins * heparin-binding proteins * aggregated forms of proteins Subject RIV: CE - Biochemistry Impact factor: 1.062, year: 2004

  17. Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2011-02-01

    Full Text Available Abstract Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D. As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might

  18. Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

    Science.gov (United States)

    Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

    2012-09-25

    Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

  19. Quality control of Photosystem II: reversible and irreversible protein aggregation decides the fate of Photosystem II under excessive illumination

    Directory of Open Access Journals (Sweden)

    Yasusi eYamamoto

    2013-10-01

    Full Text Available In response to excessive light, the thylakoid membranes of higher plant chloroplasts show dynamic changes including the degradation and reassembly of proteins, a change in the distribution of proteins, and large-scale structural changes such as unstacking of the grana. Here, we examined the aggregation of light-harvesting chlorophyll-protein complexes and Photosystem II core subunits of spinach thylakoid membranes under light stress with 77K chlorophyll fluorescence; aggregation of these proteins was found to proceed with increasing light intensity. Measurement of changes in the fluidity of thylakoid membranes with fluorescence polarization of diphenylhexatriene showed that membrane fluidity increased at a light intensity of 500–1,000 µmol photons m-2 s-1, and decreased at very high light intensity (1,500 µmol photons m-2 s-1. The aggregation of light-harvesting complexes at moderately high light intensity is known to be reversible, while that of Photosystem II core subunits at extremely high light intensity is irreversible. It is likely that the reversibility of protein aggregation is closely related to membrane fluidity: increases in fluidity should stimulate reversible protein aggregation, whereas irreversible protein aggregation might decrease membrane fluidity. When spinach leaves were pre-illuminated with moderately high light intensity, the qE component of non-photochemical quenching and the optimum quantum yield of Photosystem II increased, indicating that Photosystem II/ light-harvesting complexes rearranged in the thylakoid membranes to optimize Photosystem II activity. Transmission electron microscopy revealed that the thylakoids underwent partial unstacking under these light stress conditions. Thus, protein aggregation is involved in thylakoid dynamics and regulates photochemical reactions, thereby deciding the fate of Photosystem II.

  20. Maillard-reaction-induced modification and aggregation of proteins and hardening of texture in protein bar model systems.

    Science.gov (United States)

    Zhou, Peng; Guo, Mufan; Liu, Dasong; Liu, Xiaoming; Labuza, Teodore P

    2013-03-01

    The hardening of high-protein bars causes problems in their acceptability to consumers. The objective of this study was to determine the progress of the Maillard reaction in model systems of high-protein nutritional bars containing reducing sugars, and to illustrate the influences of the Maillard reaction on the modification and aggregation of proteins and the hardening of bar matrices during storage. The progress of the Maillard reaction, glycation, and aggregation of proteins, and textural changes in bar matrices were investigated during storage at 25, 35, and 45 °C. The initial development of the Maillard reaction caused little changes in hardness; however, further storage resulted in dramatic modification of protein with formation of high-molecular-weight polymers, resulting in the hardening in texture. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula minimized the changes in texture. The hardening of high-protein bars causes problems in their acceptability to consumers. Maillard reaction is one of the mechanisms contributing to the hardening of bar matrix, particularly for the late stage of storage. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula will minimize the changes in texture. © 2013 Institute of Food Technologists®

  1. Role of the disaggregase ClpB in processing of proteins aggregated as inclusion bodies.

    Science.gov (United States)

    Zblewska, Kamila; Krajewska, Joanna; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

    2014-08-01

    Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

    Science.gov (United States)

    Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

    2000-01-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

  3. Model for amorphous aggregation processes

    Science.gov (United States)

    Stranks, Samuel D.; Ecroyd, Heath; van Sluyter, Steven; Waters, Elizabeth J.; Carver, John A.; von Smekal, Lorenz

    2009-11-01

    The amorphous aggregation of proteins is associated with many phenomena, ranging from the formation of protein wine haze to the development of cataract in the eye lens and the precipitation of recombinant proteins during their expression and purification. While much literature exists describing models for linear protein aggregation, such as amyloid fibril formation, there are few reports of models which address amorphous aggregation. Here, we propose a model to describe the amorphous aggregation of proteins which is also more widely applicable to other situations where a similar process occurs, such as in the formation of colloids and nanoclusters. As first applications of the model, we have tested it against experimental turbidimetry data of three proteins relevant to the wine industry and biochemistry, namely, thaumatin, a thaumatinlike protein, and α -lactalbumin. The model is very robust and describes amorphous experimental data to a high degree of accuracy. Details about the aggregation process, such as shape parameters of the aggregates and rate constants, can also be extracted.

  4. ALPHA-SYNUCLEIN STRUCTURE, AGGREGATION AND MODULATORS

    Directory of Open Access Journals (Sweden)

    Pinakin K. Makwana

    2016-06-01

    Full Text Available Alpha-synuclein is an intrinsically unstructured protein, involved in various neurodegenerative disorders. In vitro/in vivo experiments, as well as genetic mutation studies establish a direct link between alphasynuclein and synucleinopathies. Due to its natively unfolded state, alpha synuclein can adopt numerous conformations upon interaction with its partners and cellular factors, offering explanation for its diverse interactions. Aggregated form of alpha-synuclein has been observed in the brain of patients with synucleinopathies, a hallmark of neurodegeneration, and cell death has been attributed to aggregation induced toxicity. The process of aggregation involves nucleation, followed by intermediate oligomeric states, and finally the fibrillar amyloids. Of the various conformations/species that alpha-synuclein assumes before it transforms into mature amyloid fibrils, the oligomeric species is the most toxic. Thus, an effective way to limit disease progression is by modifying/slowing down protein aggregation/deposition in the brain. Various small natural products, synthetic chemicals, peptides and antibodies specific to alpha-synuclein have been designed/identified to reduce its rate of aggregation. Unfortunately, not even a handful of the molecules have cleared the clinical trials. Even today, medications available for Parkinson’s patients are mostly the drugs that adjust for loss of dopamine in the brain, and hence do not stop the progression of the disease or cure the symptoms. Thus, more molecular level studies are warranted to fully elucidate the process of alpha-synuclein aggregation, which in turn could help in identifying novel therapeutics and preventives. The present review summarizes the insights gained into the structure, in vitro aggregation and inhibitors/modulators of alpha-synuclein aggregation, that can be used to design better and effective inhibitors against the diseases.

  5. Altering protein surface charge with chemical modification modulates protein–gold nanoparticle aggregation

    International Nuclear Information System (INIS)

    Jamison, Jennifer A.; Bryant, Erika L.; Kadali, Shyam B.; Wong, Michael S.; Colvin, Vicki L.; Matthews, Kathleen S.; Calabretta, Michelle K.

    2011-01-01

    Gold nanoparticles (AuNP) can interact with a wide range of molecules including proteins. Whereas significant attention has focused on modifying the nanoparticle surface to regulate protein–AuNP assembly or influence the formation of the protein “corona,” modification of the protein surface as a mechanism to modulate protein–AuNP interaction has been less explored. Here, we examine this possibility utilizing three small globular proteins—lysozyme with high isoelectric point (pI) and established interactions with AuNP; α-lactalbumin with similar tertiary fold to lysozyme but low pI; and myoglobin with a different globular fold and an intermediate pI. We first chemically modified these proteins to alter their charged surface functionalities, and thereby shift protein pI, and then applied multiple methods to assess protein–AuNP assembly. At pH values lower than the anticipated pI of the modified protein, AuNP exposure elicits changes in the optical absorbance of the protein–NP solutions and other properties due to aggregate formation. Above the expected pI, however, protein–AuNP interaction is minimal, and both components remain isolated, presumably because both species are negatively charged. These data demonstrate that protein modification provides a powerful tool for modulating whether nanoparticle–protein interactions result in material aggregation. The results also underscore that naturally occurring protein modifications found in vivo may be critical in defining nanoparticle–protein corona compositions.

  6. Hydrolysis of Whey Protein Isolate with Bacillus licheniformis Protease: Fractionation and Identification of Aggregating Peptides

    NARCIS (Netherlands)

    Creusot, N.P.; Gruppen, H.

    2007-01-01

    The objective of this work was to identify the dominant aggregating peptides from a whey protein hydrolysate (degree of hydrolysis of 6.8%) obtained with Bacillus licheniformis protease. The aggregating peptides were fractionated with preparative reversed-phase chromatography and identified with

  7. Influence of hemodilution of plasma proteins on erythrocyte aggregability : An in vivo study in patients undergoing cardiopulmonary bypass

    NARCIS (Netherlands)

    Gu, YJ; Graaff, R; de Hoog, E; Veeger, NJGM; Panday, G; Boonstra, PW; van Oeveren, W

    2005-01-01

    Erythrocyte aggregation is known to be affected by a number of factors including the concentration of various plasma proteins. This study was performed to examine the in vivo effect of hemodilution of plasma proteins on erythrocyte aggregation in patients undergoing cardiopulmonary bypass (CPB)

  8. The Nucleation of Protein Aggregates - From Crystals to Amyloid Fibrils.

    Science.gov (United States)

    Buell, Alexander K

    2017-01-01

    The condensation and aggregation of individual protein molecules into dense insoluble phases is of relevance in such diverse fields as materials science, medicine, structural biology and pharmacology. A common feature of these condensation phenomena is that they usually are nucleated processes, i.e. the first piece of the condensed phase is energetically costly to create and hence forms slowly compared to its subsequent growth. Here we give a compact overview of the differences and similarities of various protein nucleation phenomena, their theoretical description in the framework of colloid and polymer science and their experimental study. Particular emphasis is put on the nucleation of a specific type of filamentous protein aggregates, amyloid fibrils. The current experimentally derived knowledge on amyloid fibril nucleation is critically assessed, and we argue that it is less advanced than is generally believed. This is due to (I) the lack of emphasis that has been put on the distinction between homogeneous and heterogeneous nucleation in experimental studies (II) the use of oversimplifying and/or inappropriate theoretical frameworks for the analysis of kinetic data of amyloid fibril nucleation. A strategy is outlined and advocated of how our understanding of this important class of processes can be improved in the future. © 2017 Elsevier Inc. All rights reserved.

  9. UV-Visible intensity ratio (aggregates/single particles) as a measure to obtain stability of gold nanoparticles conjugated with protein A

    Energy Technology Data Exchange (ETDEWEB)

    Rios-Corripio, M. A. [Instituto Politecnico Nacional, CIBA-Tlaxcala (Mexico); Garcia-Perez, B. E. [Instituto Politecnico Nacional, Departamento de Inmunologia, ENCB (Mexico); Jaramillo-Flores, M. E. [Instituto Politecnico Nacional, Departamento de Ingenieria Bioquimica, ENCB (Mexico); Gayou, V. L.; Rojas-Lopez, M., E-mail: marlonrl@yahoo.com.mx [Instituto Politecnico Nacional, CIBA-Tlaxcala (Mexico)

    2013-05-15

    We have analyzed the titration process of gold nanoparticles with several amounts of protein A (0.3, 0.5, 1, 3, 6, and 9 {mu}g/ml) in the presence of NaCl, which induces aggregation if the surface of particles is not fully covered with protein A. The colloidal solutions with different particle size (16, 18, 20, 33 nm) were synthesized by citrate reduction to be conjugated with protein A. UV-Visible spectroscopy was used to measure the absorption of the surface plasmon resonance of gold nanoparticles as a function of the concentration of protein A. Such dependence shows an aggregation region (0 < x<6 {mu}g/ml), where the amount of protein A was insufficient to cover the surface of particles, obtaining aggregation caused by NaCl. The next part is the stability region (x {>=} 6 {mu}g/ml), where the amount of protein used covers the surface of particles and protects it from the aggregation. In addition to that the ratio between the intensities of both: the aggregates and of the gold nanoparticle bands was plotted as a function of the concentration of protein A. It was determined that 6 {mu}g/ml is a sufficient value of protein A to stabilize the gold nanoparticle-protein A system. This method provides a simple way to stabilize gold nanoparticles obtained by citrate reduction, with protein A.

  10. Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases.

    Science.gov (United States)

    Chiti, Fabrizio; Calamai, Martino; Taddei, Niccolo; Stefani, Massimo; Ramponi, Giampietro; Dobson, Christopher M

    2002-12-10

    Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimer's disease, Parkinson's disease, and the Creutzfeldt-Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain. A highly significant inverse correlation was found to exist between the rates of aggregation of the protein variants under denaturing conditions and their overall net charge. This result indicates that aggregation is generally favored by mutations that bring the net charge of the protein closer to neutrality. In light of this finding, we have analyzed natural mutations associated with familial forms of amyloid diseases that involve alteration of the net charge of the proteins or protein fragments associated with the diseases. Sixteen mutations have been identified for which the mechanism of action that causes the pathological condition is not yet known or fully understood. Remarkably, 14 of these 16 mutations cause the net charge of the corresponding peptide or protein that converts into amyloid deposits to be reduced. This result suggests that charge has been a key parameter in molecular evolution to ensure the avoidance of protein aggregation and identifies reduction of the net charge as an important determinant in at least some forms of protein deposition diseases.

  11. Formation of truncated proteins and high-molecular-mass aggregates upon soft illumination of photosynthetic proteins

    DEFF Research Database (Denmark)

    Rinalducci, Sara; Campostrini, Natascia; Antonioli, Paolo

    2005-01-01

    Different spot profiles were observed in 2D gel electrophoresis of thylakoid membranes performed either under complete darkness or by leaving the sample for a short time to low visible light. In the latter case, a large number of new spots with lower molecular masses, ranging between 15,000 and 25......,000 Da, were observed, and high-molecular-mass aggregates, seen as a smearing in the upper part of the gel, appeared in the region around 250 kDa. Identification of protein(s) contained in these new spots by MS/MS revealed that most of them are simply truncated proteins deriving from native ones...

  12. Resolving the paradox for protein aggregation diseases: NMR structure and dynamics of the membrane-embedded P56S-MSP causing ALS imply a common mechanism for aggregation-prone proteins to attack membranes [v2; ref status: indexed, http://f1000r.es/3zl

    Directory of Open Access Journals (Sweden)

    Haina Qin

    2014-07-01

    Full Text Available Paradoxically, aggregation of specific proteins is characteristic of many human diseases and aging, yet aggregates have increasingly been found to be unnecessary for initiating pathogenesis. Here we determined the NMR topology and dynamics of a helical mutant in a membrane environment transformed from the 125-residue cytosolic all-β MSP domain of vesicle-associated membrane protein-associated protein B (VAPB by the ALS-causing P56S mutation. Despite its low hydrophobicity, the P56S major sperm protein (MSP domain becomes largely embedded in the membrane environment with high backbone rigidity. Furthermore it is composed of five helices with amphiphilicity comparable to those of the partly-soluble membrane toxin mellitin and α-synuclein causing Parkinson's disease. Consequently, the mechanism underlying this chameleon transformation becomes clear: by disrupting the specific tertiary interaction network stabilizing the native all-β MSP fold to release previously-locked amphiphilic segments, the P56S mutation acts to convert the classic MSP fold into a membrane-active protein that is fundamentally indistinguishable from mellitin and α-synuclein which are disordered in aqueous solution but spontaneously partition into membrane interfaces driven by hydrogen-bond energetics gained from forming α-helix in the membrane environments. As segments with high amphiphilicity exist in all proteins, our study successfully resolves the paradox by deciphering that the proteins with a higher tendency to aggregate have a stronger potential to partition into membranes through the same mechanism as α-synuclein to initially attack membranes to trigger pathogenesis without needing aggregates. This might represent the common first step for various kinds of aggregated proteins to trigger familiar, sporadic and aging diseases. Therefore the homeostasis of aggregated proteins in vivo is the central factor responsible for a variety of human diseases including aging

  13. Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

    KAUST Repository

    Shah, Dhawal

    2011-09-21

    We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP\\'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine\\'s preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine\\'s interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

  14. Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

    KAUST Repository

    Shah, Dhawal; Li, Jianguo; Shaikh, Abdul Rajjak; Rajagopalan, Raj

    2011-01-01

    We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP's indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

  15. Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Arun K Upadhyay

    Full Text Available The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

  16. Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

    Science.gov (United States)

    Upadhyay, Arun K.; Murmu, Aruna; Singh, Anupam; Panda, Amulya K.

    2012-01-01

    The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2–3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies. PMID:22479486

  17. Aggregation of soy protein-isoflavone complexes and gel formation induced by glucono-δ-lactone in soymilk

    Science.gov (United States)

    Hsia, Sheng-Yang; Hsiao, Yu-Hsuan; Li, Wen-Tai; Hsieh, Jung-Feng

    2016-10-01

    This study investigated the glucono-δ-lactone (GDL)-induced aggregation of isoflavones and soy proteins in soymilk. High-performance liquid chromatography (HPLC) analysis indicated that isoflavones mixed with β-conglycinin (7S) and glycinin (11S) proteins formed 7S-isoflavone and 11S-isoflavone complexes in soymilk supernatant fraction (SSF). Most of the soy protein-isoflavone complexes then precipitated into the soymilk pellet fraction (SPF) following the addition of 4 mM GDL, whereupon the pH value of the soymilk dropped from 6.6 to 5.9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis suggest that the addition of 4 mM GDL induced the aggregation of most 7S (α’, α and β subunits), 11S acidic and 11S basic proteins as well as isoflavones, including most aglycones, including daidzein, glycitein, genistein and a portion of glucosides, including daidzin, glycitin, genistin, malonyldaidzin and malonylgenistin. These results provide an important reference pertaining to the effects of GDL on the aggregation of soy protein-isoflavone complexes and could benefit future research regarding the production of tofu from soymilk.

  18. Aggregation and network formation in self-assembly of protein (H3.1) by a coarse-grained Monte Carlo simulation

    Science.gov (United States)

    Pandey, R. B.; Farmer, B. L.

    2014-11-01

    Multi-scale aggregation to network formation of interacting proteins (H3.1) are examined by a knowledge-based coarse-grained Monte Carlo simulation as a function of temperature and the number of protein chains, i.e., the concentration of the protein. Self-assembly of corresponding homo-polymers of constitutive residues (Cys, Thr, and Glu) with extreme residue-residue interactions, i.e., attractive (Cys-Cys), neutral (Thr-Thr), and repulsive (Glu-Glu), are also studied for comparison with the native protein. Visual inspections show contrast and similarity in morphological evolutions of protein assembly, aggregation of small aggregates to a ramified network from low to high temperature with the aggregation of a Cys-polymer, and an entangled network of Glu and Thr polymers. Variations in mobility profiles of residues with the concentration of the protein suggest that the segmental characteristic of proteins is altered considerably by the self-assembly from that in its isolated state. The global motion of proteins and Cys polymer chains is enhanced by their interacting network at the low temperature where isolated chains remain quasi-static. Transition from globular to random coil transition, evidenced by the sharp variation in the radius of gyration, of an isolated protein is smeared due to self-assembly of interacting networks of many proteins. Scaling of the structure factor S(q) with the wave vector q provides estimates of effective dimension D of the mass distribution at multiple length scales in self-assembly. Crossover from solid aggregates (D ˜ 3) at low temperature to a ramified fibrous network (D ˜ 2) at high temperature is observed for the protein H3.1 and Cys polymers in contrast to little changes in mass distribution (D ˜ 1.6) of fibrous Glu- and Thr-chain configurations.

  19. Cholesterol impairment contributes to neuroserpin aggregation

    Science.gov (United States)

    Giampietro, Costanza; Lionetti, Maria Chiara; Costantini, Giulio; Mutti, Federico; Zapperi, Stefano; La Porta, Caterina A. M.

    2017-03-01

    Intraneural accumulation of misfolded proteins is a common feature of several neurodegenerative pathologies including Alzheimer’s and Parkinson’s diseases, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). FENIB is a rare disease due to a point mutation in neuroserpin which accelerates protein aggregation in the endoplasmic reticulum (ER). Here we show that cholesterol depletion induced either by prolonged exposure to statins or by inhibiting the sterol reg-ulatory binding-element protein (SREBP) pathway also enhances aggregation of neuroserpin proteins. These findings can be explained considering a computational model of protein aggregation under non-equilibrium conditions, where a decrease in the rate of protein clearance improves aggregation. Decreasing cholesterol in cell membranes affects their biophysical properties, including their ability to form the vesicles needed for protein clearance, as we illustrate by a simple mathematical model. Taken together, these results suggest that cholesterol reduction induces neuroserpin aggregation, even in absence of specific neuroserpin mutations. The new mechanism we uncover could be relevant also for other neurodegenerative diseases associated with protein aggregation.

  20. Detection of ubiquitinated huntingtin species in intracellular aggregates

    Directory of Open Access Journals (Sweden)

    Katrin eJuenemann

    2015-01-01

    Full Text Available Protein conformation diseases, including polyglutamine diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease is one of nine diseases caused by an expanded polyglutamine repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated huntingtin such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated huntingtin fragments offers an important possibility to understand huntingtin degradation and aggregation processes within the cell. For the identification of aggregated huntingtin and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant huntingtin. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

  1. Molecular Mechanism of the Early Stage of Amyloidogenic Hexapeptides (NFGAIL) Aggregation

    International Nuclear Information System (INIS)

    Shi Bi-Yun; Zhou Bo; Cai Zhuo-Wei; Yang Zai-Xing; Xiu Peng

    2013-01-01

    Peptides/proteins aggregation can give rise to pathological conditions of many human diseases. Small partially ordered oligomers formed in the early stage of aggregation, rather than mature fibrils, are thought to be the main toxicity agent for the living cell. Thus, understanding the pathway and the underlying physical mechanism in the early stage of aggregation is very important for prevention and treatment of these protein functional diseases. Herein we use all-atom molecular dynamics simulations to study the aggregation of four NFGAIL hexapeptides (NFGAIL peptide is a core segment of human islet amyloid polypeptide and exhibits similar aggregation kinetics as the full-length polypeptide). We observe that the peptide monomers in water mainly adopt non-structural coil configurations; the four peptides which are randomly placed in water aggregate spontaneously to partially ordered oligomer (β-sheets) through dimerization or trimerization, with the dimerization predominated. Both parallel and anti-parallel β-sheets are observed. The hydrophobic interactions drive the initial peptides associations, and the subsequent conformational fluctuations promote the formation of more hydrogen bonds between the dangling hydrogen sites in the main chains of peptides. (interdisciplinary physics and related areas of science and technology)

  2. Misfolding, degradation, and aggregation of variant proteins. The molecular pathogenesis of short chain acyl-CoA dehydrogenase (SCAD) deficiency

    DEFF Research Database (Denmark)

    Pedersen, Christina Bak; Bross, P.; Winter, V.S.

    2003-01-01

    and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate...... proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency.......Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding...

  3. From the test tube to the cell: exploring the folding and aggregation of a beta-clam protein.

    Science.gov (United States)

    Ignatova, Zoya; Krishnan, Beena; Bombardier, Jeffrey P; Marcelino, Anna Marie C; Hong, Jiang; Gierasch, Lila M

    2007-01-01

    A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model beta-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.

  4. Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles

    International Nuclear Information System (INIS)

    Chen Min; Mikecz, Anna von

    2005-01-01

    Despite of their exponentially growing use, little is known about cell biological effects of nanoparticles. Here, we report uptake of silica (SiO 2 ) nanoparticles to the cell nucleus where they induce aberrant clusters of topoisomerase I (topo I) in the nucleoplasm that additionally contain signature proteins of nuclear domains, and protein aggregation such as ubiquitin, proteasomes, cellular glutamine repeat (polyQ) proteins, and huntingtin. Formation of intranuclear protein aggregates (1) inhibits replication, transcription, and cell proliferation; (2) does not significantly alter proteasomal activity or cell viability; and (3) is reversible by Congo red and trehalose. Since SiO 2 nanoparticles trigger a subnuclear pathology resembling the one occurring in expanded polyglutamine neurodegenerative disorders, we suggest that integrity of the functional architecture of the cell nucleus should be used as a read out for cytotoxicity and considered in the development of safe nanotechnology

  5. Combined Dynamic Light Scattering and Raman Spectroscopy Approach for Characterizing the Aggregation of Therapeutic Proteins

    Directory of Open Access Journals (Sweden)

    E. Neil Lewis

    2014-12-01

    Full Text Available Determination of the physicochemical properties of protein therapeutics and their aggregates is critical for developing formulations that enhance product efficacy, stability, safety and manufacturability. Analytical challenges are compounded for materials: (1 that are formulated at high concentration, (2 that are formulated with a variety of excipients, and (3 that are available only in small volumes. In this article, a new instrument is described that measures protein secondary and tertiary structure, as well as molecular size, over a range of concentrations and formulation conditions of low volume samples. Specifically, characterization of colloidal and conformational stability is obtained through a combination of two well-established analytical techniques: dynamic light scattering (DLS and Raman spectroscopy, respectively. As the data for these two analytical modalities are collected on the same sample at the same time, the technique enables direct correlation between them, in addition to the more straightforward benefit of minimizing sample usage by providing multiple analytical measurements on the same aliquot non-destructively. The ability to differentiate between unfolding and aggregation that the combination of these techniques provides enables insights into underlying protein aggregation mechanisms. The article will report on mechanistic insights for aggregation that have been obtained from the application of this technique to the characterization of lysozyme, which was evaluated as a function of concentration and pH.

  6. A novel nuclear DnaJ protein, DNAJC8, can suppress the formation of spinocerebellar ataxia 3 polyglutamine aggregation in a J-domain independent manner

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Norie [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Kamiguchi, Kenjiro; Nakanishi, Katsuya; Sokolovskya, Alice; Hirohashi, Yoshihiko; Tamura, Yasuaki; Murai, Aiko; Yamamoto, Eri; Kanaseki, Takayuki; Tsukahara, Tomohide; Kochin, Vitaly [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Chiba, Susumu [Department of Neurology, Clinical Brain Research Laboratory, Toyokura Memorial Hall, Sapporo Yamano-ue Hospital (Japan); Shimohama, Shun [Department of Neurology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Sato, Noriyuki [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Torigoe, Toshihiko, E-mail: torigoe@sapmed.ac.jp [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan)

    2016-06-10

    Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

  7. The chaperone-like activity of α-synuclein attenuates aggregation of its alternatively spliced isoform, 112-synuclein in vitro: plausible cross-talk between isoforms in protein aggregation.

    Directory of Open Access Journals (Sweden)

    Krishna Madhuri Manda

    Full Text Available Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD. Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synuclein (112-syn in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45 °C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37 °C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates.

  8. Effect of osmolytes on the conformation and aggregation of some amyloid peptides: CD spectroscopic data

    Directory of Open Access Journals (Sweden)

    Mohammed Inayathullah

    2016-06-01

    Full Text Available Protein misfolding and aggregation are responsible for a large number of diseases called protein conformational diseases or disorders that include Alzheimer׳s disease, Huntington׳s diseases, Prion related encephalopathies and type-II diabetes (http://dx.doi.org/10.1038/35041139 (Kopito and Ron, 2000 [1]. A variety of studies have shown that some small organic molecules, known as osmolytes have the ability to stabilize native conformation of proteins and prevent misfolding and aggregation (http://www.la-press.com/article.php?article_id=447 (Zhao et al., 2008 [2]. It has been shown that certain short segment or fragment of respective proteins can also form amyloids, and the segments also promote the aggregation in the full-length protein (http://dx.doi.org/10.2174/0929867023369187 (Gazit, 2002 [3]. This article presents circular dichroism spectroscopic data on conformational analysis and effect of osmolytes on Aβ peptide fragments, different lengths of polyglutamine peptide and the amyloidogenic segment of islet amyloid polypeptide.

  9. Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake.

    Science.gov (United States)

    Lee, Jong Woo; Lee, Seonju; Jang, Sangmok; Han, Kyu Young; Kim, Younggyu; Hyun, Jaekyung; Kim, Seong Keun; Lee, Yan

    2013-05-01

    Fluorescent nanodiamonds (FNDs) are very promising fluorophores for use in biosystems due to their high biocompatibility and photostability. To overcome their tendency to aggregate in physiological solutions, which severely limits the biological applications of FNDs, we developed a new non-covalent coating method using a block copolymer, PEG-b-P(DMAEMA-co-BMA), or proteins such as BSA and HSA. By simple mixing of the block copolymer with FNDs, the cationic DMAEMA and hydrophobic BMA moieties can strongly interact with the anionic and hydrophobic moieties on the FND surface, while the PEG block can form a shell to prevent the direct contact between FNDs. The polymer-coated FNDs, along with BSA- and HSA-coated FNDs, showed non-aggregation characteristics and maintained their size at the physiological salt concentration. The well-dispersed, polymer- or protein-coated FNDs in physiological solutions showed enhanced intracellular uptake, which was confirmed by CLSM. In addition, the biocompatibility of the coated FNDs was expressly supported by a cytotoxicity assay. Our simple non-covalent coating with the block copolymer, which can be easily modified by various chemical methods, projects a very promising outlook for future biomedical applications, especially in comparison with covalent coating or protein-based coating.

  10. A Mesoscopic Model for Protein-Protein Interactions in Solution

    OpenAIRE

    Lund, Mikael; Jönsson, Bo

    2003-01-01

    Protein self-association may be detrimental in biological systems, but can be utilized in a controlled fashion for protein crystallization. It is hence of considerable interest to understand how factors like solution conditions prevent or promote aggregation. Here we present a computational model describing interactions between protein molecules in solution. The calculations are based on a molecular description capturing the detailed structure of the protein molecule using x-ray or nuclear ma...

  11. Aggregation of MBP in chronic demyelination.

    Science.gov (United States)

    Frid, Kati; Einstein, Ofira; Friedman-Levi, Yael; Binyamin, Orli; Ben-Hur, Tamir; Gabizon, Ruth

    2015-07-01

    Misfolding of key disease proteins to an insoluble state is associated with most neurodegenerative conditions, such as prion, Parkinson, and Alzheimer's diseases. In this work, and by studying animal models of multiple sclerosis, we asked whether this is also the case for myelin basic protein (MBP) in the late and neurodegenerative phases of demyelinating diseases. To this effect, we tested whether MBP, an essential myelin component, present prion-like properties in animal models of MS, as is the case for Cuprizone-induced chronic demyelination or chronic phases of Experimental Autoimmune Encephalomyelitis (EAE). We show here that while total levels of MBP were not reduced following extensive demyelination, part of these molecules accumulated thereafter as aggregates inside oligodendrocytes or around neuronal cells. In chronic EAE, MBP precipitated concomitantly with Tau, a marker of diverse neurodegenerative conditions, including MS. Most important, analysis of fractions from Triton X-100 floatation gradients suggest that the lipid composition of brain membranes in chronic EAE differs significantly from that of naïve mice, an effect which may relate to oxidative insults and subsequently prevent the appropriate insertion and compaction of new MBP in the myelin sheath, thereby causing its misfolding and aggregation. Prion-like aggregation of MBP following chronic demyelination may result from an aberrant lipid composition accompanying this pathological status. Such aggregation of MBP may contribute to neuronal damage that occurs in the progressive phase of MS.

  12. TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

    Science.gov (United States)

    Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

    2014-01-01

    In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. PMID:25287913

  13. Aggregation factor analysis for protein formulation by a systematic approach using FTIR, SEC and design of experiments techniques.

    Science.gov (United States)

    Feng, Yan Wen; Ooishi, Ayako; Honda, Shinya

    2012-01-05

    A simple systematic approach using Fourier transform infrared (FTIR) spectroscopy, size exclusion chromatography (SEC) and design of experiments (DOE) techniques was applied to the analysis of aggregation factors for protein formulations in stress and accelerated testings. FTIR and SEC were used to evaluate protein conformational and storage stabilities, respectively. DOE was used to determine the suitable formulation and to analyze both the main effect of single factors and the interaction effect of combined factors on aggregation. Our results indicated that (i) analysis at a low protein concentration is not always applicable to high concentration formulations; (ii) an investigation of interaction effects of combined factors as well as main effects of single factors is effective for improving conformational stability of proteins; (iii) with the exception of pH, the results of stress testing with regard to aggregation factors would be available for suitable formulation instead of performing time-consuming accelerated testing; (iv) a suitable pH condition should not be determined in stress testing but in accelerated testing, because of inconsistent effects of pH on conformational and storage stabilities. In summary, we propose a three-step strategy, using FTIR, SEC and DOE techniques, to effectively analyze the aggregation factors and perform a rapid screening for suitable conditions of protein formulation. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Analyzing modifiers of protein aggregation in C. elegans by native agarose gel electrophoresis

    NARCIS (Netherlands)

    Holmberg, Mats; Nollen, Ellen A A; Hatters, Danny M.; Hannan, Anthony J.

    2013-01-01

    The accumulation of specific aggregation-prone proteins during aging is thought to be involved in several diseases, most notably Alzheimer's and Parkinson's disease as well as polyglutamine expansion disorders such as Huntington's disease. Caenorhabditis elegans disease models with transgenic

  15. Glycation precedes lens crystallin aggregation

    International Nuclear Information System (INIS)

    Swamy, M.S.; Perry, R.E.; Abraham, E.C.

    1987-01-01

    Non-enzymatic glycosylation (glycation) seems to have the potential to alter the structure of crystallins and make them susceptible to thiol oxidation leading to disulfide-linked high molecular weight (HMW) aggregate formation. They used streptozotocin diabetic rats during precataract and cataract stages and long-term cell-free glycation of bovine lens crystallins to study the relationship between glycation and lens crystallin aggregation. HMW aggregates and other protein components of the water-soluble (WS) and urea-soluble (US) fractions were separated by molecular sieve high performance liquid chromatography. Glycation was estimated by both [ 3 H]NaBH 4 reduction and phenylboronate agarose affinity chromatography. Levels of total glycated protein (GP) in the US fractions were about 2-fold higher than in the WS fractions and there was a linear increase in GP in both WS and US fractions. This increase was parallelled by a corresponding increase in HMW aggregates. Total GP extracted by the affinity method from the US fraction showed a predominance of HMW aggregates and vice versa. Cell-free glycation studies with bovine crystallins confirmed the results of the animals studies. Increasing glycation caused a corresponding increase in protein insolubilization and the insoluble fraction thus formed also contained more glycated protein. It appears that lens protein glycation, HMW aggregate formation, and protein insolubilization are interrelated

  16. Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3).

    Science.gov (United States)

    Seidel, K; Meister, M; Dugbartey, G J; Zijlstra, M P; Vinet, J; Brunt, E R P; van Leeuwen, F W; Rüb, U; Kampinga, H H; den Dunnen, W F A

    2012-10-01

    A characteristic of polyglutamine diseases is the increased propensity of disease proteins to aggregate, which is thought to be a major contributing factor to the underlying neurodegeneration. Healthy cells contain mechanisms for handling protein damage, the protein quality control, which must be impaired or inefficient to permit proteotoxicity under pathological conditions. We used a quantitative analysis of immunohistochemistry of the pons of eight patients with the polyglutamine disorder spinocerebellar ataxia type 3. We employed the anti-polyglutamine antibody 1C2, antibodies against p62 that is involved in delivering ubiquitinated protein aggregates to autophagosomes, antibodies against the chaperones HSPA1A and DNAJB1 and the proteasomal stress marker UBB⁺¹. The 1C2 antibody stained neuronal nuclear inclusions (NNIs), diffuse nuclear staining (DNS), granular cytoplasmic staining (GCS) and combinations, with reproducible distribution. P62 always co-localized with 1C2 in NNI. DNS and GCS co-stained with a lower frequency. UBB⁺¹ was present in a subset of neurones with NNI. A subset of UBB⁺¹-containing neurones displayed increased levels of HSPA1A, while DNAJB1 was sequestered into the NNI. Based on our results, we propose a model for the aggregation-associated pathology of spinocerebellar ataxia type 3: GCS and DNS aggregation likely represents early stages of pathology, which progresses towards formation of p62-positive NNI. A fraction of NNI exhibits UBB⁺¹ staining, implying proteasomal overload at a later stage. Subsequently, the stress-inducible HSPA1A is elevated while DNAJB1 is recruited into NNIs. This indicates that the stress response is only induced late when all endogenous protein quality control systems have failed. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.

  17. Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

    Directory of Open Access Journals (Sweden)

    Wüstner Daniel

    2012-11-01

    Full Text Available Abstract Background Fluorescence loss in photobleaching (FLIP is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp function is fitted to fluorescence loss (FL inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP, we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ disease proteins like mutant huntingtin (mtHtt can form large aggregates called inclusion bodies (IB’s. The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and

  18. 75 FR 27643 - Prevention of Significant Deterioration (PSD) and Nonattainment New Source Review (NSR): Aggregation

    Science.gov (United States)

    2010-05-18

    ... once the delay is no longer necessary. ADDRESSES: Docket: The final rule, the petition for...): Aggregation AGENCY: Environmental Protection Agency (EPA). ACTION: Delay of effective date. SUMMARY: EPA is delaying the effective date of the final rule titled ``Prevention of Significant Deterioration (PSD) and...

  19. The HSPB8-BAG3 chaperone complex is upregulated in astrocytes in the human brain affected by protein aggregation diseases.

    Science.gov (United States)

    Seidel, K; Vinet, J; Dunnen, W F A den; Brunt, E R; Meister, M; Boncoraglio, A; Zijlstra, M P; Boddeke, H W G M; Rüb, U; Kampinga, H H; Carra, S

    2012-02-01

    HSPB8 is a small heat shock protein that forms a complex with the co-chaperone BAG3. Overexpression of the HSPB8-BAG3 complex in cells stimulates autophagy and facilitates the clearance of mutated aggregation-prone proteins, whose accumulation is a hallmark of many neurodegenerative disorders. HSPB8-BAG3 could thus play a protective role in protein aggregation diseases and might be specifically upregulated in response to aggregate-prone protein-mediated toxicity. Here we analysed HSPB8-BAG3 expression levels in post-mortem human brain tissue from patients suffering of the following protein conformation disorders: Alzheimer's disease, Parkinson's disease, Huntington's disease and spinocerebellar ataxia type 3 (SCA3). Western blotting and immunohistochemistry techniques were used to analyse HSPB8 and BAG3 expression levels in fibroblasts from SCA3 patients and post-mortem brain tissues, respectively. In all diseases investigated, we observed a strong upregulation of HSPB8 and a moderate upregulation of BAG3 specifically in astrocytes in the cerebral areas affected by neuronal damage and degeneration. Intriguingly, no significant change in the HSPB8-BAG3 expression levels was observed within neurones, irrespective of their localization or of the presence of proteinaceous aggregates. We propose that the upregulation of HSPB8 and BAG3 may enhance the ability of astrocytes to clear aggregated proteins released from neurones and cellular debris, maintain the local tissue homeostasis and/or participate in the cytoskeletal remodelling that astrocytes undergo during astrogliosis. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.

  20. Reduced Insulin/IGF-1 Signaling Restores the Dynamic Properties of Key Stress Granule Proteins during Aging

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    Marie C. Lechler

    2017-01-01

    Full Text Available Summary: Low-complexity “prion-like” domains in key RNA-binding proteins (RBPs mediate the reversible assembly of RNA granules. Individual RBPs harboring these domains have been linked to specific neurodegenerative diseases. Although their aggregation in neurodegeneration has been extensively characterized, it remains unknown how the process of aging disturbs RBP dynamics. We show that a wide variety of RNA granule components, including stress granule proteins, become highly insoluble with age in C. elegans and that reduced insulin/insulin-like growth factor 1 (IGF-1 daf-2 receptor signaling efficiently prevents their aggregation. Importantly, stress-granule-related RBP aggregates are associated with reduced fitness. We show that heat shock transcription factor 1 (HSF-1 is a main regulator of stress-granule-related RBP aggregation in both young and aged animals. During aging, increasing DAF-16 activity restores dynamic stress-granule-related RBPs, partly by decreasing the buildup of other misfolded proteins that seed RBP aggregation. Longevity-associated mechanisms found to maintain dynamic RBPs during aging could be relevant for neurodegenerative diseases. : Lechler et al. show that RNA-binding proteins (RBPs including stress granule proteins are prone to aggregate with age in C. elegans. Aggregation of stress granule RBPs with “prion-like” domains is associated with reduced fitness. Their aggregation is prevented by longevity pathways and promoted by the aggregation of other misfolded proteins. Keywords: neurodegenerative diseases, Caenorhabditis elegans, protein aggregation, aging, RNA-binding proteins, stress granules, HSF-1, DAF-2, longevity

  1. Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd).

    Science.gov (United States)

    Ruiz, Geraldine Avila; Xiao, Wukai; van Boekel, Martinus; Minor, Marcel; Stieger, Markus

    2016-10-15

    The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8 (E8), 9 (E9), 10 (E10) and 11 (E11)), followed by acid precipitation. The obtained protein isolates were freeze dried. The protein isolates E8 and E9 resulted in a lower protein yield as well as less protein denaturation. These isolates also had a higher protein purity, more protein bands at higher molecular weights, and a higher protein solubility in the pH range of 3-4.5, compared to the isolates E10 and E11. Heating the 10%w/w protein isolate suspensions E8 and E9 led to increased aggregation, and semi-solid gels with a dense microstructure were formed. The isolate suspensions E10 and E11, on the other hand, aggregated less, did not form self-supporting gels and had loose particle arrangements. We conclude that extraction pH plays an important role in determining the functionality of quinoa protein isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Light-induced aggregation of microbial exopolymeric substances.

    Science.gov (United States)

    Sun, Luni; Xu, Chen; Zhang, Saijin; Lin, Peng; Schwehr, Kathleen A; Quigg, Antonietta; Chiu, Meng-Hsuen; Chin, Wei-Chun; Santschi, Peter H

    2017-08-01

    Sunlight can inhibit or disrupt the aggregation process of marine colloids via cleavage of high molecular weight compounds into smaller, less stable fragments. In contrast, some biomolecules, such as proteins excreted from bacteria can form aggregates via cross-linking due to photo-oxidation. To examine whether light-induced aggregation can occur in the marine environment, we conducted irradiation experiments on a well-characterized protein-containing exopolymeric substance (EPS) from the marine bacterium Sagitulla stellata. Our results show that after 1 h sunlight irradiation, the turbidity level of soluble EPS was 60% higher than in the dark control. Flow cytometry also confirmed that more particles of larger sized were formed by sunlight. In addition, we determined a higher mass of aggregates collected on filter in the irradiated samples. This suggests light can induce aggregation of this bacterial EPS. Reactive oxygen species hydroxyl radical and peroxide played critical roles in the photo-oxidation process, and salts assisted the aggregation process. The observation that Sagitulla stellata EPS with relatively high protein content promoted aggregation, was in contrast to the case where no significant differences were found in the aggregation of a non-protein containing phytoplankton EPS between the dark and light conditions. This, together with the evidence that protein-to-carbohydrate ratio of aggregates formed under light condition is significantly higher than that formed under dark condition suggest that proteins are likely the important component for aggregate formation. Light-induced aggregation provides new insights into polymer assembly, marine snow formation, and the fate/transport of organic carbon and nitrogen in the ocean. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells.

    Science.gov (United States)

    Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

    2014-01-01

    We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation.

  4. Cooperative structural transitions in amyloid-like aggregation

    Science.gov (United States)

    Steckmann, Timothy; Bhandari, Yuba R.; Chapagain, Prem P.; Gerstman, Bernard S.

    2017-04-01

    Amyloid fibril aggregation is associated with several horrific diseases such as Alzheimer's, Creutzfeld-Jacob, diabetes, Parkinson's, and others. Although proteins that undergo aggregation vary widely in their primary structure, they all produce a cross-β motif with the proteins in β-strand conformations perpendicular to the fibril axis. The process of amyloid aggregation involves forming myriad different metastable intermediate aggregates. To better understand the molecular basis of the protein structural transitions and aggregation, we report on molecular dynamics (MD) computational studies on the formation of amyloid protofibrillar structures in the small model protein ccβ, which undergoes many of the structural transitions of the larger, naturally occurring amyloid forming proteins. Two different structural transition processes involving hydrogen bonds are observed for aggregation into fibrils: the breaking of intrachain hydrogen bonds to allow β-hairpin proteins to straighten, and the subsequent formation of interchain H-bonds during aggregation into amyloid fibrils. For our MD simulations, we found that the temperature dependence of these two different structural transition processes results in the existence of a temperature window that the ccβ protein experiences during the process of forming protofibrillar structures. This temperature dependence allows us to investigate the dynamics on a molecular level. We report on the thermodynamics and cooperativity of the transformations. The structural transitions that occurred in a specific temperature window for ccβ in our investigations may also occur in other amyloid forming proteins but with biochemical parameters controlling the dynamics rather than temperature.

  5. Finite size effects in simulations of protein aggregation.

    Directory of Open Access Journals (Sweden)

    Amol Pawar

    Full Text Available It is becoming increasingly clear that the soluble protofibrillar species that proceed amyloid fibril formation are associated with a range of neurodegenerative disorders such as Alzheimer's and Parkinson diseases. Computer simulations of the processes that lead to the formation of these oligomeric species are starting to make significant contributions to our understanding of the determinants of protein aggregation. We simulate different systems at constant concentration but with a different number of peptides and we study the how the finite number of proteins affects the underlying free energy of the system and therefore the relative stability of the species involved in the process. If not taken into account, this finite size effect can undermine the validity of theoretical predictions regarding the relative stability of the species involved and the rates of conversion from one to the other. We discuss the reasons that give rise to this finite size effect form both a probabilistic and energy fluctuations point of view and also how this problem can be dealt by a finite size scaling analysis.

  6. [In vitro renaturation of proteins from inclusion bodies].

    Science.gov (United States)

    Porowińska, Dorota; Marszałek, Ewelina; Wardęcka, Paulina; Komoszyński, Michał

    2012-06-11

    Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties. Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules. Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules. To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.

  7. Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo.

    Directory of Open Access Journals (Sweden)

    Leila M Luheshi

    2010-03-01

    Full Text Available Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta peptide by using a small engineered binding protein (Z(Abeta3 that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3 in the brains of Drosophila melanogaster expressing either Abeta(42 or the aggressive familial associated E22G variant of Abeta(42 abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3 not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

  8. The Role of Aggregates of Therapeutic Protein Products in Immunogenicity: An Evaluation by Mathematical Modeling

    Directory of Open Access Journals (Sweden)

    Liusong Yin

    2015-01-01

    Full Text Available Therapeutic protein products (TPP have been widely used to treat a variety of human diseases, including cancer, hemophilia, and autoimmune diseases. However, TPP can induce unwanted immune responses that can impact both drug efficacy and patient safety. The presence of aggregates is of particular concern as they have been implicated in inducing both T cell-independent and T cell-dependent immune responses. We used mathematical modeling to evaluate several mechanisms through which aggregates of TPP could contribute to the development of immunogenicity. Modeling interactions between aggregates and B cell receptors demonstrated that aggregates are unlikely to induce T cell-independent immune responses by cross-linking B cell receptors because the amount of signal transducing complex that can form under physiologically relevant conditions is limited. We systematically evaluate the role of aggregates in inducing T cell-dependent immune responses using a recently developed multiscale mechanistic mathematical model. Our analysis indicates that aggregates could contribute to T cell-dependent immune response by inducing high affinity epitopes which may not be present in the nonaggregated TPP and/or by enhancing danger signals to break tolerance. In summary, our computational analysis is suggestive of novel insights into the mechanisms underlying aggregate-induced immunogenicity, which could be used to develop mitigation strategies.

  9. ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1*

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-01-01

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Δhep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer. PMID:20007714

  10. ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1.

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-02-12

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

  11. Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

    Science.gov (United States)

    Tyedmers, Jens

    2012-01-01

    The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI+] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates. PMID:22449721

  12. Coupling of aggregation and immunogenicity in biotherapeutics: T- and B-cell immune epitopes may contain aggregation-prone regions.

    Science.gov (United States)

    Kumar, Sandeep; Singh, Satish K; Wang, Xiaoling; Rup, Bonita; Gill, Davinder

    2011-05-01

    Biotherapeutics, including recombinant or plasma-derived human proteins and antibody-based molecules, have emerged as an important class of pharmaceuticals. Aggregation and immunogenicity are among the major bottlenecks during discovery and development of biotherapeutics. Computational tools that can predict aggregation prone regions as well as T- and B-cell immune epitopes from protein sequence and structure have become available recently. Here, we describe a potential coupling between aggregation and immunogenicity: T-cell and B-cell immune epitopes in therapeutic proteins may contain aggregation-prone regions. The details of biological mechanisms behind this observation remain to be understood. However, our observation opens up an exciting potential for rational design of de-immunized novel, as well as follow on biotherapeutics with reduced aggregation propensity.

  13. Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

    International Nuclear Information System (INIS)

    Zhou, Donghua H.; Nieuwkoop, Andrew J.; Berthold, Deborah A.; Comellas, Gemma; Sperling, Lindsay J.; Tang, Ming; Shah, Gautam J.; Brea, Elliott J.; Lemkau, Luisel R.; Rienstra, Chad M.

    2012-01-01

    Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.

  14. Small heat shock proteins protect against α-synuclein-induced toxicity and aggregation

    International Nuclear Information System (INIS)

    Outeiro, Tiago Fleming; Klucken, Jochen; Strathearn, Katherine E.; Liu Fang; Nguyen, Paul; Rochet, Jean-Christophe; Hyman, Bradley T.; McLean, Pamela J.

    2006-01-01

    Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). α-Synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and αB-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messenger RNA levels are ∼2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by ∼80% in a culture model while αB-crystallin reduces toxicity by ∼20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model

  15. Single particle detection and characterization of synuclein co-aggregation

    International Nuclear Information System (INIS)

    Giese, Armin; Bader, Benedikt; Bieschke, Jan; Schaffar, Gregor; Odoy, Sabine; Kahle, Philipp J.; Haass, Christian; Kretzschmar, Hans

    2005-01-01

    Protein aggregation is the key event in a number of human diseases such as Alzheimer's and Parkinson's disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type α-synuclein forms co-aggregates with a mutant variant found in familial Parkinson's disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects

  16. Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

    Science.gov (United States)

    Mirecka, Ewa Agnieszka; Gremer, Lothar; Schiefer, Stephanie; Oesterhelt, Filipp; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2014-12-10

    Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Disrupting beta-amyloid aggregation for Alzheimer disease treatment.

    Science.gov (United States)

    Estrada, L D; Soto, C

    2007-01-01

    Alzheimer's disease is a devastating degenerative disorder for which there is no cure or effective treatment. Although the etiology of Alzheimer's disease is not fully understood, compelling evidence indicates that deposition of aggregates composed by a misfolded form of the amyloid beta peptide (Abeta) is the central event in the disease pathogenesis. Therefore, an attractive therapeutic strategy is to prevent or reverse Abeta misfolding and aggregation. Diverse strategies have been described to identify inhibitors of this process, including screening of libraries of small molecules chemical compounds, rational design of synthetic peptides, assessment of natural Abeta-binding proteins and stimulation of the immune system by vaccination. In this article we describe these different approaches, their principles and their potential strengths and weaknesses. Overall the available data suggest that the development of drugs to interfere with Abeta misfolding and aggregation is a feasible target that hold great promise for the treatment of Alzheimer's disease.

  18. Application of mid-infrared free-electron laser tuned to amide bands for dissociation of aggregate structure of protein.

    Science.gov (United States)

    Kawasaki, Takayasu; Yaji, Toyonari; Ohta, Toshiaki; Tsukiyama, Koichi

    2016-01-01

    A mid-infrared free-electron laser (FEL) is a linearly polarized, high-peak powered pulse laser with tunable wavelength within the mid-infrared absorption region. It was recently found that pathogenic amyloid fibrils could be partially dissociated to the monomer form by the irradiation of the FEL targeting the amide I band (C=O stretching vibration), amide II band (N-H bending vibration) and amide III band (C-N stretching vibration). In this study, the irradiation effect of the FEL on keratin aggregate was tested as another model to demonstrate an applicability of the FEL for dissociation of protein aggregates. Synchrotron radiation infrared microscopy analysis showed that the α-helix content in the aggregate structure decreased to almost the same level as that in the monomer state after FEL irradiation tuned to 6.06 µm (amide I band). Both irradiations at 6.51 µm (amide II band) and 8.06 µm (amide III band) also decreased the content of the aggregate but to a lesser extent than for the irradiation at the amide I band. On the contrary, the irradiation tuned to 5.6 µm (non-absorbance region) changed little the secondary structure of the aggregate. Scanning-electron microscopy observation at the submicrometer order showed that the angular solid of the aggregate was converted to non-ordered fragments by the irradiation at each amide band, while the aggregate was hardly deformed by the irradiation at 5.6 µm. These results demonstrate that the amide-specific irradiation by the FEL was effective for dissociation of the protein aggregate to the monomer form.

  19. Pressure effects on the structure, kinetic, and thermodynamic properties of heat-induced aggregation of protein studied by FT-IR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Taniguchi, Y [Applied Chemistry Department, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Okuno, A [Research Department 3, Central Research, Bridgestone Co. Kodaira, Tokyo 187-8531 (Japan); Kato, M, E-mail: taniguti@sk.ritsumei.ac.j [Pharmaceutical Sciences Department, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2010-03-01

    Pressure can retrain the heat-induced aggregation and dissociate the heat-induced aggregates. We observed the aggregation-preventing pressure effect and the aggregates-dissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by FT-IR spectroscopy. The results suggest the {alpha}-helical structure collapses at the beginning of heat-induced aggregation through the swollen structure, and then the rearrangement of structure to the intermolecular {beta}-sheet takes place through partially unfolded structure. We determined the activation volume for the heat-induced aggregation ({Delta}V'' = +93 ml/mol) and the partial molar volume difference between native state and heat-induced aggregates ({Delta}V=+32 ml/mol). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular {beta}-sheet is unfavorable under high pressure.

  20. Electrostatics promotes molecular crowding and selects the aggregation pathway in fibril-forming protein solutions

    International Nuclear Information System (INIS)

    Raccosta, S.; Martorana, V.; Manno, M.; Blanco, M.; Roberts, C.J.

    2016-01-01

    The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation are crucial aspects for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions for two proteins at acidic ph, lysozyme and α-chymotrypsinogen. By using light scattering experiments and the Kirkwood-Buff integral approach, we show how concentration fluctuations are damped even at moderate protein concentrations by the dominant long-ranged electrostatic repulsion, which determines an effective crowded environment. In denaturing conditions, electrostatic repulsion keeps the monomeric solution in a thermodynamically metastable state, which is escaped through kinetically populated conformational sub-states. This explains how electrostatics acts as a gatekeeper in selecting a specific aggregation pathway.

  1. S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site.

    Science.gov (United States)

    Kanda, Hironori; Toyama, Takashi; Shinohara-Kanda, Azusa; Iwamatsu, Akihiro; Shinkai, Yasuhiro; Kaji, Toshiyuki; Kikushima, Makoto; Kumagai, Yoshito

    2012-11-01

    We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803-808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42 kDa, thereby facilitating its aggregation. Two-dimensional SDS-polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42 kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.

  2. Superoxide dismutase 1 is positively selected to minimize protein aggregation in great apes

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Kepp, Kasper Planeta

    2017-01-01

    Positive (adaptive) selection has recently been implied in human superoxide dismutase 1 (SOD1), a highly abundant antioxidant protein with energy signaling and antiaging functions, one of very few examples of direct selection on a human protein product (exon); the molecular drivers...... and SOD1 aggregates and triggered by aging. Our study thus marks an example of direct selection for a particular chemical phenotype (high net charge and stability) in a single human protein with possible implications for the evolution of aging....... of this selection are unknown. We mapped 30 extant SOD1 sequences to the recently established mammalian species tree and inferred ancestors, key substitutions, and signatures of selection during the protein's evolution. We detected elevated substitution rates leading to great apes (Hominidae) at ~1 per 2 million...

  3. Long-term manure amendments reduced soil aggregate stability via redistribution of the glomalin-related soil protein in macroaggregates

    Science.gov (United States)

    Xie, Hongtu; Li, Jianwei; Zhang, Bin; Wang, Lianfeng; Wang, Jingkuan; He, Hongbo; Zhang, Xudong

    2015-01-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm; 2000–250 μm; 250–53 μm; and soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000–250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture. PMID:26423355

  4. Selenite reduction by anaerobic microbial aggregates: Microbial community structure, and proteins associated to the produced selenium spheres.

    Directory of Open Access Journals (Sweden)

    Graciela eGonzalez-Gil

    2016-04-01

    Full Text Available Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se0, insights into the microbial community structure and synthesis of Se0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20 % and Pseudomonadaceae (c.a.10 % were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (~200 m of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se0 (nano spheres, with some cells having high numbers of intracellular Se0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS analysis of extracted Se0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se0 spheres by the microorganisms inhabiting the granular sludge is proposed.

  5. Selenite Reduction by Anaerobic Microbial Aggregates: Microbial Community Structure, and Proteins Associated to the Produced Selenium Spheres

    KAUST Repository

    Gonzalez-Gil, Graciela

    2016-04-26

    Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se0), insights into the microbial community structure and synthesis of Se0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (∼200 μm) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se0 (nano)spheres, with some cells having high numbers of intracellular Se0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se0 spheres by the microorganisms inhabiting the granular sludge is proposed.

  6. Sensitive electrochemical detection of native and aggregated alpha-synuclein protein involved in Parkinson's disease

    Czech Academy of Sciences Publication Activity Database

    Masařík, Michal; Stobiecka, A.; Kizek, René; Jelen, František; Pechan, Zdeněk; Hoyer, W.; Jovin, T.; Subramaniam, V.; Paleček, Emil

    2004-01-01

    Roč. 16, 13-14 (2004), s. 1172-1181 ISSN 1040-0397 R&D Projects: GA ČR GA204/03/0566 Institutional research plan: CEZ:AV0Z5004920 Keywords : electrochemistry of proteins * alpha-synuclein aggregation * adsorptive transfer stripping Subject RIV: BO - Biophysics Impact factor: 2.038, year: 2004

  7. Impact of high hydrostatic pressure processing on individual cellular resuscitation times and protein aggregates in Escherichia coli.

    Science.gov (United States)

    Govers, Sander K; Aertsen, Abram

    2015-11-20

    Live cell biology approaches can contribute to a more comprehensive understanding of heterogeneous injury and resuscitation phenomena in stressed populations of foodborne pathogens and spoilage microorganisms, and in turn lead to better insights in the mechanisms and dynamics of inactivation that can improve food safety and preservation measures. Especially in the context of designing minimal processing strategies, which depend on a synergistic combination of different mild stresses to ensure sufficient microbial reduction, a more profound understanding of the impact of each such stress or hurdle is mandatory. High hydrostatic pressure (HHP) stress is an interesting hurdle in this concept since cells that manage to survive this stress nevertheless tend to be injured and sensitized to subsequent stresses. In this study, populations of Escherichia coli were subjected to different HHP intensities and studied at the single-cell level with time-lapse fluorescence microscopy while monitoring resuscitation times and protein aggregate integrity at the single-cell level. This approach revealed that higher pressure intensities lead to longer and more variable resuscitation times of surviving cells as well as an increased dispersal of intracellular protein aggregates. Interestingly, at mild HHP exposure, cells within the population incurring less dispersion of protein aggregates appeared to have a higher probability of survival. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. 17-AAG induces cytoplasmic alpha-synuclein aggregate clearance by induction of autophagy.

    Science.gov (United States)

    Riedel, Michael; Goldbaum, Olaf; Schwarz, Lisa; Schmitt, Sebastian; Richter-Landsberg, Christiane

    2010-01-18

    The accumulation and aggregation of alpha-synuclein in nerve cells and glia are characteristic features of a number of neurodegenerative diseases termed synucleinopathies. alpha-Synuclein is a highly soluble protein which in a nucleation dependent process is capable of self-aggregation. The causes underlying aggregate formation are not yet understood, impairment of the proteolytic degradation systems might be involved. In the present study the possible aggregate clearing effects of the geldanamycin analogue 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) was investigated. Towards this, an oligodendroglial cell line (OLN-93 cells), stably expressing human alpha-synuclein (A53T mutation) was used. In these cells small punctate aggregates, not staining with thioflavine S, representing prefibrillary aggregates, occur characteristically. Our data demonstrate that 17-AAG attenuated the formation of alpha-synuclein aggregates by stimulating macroautophagy. By blocking the lysosomal compartment with NH(4)Cl the aggregate clearing effects of 17-AAG were abolished and alpha-synuclein deposits were enlarged. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that 17-AAG led to the recruitment of LC3-II and to the formation of LC3 positive puncta. This effect was also observed in cultured oligodendrocytes derived from the brains of newborn rats. Inhibition of macroautophagy by 3-methyladenine prevented 17-AAG induced occurrence of LC3 positive puncta as well as the removal of alpha-synuclein aggregates in OLN-A53T cells. Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which alpha-synuclein can be removed. Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.

  9. Morphology-Variable Aggregates Prepared from Cholesterol-Containing Amphiphilic Glycopolymers: Their Protein Recognition/Adsorption and Drug Delivery Applications

    Directory of Open Access Journals (Sweden)

    Zhao Wang

    2018-02-01

    Full Text Available In this study, a series of diblock glycopolymers, poly(6-O-methacryloyl-d-galactopyranose-b-poly(6-cholesteryloxyhexyl methacrylate (PMAgala-b-PMAChols, with cholesterol/galactose grafts were prepared through a sequential reversible addition-fragmentation chain transfer (RAFT polymerization and deprotection process. The glycopolymers could self-assemble into aggregates with various morphologies depending on cholesterol/galactose-containing block weight ratios, as determined by transmission electronic microscopy (TEM and dynamic laser light scattering (DLS. In addition, the lectin (Ricinus communis agglutinin II, RCA120 recognition and bovine serum albumin (BSA adsorption of the PMAgala-b-PMAChol aggregates were evaluated. The SK-Hep-1 tumor cell inhibition properties of the PMAgala-b-PMAChol/doxorubicin (DOX complex aggregates were further examined in vitro. Results indicate that the PMAgala-b-PMAChol aggregates with various morphologies showed different interaction/recognition features with RCA120 and BSA. Spherical aggregates (d ≈ 92 nm possessed the highest RCA120 recognition ability and lowest BSA protein adsorption. In addition, the DOX-loaded spherical complex aggregates exhibited a better tumor cell inhibition property than those of nanofibrous complex aggregates. The morphology-variable aggregates derived from the amphiphilic glycopolymers may serve as multifunctional biomaterials with biomolecular recognition and drug delivery features.

  10. Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

    International Nuclear Information System (INIS)

    Kanapathipillai, Mathumai; Ku, Sook Hee; Girigoswami, Koyeli; Park, Chan Beum

    2008-01-01

    In prion diseases, the posttranslational modification of host-encoded prion protein PrP c yields a high β-sheet content modified protein PrP sc , which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP c to PrP sc . Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs-ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity

  11. Aggregation of endosomal-vacuolar compartments in the Aovps24-deleted strain in the filamentous fungus Aspergillus oryzae

    International Nuclear Information System (INIS)

    Tatsumi, Akinori; Shoji, Jun-ya; Kikuma, Takashi; Arioka, Manabu; Kitamoto, Katsuhiko

    2007-01-01

    Previously, we found that deletion of Aovps24, an ortholog of Saccharomyces cerevisiae VPS24, that encodes an ESCRT (endosomal sorting complex required for transport)-III component required for late endosomal function results in fragmented and aggregated vacuoles. Although defective late endosomal function is likely responsible for this phenotype, critical lack of our knowledge on late endosomes in filamentous fungi prevented us from further characterization. In this study, we identified late endosomes of Aspergillus oryzae, by expressing a series of fusion proteins of fluorescent proteins with orthologs of late endosomal proteins. Using these fusion proteins as markers, we observed late endosomes in the wild type strain and the Aovps24 disruptant and demonstrated that late endosomes are aberrantly aggregated in the Aovps24 disruptant. Moreover, we revealed that the aggregated late endosomes have features of vacuoles as well. As deletion of another ESCRT-III component-encoding gene, Aovps2, resulted in similar phenotypes to that in the Aovps24 disruptant, phenotypes of the Aovps24 disruptant are probably due to defective late endosomal function

  12. Effects of the size and content of protein aggregates on the rheological and structural properties of soy protein isolate emulsion gels induced by CaSO4.

    Science.gov (United States)

    Wang, Xufeng; He, Zhiyong; Zeng, Maomao; Qin, Fang; Adhikari, Benu; Chen, Jie

    2017-04-15

    The effects of the size and content of soy protein isolate (SPI) aggregates on the rheological and textural properties of CaSO 4 -induced SPI emulsion gels were investigated. Considerable differences in the rheological, water-holding, and micro-structural properties were observed. The gels with larger and/or more SPI aggregates showed substantial increase in the elastic modulus and had lower gelation temperatures. Creep data suggested that the size of the SPI aggregates contributed more to the elastic modulus, whereas the increase of aggregate content enhanced the elastic modulus and viscous component of the gels. The water-holding capacity was markedly enhanced (pemulsions and emulsion gels. Copyright © 2016. Published by Elsevier Ltd.

  13. The molecular chaperone α-crystallin inhibits UV-induced protein aggregation

    International Nuclear Information System (INIS)

    Borkman, R.F.; Knight, Grady; Obi, Bettie

    1996-01-01

    Solutions of γ-crystallin, and various enzymes, at neutral pH and 24-26 o C, became turbid upon exposure to UV radiation at 295 or 308 nm. SDS-PAGE analysis revealed interchain cross-linking and aggregate formation compared to dark control solutions as reported previously. When α-crystallin was added to the protein solutions in stoichiometric amounts. UV irradiation resulted in significantly less turbidity than in the absence of α-crystallin. For example, addition of 0.5 mg of α-crystallin to 0.5 mg of γ-crystallin in 1.0 ml solution yielded only 25% of the turbidity seen in the absence of α-crystallin. Addition of 2.0 mg of α-crystallin resulted in 20% of the turbidity. Given the molecular weights of α- and γ-crystallin (about 800 kDa and 20 kDa, respectively), A γ/α 1:1 weight ratio corresponds to a 40:1 molar ratio, and a γ-/α 1:4 weight ratio corresponds to a 10:1 molar ratio. Hence, the molar ratio of α-crystallin needed to effectively protect γ-crystallin from photochemical opacification was γ/α = n:1, where n was in the range 10-40. In terms of subunits, this ratio is γ/α = 1:m, where m = 1-4. Thus, each γ-crystallin molecule needs 1-4 α subunits for protection. Similar stoichiometries were observed for protection of the other proteins studied. The protection stems in part from screening of UV radiation by α-crystallin but more importantly from a chaperone effect analogous to that seen in thermal aggregation experiments. (author)

  14. Spinocerebellar Ataxia Type 6 Protein Aggregates Cause Deficits in Motor Learning and Cerebellar Plasticity

    NARCIS (Netherlands)

    Mark, Melanie D; Krause, Martin; Boele, Henk-Jan; Kruse, Wolfgang; Pollok, Stefan; Kuner, Thomas; Dalkara, Deniz; Koekkoek, Sebastiaan; De Zeeuw, Chris I; Herlitze, Stefan

    2015-01-01

    Spinocerebellar ataxia type 6 (SCA6) is linked to poly-glutamine (polyQ) within the C terminus (CT) of the pore-forming subunits of P/Q-type Ca(2+) channels (Cav2.1) and is characterized by CT protein aggregates found in cerebellar Purkinje cells (PCs). One hypothesis regarding SCA6 disease is that

  15. Aluminum induces lipid peroxidation and aggregation of human blood platelets

    Directory of Open Access Journals (Sweden)

    T.J.C. Neiva

    1997-05-01

    Full Text Available Aluminum (Al3+ intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al3+-induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 µM stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA (100 µM and n-propyl gallate (NPG (100 µM, inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA (100 µM, an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation

  16. The choice of a suitable oligosaccharide to prevent aggregation of PEGylated nanoparticles during freeze thawing and freeze drying

    NARCIS (Netherlands)

    Hinrichs, Wouter; Manceñido, F A; Sanders, N N; Braeckmans, K; De Smedt, S C; Demeester, J; Frijlink, H W

    2006-01-01

    In a previous Study we have shown that the oligosaccharide inulin can prevent aggregation of poly(ethylene glycol) (PEG) coated plasmid DNA/cationic liposome complexes ('' PEGylated lipoplexes '') during freeze thawing and freeze drying [Hinrichs et al., 2005. J. Control. Release 103, 465]. By

  17. Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets.

    Science.gov (United States)

    Marreiros, Rita; Müller-Schiffmann, Andreas; Bader, Verian; Selvarajah, Suganya; Dey, Debendranath; Lingappa, Vishwanath R; Korth, Carsten

    2015-09-02

    Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation

  18. Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates.

    Directory of Open Access Journals (Sweden)

    Junghyun Lim

    Full Text Available Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1 linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt induces p62 phosphorylation at its ubiquitin-association (UBA domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington's disease.

  19. Sorbitol crystallization-induced aggregation in frozen mAb formulations.

    Science.gov (United States)

    Piedmonte, Deirdre Murphy; Hair, Alison; Baker, Priti; Brych, Lejla; Nagapudi, Karthik; Lin, Hong; Cao, Wenjin; Hershenson, Susan; Ratnaswamy, Gayathri

    2015-02-01

    Sorbitol crystallization-induced aggregation of mAbs in the frozen state was evaluated. The effect of protein aggregation resulting from sorbitol crystallization was measured as a function of formulation variables such as protein concentration and pH. Long-term studies were performed on both IgG1 and IgG2 mAbs over the protein concentration range of 0.1-120 mg/mL. Protein aggregation was measured by size-exclusion HPLC (SE-HPLC) and further characterized by capillary-electrophoresis SDS. Sorbitol crystallization was monitored and characterized by subambient differential scanning calorimetry and X-ray diffraction. Aggregation due to sorbitol crystallization is inversely proportional to both protein concentration and formulation pH. At high protein concentrations, sorbitol crystallization was suppressed, and minimal aggregation by SE-HPLC resulted, presumably because of self-stabilization of the mAbs. The glass transition temperature (Tg ') and fragility index measurements were made to assess the influence of molecular mobility on the crystallization of sorbitol. Tg ' increased with increasing protein concentration for both mAbs. The fragility index decreased with increasing protein concentration, suggesting that it is increasingly difficult for sorbitol to crystallize at high protein concentrations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  20. Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

    International Nuclear Information System (INIS)

    Gupta, Preeti; Deep, Shashank

    2014-01-01

    Highlights: • HCAII forms amyloid-like aggregates at moderate concentration of trifluoroethanol. • Protein adopts a state between β-sheet and α-helix at moderate % of TFE. • Hydrophobic surface(s) of partially structured conformation forms amyloid. • High % of TFE induces stable α-helical state preventing aggregation. - Abstract: In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII

  1. Photodamaging mechanisms of the eye structure: the aggregates appearance at UV-illumination of lens proteins

    International Nuclear Information System (INIS)

    El'chaninov, V.V.; Fedorovich, I.B.

    1989-01-01

    UV-light injury of individual crystallines (water soluble proteins of the cattle eye crystalline lens) were studied by SDS PSSG technique. Photodamage resulted in olygomer formation. The appearance of high molecular aggregates with the molecular mass as large as 10 5 D were seen in all fractions of the crystalline

  2. An overview on the small heat shock proteins | Mahmood | African ...

    African Journals Online (AJOL)

    In eukaryotes, different heat shock genes are expressed uncoordinatedly, whereas in prokaryote, heat shock genes form a regulon and appear simultaneously. sHSPs are associated with nuclei, cytoskeleton and membranes. They bind partially to denatured proteins, preventing irreversible protein aggregation during stress.

  3. Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases

    OpenAIRE

    Parvez Alam; Sumit Kumar Chaturvedi; Mohammad Khursheed Siddiqi; Ravi Kant Rajpoot; Mohd Rehan Ajmal; Masihuz Zaman; Rizwan Hasan Khan

    2016-01-01

    Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer?s, Parkinson?s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and A?-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demons...

  4. Aggregation in charged nanoparticles solutions induced by different interactions

    Energy Technology Data Exchange (ETDEWEB)

    Abbas, S.; Kumar, Sugam; Aswal, V. K., E-mail: vkaswal@barc.gov.in [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Kohlbrecher, J. [Laboratory for Neutron Scattering, Paul Scherrer Institut, CH-5232 PSI Villigen (Switzerland)

    2016-05-23

    Small-angle neutron scattering (SANS) has been used to study the aggregation of anionic silica nanoparticles as induced through different interactions. The nanoparticle aggregation is induced by addition of salt (NaCl), cationic protein (lysozyme) and non-ionic surfactant (C12E10) employing different kind of interactions. The results show that the interaction in presence of salt can be explained using DLVO theory whereas non-DLVO forces play important role for interaction of nanoparticles with protein and surfactant. The presence of salt screens the repulsion between charged nanoparticles giving rise to a net attraction in the DLVO potential. On the other hand, strong electrostatic attraction between nanoparticle and oppositely charged protein leads to protein-mediated nanoparticle aggregation. In case of non-ionic surfactant, the relatively long-range attractive depletion interaction is found to be responsible for the particle aggregation. Interestingly, the completely different interactions lead to similar kind of aggregate morphology. The nanoparticle aggregates formed are found to have mass fractal nature having a fractal dimension (~2.5) consistent with diffusion limited type of fractal morphology in all three cases.

  5. The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter

    Directory of Open Access Journals (Sweden)

    Martinez Lucia

    2006-09-01

    Full Text Available Abstract Background The yields of soluble recombinant proteins expressed in bacteria are often low due to the tendency of the heterologous proteins to form aggregates. Therefore, aggregation reporters have been envisaged to simplify the comparison among different expression conditions and to speed up the identification of suitable protocols that improve the solubility. The probe we used is composed by an IbpAB promoter specifically activated by protein aggregates fused to a sequence coding the β-galactosidase, the activity of which becomes, therefore, indicative of the aggregation degree. Results The collected data show that the probe is reliable in terms of reproducibility inside a range of experimental conditions and faster and more sensitive than the analysis methods based on SDS-PAGE and successive western blot. The β-galactosidase probe was useful to identify which parameters could influence the aggregation of the model proteins and to set up an optimized expression protocol. The effect of growth temperature, induction modality, co-expression with molecular chaperones and addition of osmolytes on the accumulation of aggregates were evaluated following the β-galactosidase activity. Interestingly, a significant correlation was observed between estimated decreased aggregation and higher yields of soluble protein. We also compared a set of expression vectors with various regulative features and found that the single characteristics, like promoter, copy number or polymerase, were not relevant for controlling the recombinant protein aggregation whilst the crucial factor resulted being the total expression rate of the system. Conclusion The aggregation reporter used in our experiments represents a useful tool to evaluate the different factors that can be modulated to optimize a recombinant expression protocol. Furthermore, the rapid estimation of the aggregation degree enables to discriminate this from other causes responsible for scarce

  6. Influence of heat and shear induced protein aggregation on the in vitro digestion rate of whey proteins.

    Science.gov (United States)

    Singh, Tanoj K; Øiseth, Sofia K; Lundin, Leif; Day, Li

    2014-11-01

    Protein intake is essential for growth and repair of body cells, the normal functioning of muscles, and health related immune functions. Most food proteins are consumed after undergoing various degrees of processing. Changes in protein structure and assembly as a result of processing impact the digestibility of proteins. Research in understanding to what extent the protein structure impacts the rate of proteolysis under human physiological conditions has gained considerable interest. In this work, four whey protein gels were prepared using heat processing at two different pH values, 6.8 and 4.6, with and without applied shear. The gels showed different protein network microstructures due to heat induced unfolding (at pH 6.8) or lack of unfolding, thus resulting in fine stranded protein networks. When shear was applied during heating, particulate protein networks were formed. The differences in the gel microstructures resulted in considerable differences in their rheological properties. An in vitro gastric and intestinal model was used to investigate the resulting effects of these different gel structures on whey protein digestion. In addition, the rate of digestion was monitored by taking samples at various time points throughout the in vitro digestion process. The peptides in the digesta were profiled using SDS-polyacrylamide gel electrophoresis, reversed-phase-HPLC and LC-MS. Under simulated gastric conditions, whey proteins in structured gels were hydrolysed faster than native proteins in solution. The rate of peptides released during in vitro digestion differed depending on the structure of the gels and extent of protein aggregation. The outcomes of this work highlighted that changes in the network structure of the protein can influence the rate and pattern of its proteolysis under gastrointestinal conditions. Such knowledge could assist the food industry in designing novel food formulations to control the digestion kinetics and the release of biologically

  7. Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases

    Science.gov (United States)

    Salahuddin, Parveen; Siddiqi, Mohammad Khursheed; Khan, Sanaullah; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

    2016-11-01

    In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially misfolded intermediates aggregate via the interaction with the complementary intermediates and consequently enhance oligomers formation that grows into fibrils and proto-fibrils. The amyloid fibrils for example, accumulate in the brain and central nervous system (CNS) as amyloid deposits in the Parkinson's disease (PD), Alzheimer's disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other neurodegenerative diseases. Treatment of such misfolding maladies is considered as one of the most important challenges of the 21st century. Currently, several treatments strategies have been and are being discovered. These therapeutic interventions partly reversed or prevented the pathological state. More recently, a new approach was discovered, which employs nanobodies that targets multisteps in fibril formation pathway that may possibly completely cure these misfolding diseases. Keeping the above views in mind in the current review, we have comprehensively discussed the different mechanisms underlying protein misfolding thereby leading to diseases conditions and their therapeutic interventions.

  8. Heterologous expression of a plastid EF-Tu reduces protein thermal aggregation and enhances CO2 fixation in wheat (Triticum aestivum) following heat stress.

    Science.gov (United States)

    Fu, Jianming; Momcilović, Ivana; Clemente, Thomas E; Nersesian, Natalya; Trick, Harold N; Ristic, Zoran

    2008-10-01

    Heat stress is a major constraint to wheat production and negatively impacts grain quality, causing tremendous economic losses, and may become a more troublesome factor due to global warming. At the cellular level, heat stress causes denaturation and aggregation of proteins and injury to membranes leading to alterations in metabolic fluxes. Protein aggregation is irreversible, and protection of proteins from thermal aggregation is a strategy a cell uses to tolerate heat stress. Here we report on the development of transgenic wheat (Triticum aestivum) events, expressing a maize gene coding for plastidal protein synthesis elongation factor (EF-Tu), which, compared to non-transgenic plants, display reduced thermal aggregation of leaf proteins, reduced heat injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation after exposure to heat stress. The results support the concept that EF-Tu ameliorates negative effects of heat stress by acting as a molecular chaperone. This is the first demonstration of the introduction of a plastidal EF-Tu in plants that leads to protection against heat injury and enhanced photosynthesis after heat stress. This is also the first demonstration that a gene other than HSP gene can be used for improvement of heat tolerance and that the improvement is possible in a species that has a complex genome, hexaploid wheat. The results strongly suggest that heat tolerance of wheat, and possibly other crop plants, can be improved by modulating expression of plastidal EF-Tu and/or by selection of genotypes with increased endogenous levels of this protein.

  9. Intrinsically disordered proteins as molecular shields†

    Science.gov (United States)

    Chakrabortee, Sohini; Tripathi, Rashmi; Watson, Matthew; Kaminski Schierle, Gabriele S.; Kurniawan, Davy P.; Kaminski, Clemens F.; Wise, Michael J.; Tunnacliffe, Alan

    2017-01-01

    The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 – while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins – HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability

  10. Intrinsically disordered proteins as molecular shields.

    Science.gov (United States)

    Chakrabortee, Sohini; Tripathi, Rashmi; Watson, Matthew; Schierle, Gabriele S Kaminski; Kurniawan, Davy P; Kaminski, Clemens F; Wise, Michael J; Tunnacliffe, Alan

    2012-01-01

    The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 - while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins - HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability

  11. Data on correlation between Aβ42 structural aggregation propensity and toxicity in bacteria

    Directory of Open Access Journals (Sweden)

    Anita Carija

    2016-06-01

    Full Text Available Protein aggregation and amyloid formation is a hallmark of an increasing number of human disorders. Because protein aggregation is deleterious for the cell physiology and results in a decrease in overall cell fitness, it is thought that natural selection acts to purify aggregating proteins during evolution. This data article contains complementary figures and results related to the research article entitled “Selection against toxic aggregation-prone protein sequences in bacteria” (Navarro et al., 2014 [1]. Here, we used the AGGRESCAN3D (A3D server, a novel in house predictor that forecasts protein aggregation properties in protein structures to illustrate a striking correlation between the structure-based predictions of aggregation propensities for Alzheimer’s Aβ42 peptide variants and their previously reported deleterious effects in bacteria.

  12. Highly Stable Trypsin-Aggregate Coatings on Polymer Nanofibers for Repeated Protein Digestion

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Byoung Chan; Lopez-Ferrer, Daniel; Lee, Sang-mok; Ahn, Hye-kyung; Nair, Sujith; Kim, Seong H.; Kim, Beom S.; Petritis, Konstantinos; Camp, David G.; Grate, Jay W.; Smith, Richard D.; Koo, Yoon-mo; Gu, Man Bock; Kim, Jungbae

    2009-04-01

    A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This new process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was also resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with enzyme aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

  13. Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3)

    NARCIS (Netherlands)

    Seidel, K.; Meister, M.; Dugbartey, G. J.; Zijlstra, M. P.; Vinet, J.; Brunt, E. R. P.; van Leeuwen, F. W.; Rueb, U.; Kampinga, H. H.; den Dunnen, W. F. A.

    2012-01-01

    K. Seidel, M. Meister, G. J. Dugbartey, M. P. Zijlstra, J. Vinet, E. R. P. Brunt, F. W. van Leeuwen, U. Rub, H. H. Kampinga and W. F. A. den Dunnen (2012) Neuropathology and Applied Neurobiology38, 548558 Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type

  14. Effect of metal ions on de novo aggregation of full-length prion protein

    International Nuclear Information System (INIS)

    Giese, Armin; Levin, Johannes; Bertsch, Uwe; Kretzschmar, Hans

    2004-01-01

    It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu 2+ , Mn 2+ , Ni 2+ , Co 2+ , and Zn 2+ ) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluorescently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn 2+ at low micromolar concentrations that could be blocked by nanomolar concentration of Cu 2+ . These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo

  15. Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd)

    NARCIS (Netherlands)

    Ruiz, Geraldine Avila; Xiao, Wukai; Boekel, van Tiny; Minor, Marcel; Stieger, Markus

    2016-01-01

    The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8

  16. Utilization of unbound aggregates for road construction

    OpenAIRE

    Fladvad, Marit

    2017-01-01

    Crushed rock aggregate is a non-renewable resource of great interest in road construction and other branches of the construction industry. To prevent resource scarcity, utilization of aggregates should be considered carefully. © 2016 Norsk Bergforening

  17. Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding

    NARCIS (Netherlands)

    Hageman, Jurre; Vos, Michel J.; van Waarde, Maria A. W. H.; Kampinga, Harm H.

    2007-01-01

    Molecular chaperones are essential for cells to prevent that partially unfolded proteins form non-functional, toxic aggregates. This requirement is increased when cells experience protein unfolding stresses and such could affect all compartments in the eukaryotic cell. Whether all organelles are

  18. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice; Smit, John; Jiao, Yongqin

    2016-09-23

    ABSTRACT

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in

  19. NABi, a novel β-sheet breaker, inhibits Aβ aggregation and neuronal toxicity: Therapeutic implications for Alzheimer's disease.

    Science.gov (United States)

    Jang, Ja-Young; Rhim, Hyangshuk; Kang, Seongman

    2018-01-01

    Amyloid beta (Aβ) aggregates are an important therapeutic target for Alzheimer's disease (AD), a fatal neurodegenerative disease. To date, AD still remains a big challenge due to no effective treatments. Based on the property that Aβ aggregates have the cross-β-structure, a common structural feature in amyloids, we systemically designed the Aβ-aggregation inhibitor that maintains Aβ-interacting ability but removes toxic part from SOD1 (superoxide dismutase 1)-G93A. We identified NABi (Natural Aβ Binder and Aβ-aggregation inhibitor) composed of β2-3 strands, a novel breaker of Aβ aggregation, which does not self-aggregate and has no cytotoxicity at all. The NABi blocks Aβ-fibril formation in vitro and in vivo and prevents neuronal cell death, a hallmark of AD pathogenesis. Such anti-amyloidogenic properties can provide novel strategies for treating AD. Furthermore, our study provides molecular insights into the design of amyloidogenic inhibitors to cure various neurodegenerative and amyloid-associated diseases, as NABi would regulate aggregation of other toxic β-sheet proteins other than Aβ. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Aggregation and conformational stability evaluation of myoglobin in the presence of ionic surfactant

    Directory of Open Access Journals (Sweden)

    Mohammad A. Alsenaidy

    2018-05-01

    Full Text Available Sodium lauroyl sarcosinate (SLS is frequently used for the solubilization of inclusion bodies in vitro due to its structural similarity to lipid plasma membrane. There are many factors that could influence protein aggregation propensity, including overall protein surface charge and hydrophobicity. Here, the aggregation pathway of myoglobin protein was studied under different conditions (pH 3.5 and 7.4 in the presence of varying concentrations of SLS to evaluate the underlying forces dictating protein aggregation. Data obtained from Rayleigh light scattering, ThT binding assay, and far-UV CD indicated that SLS have different effects on the protein depending on its concentration and environmental conditions. In the presence of low concentrations of SLS (0.05–0.1 mM, no aggregation was detected at both pH conditions tested. Whereas, as we reach higher SLS concentrations (0.5–10.0 mM, myoglobin started forming larger-sized aggregates at pH 3.5 and not pH 7.4. These results suggest that electrostatics interactions as well as hydrophobic forces play an important role in SLS-induced myoglobin aggregation. Keywords: Sodium lauroyl sarcosinate, Surfactant, Myoglobin, Protein aggregation, Amorphous aggregates, pH

  1. SOD1 aggregation in ALS mice shows simplistic test tube behavior.

    Science.gov (United States)

    Lang, Lisa; Zetterström, Per; Brännström, Thomas; Marklund, Stefan L; Danielsson, Jens; Oliveberg, Mikael

    2015-08-11

    A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.

  2. Formation of thermally induced aggregates of the soya globulin beta-conglycinin.

    Science.gov (United States)

    Mills, E N; Huang, L; Noel, T R; Gunning, A P; Morris, V J

    2001-06-11

    The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.

  3. Aggregates in monoclonal antibody manufacturing processes.

    Science.gov (United States)

    Vázquez-Rey, María; Lang, Dietmar A

    2011-07-01

    Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Large-scale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. If a therapeutic protein cannot be stabilized adequately, it will lose partially or totally its therapeutic properties or even cause immunogenic reactions thus potentially further endangering the patients' health. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and efficacy of antibodies and can happen at different steps of the manufacturing process, including fermentation, purification, final formulation, and storage. Aggregate levels in drug substance and final drug product are a key factor when assessing quality attributes of the molecule, since aggregation might impact biological activity of the biopharmaceutical. In this review it is analyzed how aggregates are formed during monoclonal antibody industrial production, why they have to be removed and the manufacturing process steps that are designed to either minimize or remove aggregates in the final product. Copyright © 2011 Wiley Periodicals, Inc.

  4. Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models

    Directory of Open Access Journals (Sweden)

    Kung PJ

    2014-10-01

    Full Text Available Pin-Jui Kung,1,* Yu-Chen Tao,1,* Ho-Chiang Hsu,1 Wan-Ling Chen,1 Te-Hsien Lin,1 Donala Janreddy,2 Ching-Fa Yao,2 Kuo-Hsuan Chang,3 Jung-Yaw Lin,1 Ming-Tsan Su,1 Chung-Hsin Wu,1 Guey-Jen Lee-Chen,1 Hsiu-Mei Hsieh-Li1 1Department of Life Science, 2Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan; 3Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan *These authors contributed equally to this work Abstract: In spinocerebellar ataxia type 17 (SCA17, the expansion of a translated CAG repeat in the TATA box binding protein (TBP gene results in a long polyglutamine (polyQ tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment. Keywords: spinocerebellar ataxia type 17, TATA box binding protein, polyQ aggregation, indole and derivative, therapeutics

  5. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    International Nuclear Information System (INIS)

    Valdes, Iris; Bernardo, Lidice; Gil, Lazaro; Pavon, Alekis; Lazo, Laura; Lopez, Carlos; Romero, Yaremis; Menendez, Ivon; Falcon, Viviana; Betancourt, Lazaro; Martin, Jorge; Chinea, Glay; Silva, Ricardo; Guzman, Maria G.; Guillen, Gerardo; Hermida, Lisset

    2009-01-01

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-γ secretion and protection experiments, mediated by CD4 + and CD8 + cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  6. SUMO-1 is associated with a subset of lysosomes in glial protein aggregate diseases.

    Science.gov (United States)

    Wong, Mathew B; Goodwin, Jacob; Norazit, Anwar; Meedeniya, Adrian C B; Richter-Landsberg, Christiane; Gai, Wei Ping; Pountney, Dean L

    2013-01-01

    that was immunopositive for Hsp90 and immunoprecipitated with an anti-SUMO-1 antibody. That SUMO-1 co-localizes with a subset of lysosomes in neurodegenerative diseases with glial protein aggregates and in glial cell culture models of protein aggregation suggests a role for SUMO-1 in lysosome function.

  7. An essential nonredundant role for mycobacterial DnaK in native protein folding.

    Directory of Open Access Journals (Sweden)

    Allison Fay

    2014-07-01

    Full Text Available Protein chaperones are essential in all domains of life to prevent and resolve protein misfolding during translation and proteotoxic stress. HSP70 family chaperones, including E. coli DnaK, function in stress induced protein refolding and degradation, but are dispensable for cellular viability due to redundant chaperone systems that prevent global nascent peptide insolubility. However, the function of HSP70 chaperones in mycobacteria, a genus that includes multiple human pathogens, has not been examined. We find that mycobacterial DnaK is essential for cell growth and required for native protein folding in Mycobacterium smegmatis. Loss of DnaK is accompanied by proteotoxic collapse characterized by the accumulation of insoluble newly synthesized proteins. DnaK is required for solubility of large multimodular lipid synthases, including the essential lipid synthase FASI, and DnaK loss is accompanied by disruption of membrane structure and increased cell permeability. Trigger Factor is nonessential and has a minor role in native protein folding that is only evident in the absence of DnaK. In unstressed cells, DnaK localizes to multiple, dynamic foci, but relocalizes to focal protein aggregates during stationary phase or upon expression of aggregating peptides. Mycobacterial cells restart cell growth after proteotoxic stress by isolating persistent DnaK containing protein aggregates away from daughter cells. These results reveal unanticipated essential nonredunant roles for mycobacterial DnaK in mycobacteria and indicate that DnaK defines a unique susceptibility point in the mycobacterial proteostasis network.

  8. Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3ζ protein

    International Nuclear Information System (INIS)

    Sadik, Golam; Tanaka, Toshihisa; Kato, Kiyoko; Yanagi, Kentaro; Kudo, Takashi; Takeda, Masatoshi

    2009-01-01

    Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3ζ. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3ζ is ∼3-folds higher than that between unphosphorylated 4R-tau and 14-3-3ζ. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3ζ to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3ζ. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3ζ exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3ζ suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

  9. A microliter-scale high-throughput screening system with quantum-dot nanoprobes for amyloid-β aggregation inhibitors.

    Directory of Open Access Journals (Sweden)

    Yukako Ishigaki

    Full Text Available The aggregation of amyloid β protein (Aβ is a key step in the pathogenesis of Alzheimer's disease (AD, and therefore inhibitory substances for Aβ aggregation may have preventive and/or therapeutic potential for AD. Here we report a novel microliter-scale high-throughput screening system for Aβ aggregation inhibitors based on fluorescence microscopy-imaging technology with quantum-dot Nanoprobes. This screening system could be analyzed with a 5-µl sample volume when a 1536-well plate was used, and the inhibitory activity could be estimated as half-maximal effective concentrations (EC50. We attempted to comprehensively screen Aβ aggregation inhibitors from 52 spices using this system to assess whether this novel screening system is actually useful for screening inhibitors. Screening results indicate that approximately 90% of the ethanolic extracts from the spices showed inhibitory activity for Aβ aggregation. Interestingly, spices belonging to the Lamiaceae, the mint family, showed significantly higher activity than the average of tested spices. Furthermore, we tried to isolate the main inhibitory compound from Saturejahortensis, summer savory, a member of the Lamiaceae, using this system, and revealed that the main active compound was rosmarinic acid. These results demonstrate that this novel microliter-scale high-throughput screening system could be applied to the actual screening of Aβ aggregation inhibitors. Since this system can analyze at a microscopic scale, it is likely that further minimization of the system would easily be possible such as protein microarray technology.

  10. Computational Selection of Inhibitors of A-beta Aggregation and Neuronal Toxicity

    Science.gov (United States)

    Chen, Deliang; Martin, Zane S.; Soto, Claudio; Schein, Catherine H.

    2009-01-01

    Alzheimer’s Disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-β protein (Aβ). Disease symptoms can be alleviated, in vitro and in vivo, by “β-sheet breaker” pentapeptides that reduce plaque volume. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related β-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Aβ. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features in a conformation similar to the active peptides were selected, ranked by docking solubility parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Aβ aggregation at 2–3 μM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Aβ on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD. PMID:19540126

  11. Protein aggregation in aqueous casein solution. Effect of irradiation, dose level, concentration, storage and additives (carbohydrate and lipid)

    Energy Technology Data Exchange (ETDEWEB)

    Yousri, R M

    1980-06-01

    From the vast amount of research efforts dealing with various aspects of radiation effects on foods and food components, it is apparent up to now that much remains to be studied in depth, much may have to be added or corrected about radiation-induced physico-chemical changes in foods. A great many reactions that take place when foodstuffs are subjected to ionizing radiation are still not fully understood. The better understanding of some of the radiation-induced changes in pure proteins as such or in mixture with other food constituents could yield much data which could be meaningfully extrapolated to intact foods and consequently could help to improve the assessment of the wholesomeness of irradiated foods. It was the purpose of our investigations to elucidate some of the changes in the chemical structure of a pure protein (casein), irradiated as such or with added carbohydrate and/or lipid. The effect of subsequent storage of the irradiated solutions has been also examined. The formation of protein aggregates was studied by gel filtration technique. The application of thin-layer gel filtration, its speed and adaptability to very small samples facilitated the measurements of the extent of aggregation which occurred in protein molecules after irradiation.

  12. The protein DIIIC-2, aggregated with a specific oligodeoxynucleotide and adjuvanted in alum, protects mice and monkeys against DENV-2.

    Science.gov (United States)

    Gil, Lázaro; Marcos, Ernesto; Izquierdo, Alienys; Lazo, Laura; Valdés, Iris; Ambala, Peris; Ochola, Lucy; Hitler, Rikoi; Suzarte, Edith; Álvarez, Mayling; Kimiti, Prisilla; Ndung'u, James; Kariuki, Thomas; Guzmán, María Guadalupe; Guillén, Gerardo; Hermida, Lisset

    2015-01-01

    Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum.

  13. Anisotropy of the Coulomb Interaction between Folded Proteins: Consequences for Mesoscopic Aggregation of Lysozyme

    Science.gov (United States)

    Chan, Ho Yin; Lankevich, Vladimir; Vekilov, Peter G.; Lubchenko, Vassiliy

    2012-01-01

    Toward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. PMID:22768950

  14. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    International Nuclear Information System (INIS)

    Zanzoni, Serena; D’Onofrio, Mariapina; Molinari, Henriette; Assfalg, Michael

    2012-01-01

    Highlights: ► Bile acid binding proteins from different constructs retain structural integrity. ► NMR 15 N-T 1 relaxation data of BABPs show differences if LVPR extension is present. ► Deviations from a 15 N-T 1 /molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  15. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zanzoni, Serena; D' Onofrio, Mariapina; Molinari, Henriette [Department of Biotechnology, University of Verona, 37134 Verona (Italy); Assfalg, Michael, E-mail: michael.assfalg@univr.it [Department of Biotechnology, University of Verona, 37134 Verona (Italy)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  16. Applying chaperones to protein-misfolding disorders: molecular chaperones against α-synuclein in Parkinson's disease.

    Science.gov (United States)

    Chaari, Ali; Hoarau-Véchot, Jessica; Ladjimi, Moncef

    2013-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of a protein called α-synuclein (α-syn) into inclusions known as lewy bodies (LB) within neurons. This accumulation is also due to insufficient formation and activity of dopamine produced in certain neurons within the substantia nigra. Lewy bodies are the pathological hallmark of the idiopathic disorder and the cascade that allows α-synuclein to misfold, aggregate and form these inclusions has been the subject of intensive research. Targeting these early steps of oligomerization is one of the main therapeutic approaches in order to develop neurodegenerative-modifying agents. Because the folding and refolding of alpha synuclein is the key point of this cascade, we are interested in this review to summarize the role of some molecular chaperones proteins such as Hsp70, Hsp90 and small heat shock proteins (sHsp) and Hsp 104. Hsp70 and its co-chaperone, Hsp70 and small heat shock proteins can prevent neurodegeneration by preventing α-syn misfolding, oligomerization and aggregation in vitro and in Parkinson disease animal models. Hsp104 is able to resolve disordered protein aggregates and cross beta amyloid conformers. Together, these chaperones have a complementary effect and can be a target for therapeutic intervention in PD. Published by Elsevier B.V.

  17. A Fragment-Based Method of Creating Small-Molecule Libraries to Target the Aggregation of Intrinsically Disordered Proteins.

    Science.gov (United States)

    Joshi, Priyanka; Chia, Sean; Habchi, Johnny; Knowles, Tuomas P J; Dobson, Christopher M; Vendruscolo, Michele

    2016-03-14

    The aggregation process of intrinsically disordered proteins (IDPs) has been associated with a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Currently, however, no drug in clinical use targets IDP aggregation. To facilitate drug discovery programs in this important and challenging area, we describe a fragment-based approach of generating small-molecule libraries that target specific IDPs. The method is based on the use of molecular fragments extracted from compounds reported in the literature to inhibit of the aggregation of IDPs. These fragments are used to screen existing large generic libraries of small molecules to form smaller libraries specific for given IDPs. We illustrate this approach by describing three distinct small-molecule libraries to target, Aβ, tau, and α-synuclein, which are three IDPs implicated in Alzheimer's and Parkinson's diseases. The strategy described here offers novel opportunities for the identification of effective molecular scaffolds for drug discovery for neurodegenerative disorders and to provide insights into the mechanism of small-molecule binding to IDPs.

  18. Modeling the Aggregation Propensity and Toxicity of Amyloid-β Variants

    DEFF Research Database (Denmark)

    Tiwari, Manish Kumar; Kepp, Kasper Planeta

    2015-01-01

    Protein aggregation is a hallmark of many neurodegenerative disorders. Alzheimer’s disease (AD) is directly linked to deposits of amyloid-β (Aβ) derived from the amyloid-β protein precursor (AβPP), and multiple experimental studies have investigated the aggregation behavior of these amyloids...

  19. Aggregation and fibrillation of bovine serum albumin

    DEFF Research Database (Denmark)

    Holm, NK; Jespersen, SK; Thomassen, LV

    2007-01-01

    The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology...

  20. Dietary Protein in the Prevention of Diet-Induced Obesity and Co-Morbidities

    DEFF Research Database (Denmark)

    Tastesen, Hanne Sørup

    mice were fed obesity‐promoting diets with protein from different sources, in different forms and at different levels to evaluate the affect on development of obesity, glucose intolerance and dyslipidemia. Results: In the present study the dietary level of protein, 16 versus 32 percent energy from...... protein, was found to be negligible in development of obesity and co‐morbidities in mice. Seafood protein with high endogenous taurine and glycine contents was found to prevent diet‐induced adiposity and dyslipidemia, both in ad libitum and pair‐fed settings. The ability of seafood proteins to prevent...... that the source and form of protein has great impact on development and prevention of diet‐induced adiposity, dyslipidemia, hyperinsulinemia and impairment of glucose tolerance through modulations of voluntary locomotor activity, energy expenditure and energy substrate metabolism in mice...

  1. Effect of homogenisation in formation of thermally induced aggregates in a non- and low- fat milk model system with microparticulated whey proteins.

    Science.gov (United States)

    Torres, Isabel Celigueta; Nieto, Gema; Nylander, Tommy; Simonsen, Adam Cohen; Tolkach, Alexander; Ipsen, Richard

    2017-05-01

    The objective of the research presented in this paper was to investigate how different characteristics of whey protein microparticles (MWP) added to milk as fat replacers influence intermolecular interactions occurring with other milk proteins during homogenisation and heating. These interactions are responsible for the formation of heat-induced aggregates that influence the texture and sensory characteristics of the final product. The formation of heat-induced complexes was studied in non- and low-fat milk model systems, where microparticulated whey protein (MWP) was used as fat replacer. Five MWP types with different particle characteristics were utilised and three heat treatments used: 85 °C for 15 min, 90 °C for 5 min and 95 °C for 2 min. Surface characteristics of the protein aggregates were expressed as the number of available thiol groups and the surface net charge. Intermolecular interactions involved in the formation of protein aggregates were studied by polyacrylamide gel electrophoresis and the final complexes visualised by darkfield microscopy. Homogenisation of non-fat milk systems led to partial adsorption of caseins onto microparticles, independently of the type of microparticle. On the contrary, homogenisation of low-fat milk resulted in preferential adsorption of caseins onto fat globules, rather than onto microparticles. Further heating of the milk, led to the formation of heat induced complexes with different sizes and characteristics depending on the type of MWP and the presence or not of fat. The results highlight the importance of controlling homogenisation and heat processing in yoghurt manufacture in order to induce desired changes in the surface reactivity of the microparticles and thereby promote effective protein interactions.

  2. Assays for alpha-synuclein aggregation

    DEFF Research Database (Denmark)

    Giehm, Lise; Lorenzen, Nikolai; Otzen, Daniel

    2011-01-01

    Over the last few decades, protein aggregation gone from being an irritating side product in the test tube to becoming a subject of great interest. This has been stimulated by the realization that a large and growing number of diseases is associated with the formation and accumulation of proteins...

  3. Adaptive enhanced sampling with a path-variable for the simulation of protein folding and aggregation

    Science.gov (United States)

    Peter, Emanuel K.

    2017-12-01

    In this article, we present a novel adaptive enhanced sampling molecular dynamics (MD) method for the accelerated simulation of protein folding and aggregation. We introduce a path-variable L based on the un-biased momenta p and displacements dq for the definition of the bias s applied to the system and derive 3 algorithms: general adaptive bias MD, adaptive path-sampling, and a hybrid method which combines the first 2 methodologies. Through the analysis of the correlations between the bias and the un-biased gradient in the system, we find that the hybrid methodology leads to an improved force correlation and acceleration in the sampling of the phase space. We apply our method on SPC/E water, where we find a conservation of the average water structure. We then use our method to sample dialanine and the folding of TrpCage, where we find a good agreement with simulation data reported in the literature. Finally, we apply our methodologies on the initial stages of aggregation of a hexamer of Alzheimer's amyloid β fragment 25-35 (Aβ 25-35) and find that transitions within the hexameric aggregate are dominated by entropic barriers, while we speculate that especially the conformation entropy plays a major role in the formation of the fibril as a rate limiting factor.

  4. Protein kinase C involvement in the acetylcholine release reduction induced by amyloid-beta(25-35) aggregates on neuromuscular synapses.

    Science.gov (United States)

    Tomàs, Marta; Garcia, Neus; Santafé, Manuel M; Lanuza, Maria; Tomàs, Josep

    2009-01-01

    Using intracellular recording of the diaphragm muscle of adult rats, we have investigated the short-term functional effects of amyloid-beta (Abeta(25-35) peptide aggregates on the modulation of acetylcholine (ACh) release and the involvement of protein kinase C (PKC). The non-aggregated form of this peptide does not change the evoked and spontaneous transmitter release parameters on the neuromuscular synapse. However, the aggregated form of Abeta(25-35) acutely interferes with evoked quantal ACh release (approximately 40% reduction) when synaptic activity in the ex vivo neuromuscular preparation is maintained by low frequency (1 Hz) electrical stimulation. This effect is partially dependent on the activity of PKC that may have a permissive action. The end result of Abeta(25-35) is in opposition to the PKC-dependent maintenance effect on ACh release manifested in active synapses.

  5. Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors.

    Science.gov (United States)

    Abskharon, Romany; Dang, Johnny; Elfarash, Ameer; Wang, Zerui; Shen, Pingping; Zou, Lewis S; Hassan, Sedky; Wang, Fei; Fujioka, Hisashi; Steyaert, Jan; Mulaj, Mentor; Surewicz, Witold K; Castilla, Joaquín; Wohlkonig, Alexandre; Zou, Wen-Quan

    2017-10-04

    The infectious prion protein (PrP Sc or prion) is derived from its cellular form (PrP C ) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrP C to PrP Sc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrP C (BVPrP) is highly susceptible to PrP Sc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease. To explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion. Our results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

  6. A Protein Aggregation Inhibitor, Leuco-Methylthioninium Bis(Hydromethanesulfonate, Decreases α-Synuclein Inclusions in a Transgenic Mouse Model of Synucleinopathy

    Directory of Open Access Journals (Sweden)

    Karima Schwab

    2018-01-01

    Full Text Available α-Synuclein (α-Syn aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson’s disease (PD. We have tested whether N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminium bis(hydromethanesulfonate (leuco-methylthioninium bis(hydromethanesulfonate; LMTM, a tau aggregation inhibitor, affects α-Syn aggregation in vitro and in vivo. Both cellular and transgenic models in which the expression of full-length human α-Syn (h-α-Syn fused with a signal sequence peptide to promote α-Syn aggregation were used. Aggregated α-Syn was observed following differentiation of N1E-115 neuroblastoma cells transfected with h-α-Syn. The appearance of aggregated α-Syn was inhibited by LMTM, with an EC50 of 1.1 μM, with minimal effect on h-α-Syn mRNA levels being observed. Two independent lines of mice (L58 and L62 transgenic for the same fusion protein accumulated neuronal h-α-Syn that, with aging, developed into fibrillary inclusions characterized by both resistance to proteinase K (PK-cleavage and their ability to bind thiazin red. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following oral treatment of male and female mice with LMTM administered daily for 6 weeks at 5 and 15 mg MT/kg. The early aggregates of α-Syn and the late-stage fibrillar inclusions were both susceptible to inhibition by LMTM, a treatment that also resulted in the rescue of movement and anxiety-related traits in these mice. The results suggest that LMTM may provide a potential disease modification therapy in PD and other synucleinopathies through the inhibition of α-Syn aggregation.

  7. Markov chain aggregation and its applications to combinatorial reaction networks.

    Science.gov (United States)

    Ganguly, Arnab; Petrov, Tatjana; Koeppl, Heinz

    2014-09-01

    We consider a continuous-time Markov chain (CTMC) whose state space is partitioned into aggregates, and each aggregate is assigned a probability measure. A sufficient condition for defining a CTMC over the aggregates is presented as a variant of weak lumpability, which also characterizes that the measure over the original process can be recovered from that of the aggregated one. We show how the applicability of de-aggregation depends on the initial distribution. The application section is devoted to illustrate how the developed theory aids in reducing CTMC models of biochemical systems particularly in connection to protein-protein interactions. We assume that the model is written by a biologist in form of site-graph-rewrite rules. Site-graph-rewrite rules compactly express that, often, only a local context of a protein (instead of a full molecular species) needs to be in a certain configuration in order to trigger a reaction event. This observation leads to suitable aggregate Markov chains with smaller state spaces, thereby providing sufficient reduction in computational complexity. This is further exemplified in two case studies: simple unbounded polymerization and early EGFR/insulin crosstalk.

  8. Molecular origin of polyglutamine aggregation in neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available Expansion of polyglutamine (polyQ tracts in proteins results in protein aggregation and is associated with cell death in at least nine neurodegenerative diseases. Disease age of onset is correlated with the polyQ insert length above a critical value of 35-40 glutamines. The aggregation kinetics of isolated polyQ peptides in vitro also shows a similar critical-length dependence. While recent experimental work has provided considerable insights into polyQ aggregation, the molecular mechanism of aggregation is not well understood. Here, using computer simulations of isolated polyQ peptides, we show that a mechanism of aggregation is the conformational transition in a single polyQ peptide chain from random coil to a parallel beta-helix. This transition occurs selectively in peptides longer than 37 glutamines. In the beta-helices observed in simulations, all residues adopt beta-strand backbone dihedral angles, and the polypeptide chain coils around a central helical axis with 18.5 +/- 2 residues per turn. We also find that mutant polyQ peptides with proline-glycine inserts show formation of antiparallel beta-hairpins in their ground state, in agreement with experiments. The lower stability of mutant beta-helices explains their lower aggregation rates compared to wild type. Our results provide a molecular mechanism for polyQ-mediated aggregation.

  9. Computational design and biophysical characterization of aggregation-resistant point mutations for γD crystallin illustrate a balance of conformational stability and intrinsic aggregation propensity.

    Science.gov (United States)

    Sahin, Erinc; Jordan, Jacob L; Spatara, Michelle L; Naranjo, Andrea; Costanzo, Joseph A; Weiss, William F; Robinson, Anne Skaja; Fernandez, Erik J; Roberts, Christopher J

    2011-02-08

    γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have ΔΔG(un) ≫ 0, without significantly affecting IAP. S130P was predicted to have ΔΔG(un) ∼ 0 but with reduced IAP. The stability, conformation, and aggregation kinetics in acidic solution were experimentally characterized and compared for the variants and wild-type (WT) protein using circular dichroism and intrinsic fluorescence spectroscopy, calorimetric and chemical unfolding, thioflavin-T binding, chromatography, static laser light scattering, and kinetic modeling. Monomer secondary and tertiary structures of both variants were indistinguishable from WT, while ΔΔG(un) > 0 for M69Q and ΔΔG(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q.

  10. Aggregation of trypsin and trypsin inhibitor by Al cation.

    Science.gov (United States)

    Chanphai, P; Kreplak, L; Tajmir-Riahi, H A

    2017-04-01

    Al cation may trigger protein structural changes such as aggregation and fibrillation, causing neurodegenerative diseases. We report the effect of Al cation on the solution structures of trypsin (try) and trypsin inhibitor (tryi), using thermodynamic analysis, UV-Visible, Fourier transform infrared (FTIR) spectroscopic methods and atomic force microscopy (AFM). Thermodynamic parameters showed Al-protein bindings occur via H-bonding and van der Waals contacts for trypsin and trypsin inhibitor. AFM showed that Al cations are able to force trypsin into larger or more robust aggregates than trypsin inhibitor, with trypsin 5±1 SE (n=52) proteins per aggregate and for trypsin inhibitor 8.3±0.7 SE (n=118). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced more alterations of trypsin inhibitor conformation than trypsin. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Conformational Compatibility Is Essential for Heterologous Aggregation of α-Synuclein

    NARCIS (Netherlands)

    Sidhu, Arshdeep; Segers-Nolten, Ine; Subramaniam, Vinod

    2016-01-01

    Under aggregation-prone conditions, soluble amyloidogenic protein monomers can self-assemble into fibrils or they can fibrillize on preformed fibrillar seeds (seeded aggregation). Seeded aggregations are known to propagate the morphology of the seeds in the event of cross-seeding. However, not all

  12. A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom

    Directory of Open Access Journals (Sweden)

    Bruna Barbosa de Sousa

    2017-01-01

    Full Text Available This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP. BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

  13. Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.

    Science.gov (United States)

    Fujita-Yoshigaki, Junko; Matsuki-Fukushima, Miwako; Yokoyama, Megumi; Katsumata-Kato, Osamu

    2013-11-15

    The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.

  14. Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

    Science.gov (United States)

    Senis, Y A; Sangrar, W; Zirngibl, R A; Craig, A W B; Lee, D H; Greer, P A

    2003-05-01

    Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.

  15. Acetylcholinesterase triggers the aggregation of PrP 106-126

    International Nuclear Information System (INIS)

    Pera, M.; Roman, S.; Ratia, M.; Camps, P.; Munoz-Torrero, D.; Colombo, L.; Manzoni, C.; Salmona, M.; Badia, A.; Clos, M.V.

    2006-01-01

    Acetylcholinesterase (AChE), a senile plaque component, promotes amyloid-β-protein (Aβ) fibril formation in vitro. The presence of prion protein (PrP) in Alzheimer's disease (AD) senile plaques prompted us to assess if AChE could trigger the PrP peptides aggregation as well. Consequently, the efficacy of AChE on the PrP peptide spanning-residues 106-126 aggregation containing a coumarin fluorescence probe (coumarin-PrP 106-126) was studied. Kinetics of coumarin-PrP 106-126 aggregation showed a significant increase of maximum size of aggregates (MSA), which was dependent on AChE concentration. AChE-PrP 106-126 aggregates showed the tinctorial and optical amyloid properties as determined by polarized light and electronic microscopy analysis. A remarkable inhibition of MSA was obtained with propidium iodide, suggesting that AChE triggers PrP 106-126 and Aβ aggregation through a similar mechanism. Huprines (AChE inhibitors) also significantly decreased MSA induced by AChE as well, unveiling the potential interest for some AChE inhibitors as a novel class of potential anti-prion drugs

  16. A comparative study on the aggregating effects of guanidine thiocyanate, guanidine hydrochloride and urea on lysozyme aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Emadi, Saeed, E-mail: emadi@iasbs.ac.ir; Behzadi, Maliheh

    2014-08-08

    Highlights: • Lysozyme aggregated in guanidine thiocyanate (1.0 and 2.0 M). • Lysozyme aggregated in guanidine hydrochloride (4 and 5 M). • Lysozyme did not aggregated at any concentration (0.5–5 M) of urea. • Unfolding pathway is more important than unfolding per se in aggregation. - Abstract: Protein aggregation and its subsequent deposition in different tissues culminate in a diverse range of diseases collectively known as amyloidoses. Aggregation of hen or human lysozyme depends on certain conditions, namely acidic pH or the presence of additives. In the present study, the effects on the aggregation of hen egg-white lysozyme via incubation in concentrated solutions of three different chaotropic agents namely guanidine thiocyanate, guanidine hydrochloride and urea were investigated. Here we used three different methods for the detection of the aggregates, thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy. Our results showed that upon incubation with different concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 5.0 M) of the chemical denaturants, lysozyme was aggregated at low concentrations of guanidine thiocyanate (1.0 and 2.0 M) and at high concentrations of guanidine hydrochloride (4 and 5 M), although no fibril formation was detected. In the case of urea, no aggregation was observed at any concentration.

  17. Effect of initial protein concentration and pH on in vitro gastric digestion of heated whey proteins.

    Science.gov (United States)

    Zhang, Sha; Vardhanabhuti, Bongkosh

    2014-02-15

    The in vitro digestion of heated whey protein aggregates having different structure and physicochemical properties was evaluated under simulated gastric conditions. Aggregates were formed by heating whey protein isolates (WPI) at 3-9% w/w initial protein concentration and pH 3.0-7.0. Results showed that high protein concentration led to formation of larger WPI aggregates with fewer remaining monomers. Aggregates formed at high protein concentrations showed slower degradation rate compared to those formed at low protein concentration. The effect of initial protein concentration on peptide release pattern was not apparent. Heating pH was a significant factor affecting digestion pattern. At pH above the isoelectric point, the majority of the proteins involved in the aggregation, and aggregates formed at pH 6.0 were more susceptible to pepsin digestion than at pH 7.0. At acidic conditions, only small amount of proteins was involved in the aggregation and heated aggregates were easily digested by pepsin, while the remaining unaggregated proteins were very resistant to gastric digestion. The potential physiological implication of these results on satiety was discussed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Aged Lewis rats exposed to low and moderate doses of rotenone are a good model for studying the process of protein aggregation and its effects upon central nervous system cell physiology

    Directory of Open Access Journals (Sweden)

    Michael F. Almeida

    Full Text Available ABSTRACT Cell physiology is impaired before protein aggregation and this may be more relevant than inclusions themselves for neurodegeneration. The present study aimed to characterize an animal model to enable the analysis of the cell biology before and after protein aggregation. Ten-month-old Lewis rats were exposed either to 1 or 2 mg/kg/day of rotenone, delivered subcutaneously through mini-pumps, for one month. Hyperphosphorylated TAU, alpha-synuclein, amyloid-beta peptide and protein carbonylation (indicative of oxidative stress were evaluated in the hippocampus, substantia nigra and locus coeruleus through immunohistochemistry or western blot. It was found that 2 mg/kg/day rotenone increased amyloid-beta peptide, hyperphosphorylation of TAU and alpha-synuclein. Rotenone at 1mg/kg/day did not alter protein levels. Protein carbonylation remained unchanged. This study demonstrated that aged Lewis rats exposed to a low dose of rotenone is a useful model to study cellular processes before protein aggregation, while the higher dose makes a good model to study the effects of protein inclusions.

  19. Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL.

    Science.gov (United States)

    Wang, Xia; An, Qiang; Zhao, Bin; Guo, Jin Song; Huang, Yuan Sheng; Tian, Meng

    2018-02-01

    Enterobacter sp. strain FL was newly isolated from activated sludge and exhibited significant capability of auto-aggregation as well as aerobic denitrification. The removal efficiencies of NO 3 - -N, total nitrogen (TN), and TOC by strain FL in batch culture reached 94.6, 63.9, and 72.5% in 24 h, respectively. The production of N 2 O and N 2 in the presence of oxygen demonstrated the occurrence of aerobic denitrification. The auto-aggregation index of strain FL reached 54.3%, suggesting a high tendency that the cells would agglomerate into aggregates. The production of extracellular polymeric substances (EPSs), which were mainly composed of proteins followed by polysaccharides, was considered to be related to the cell aggregation according to Fourier transform infrared (FT-IR) and confocal laser scanning microscopy (CLSM). The proteins in EPS were evenly and tightly combined to cells and altered the protein secondary structures of cell surface from random coils to β-sheets and three-turn helices. The alteration of protein secondary structures of cell surface caused by the proteins in EPS might play a dominant role in the auto-aggregation of strain FL. To further assess the feasibility of strain FL for synthetic wastewater treatment, a sequencing batch reactor (SBR), solely inoculated with strain FL, was conducted. During the 16 running cycles, the removal efficiency of NO 3 - -N was 90.2-99.7% and the auto-aggregation index was stabilized at 35.0-41.5%. The EPS promoted the biomass of strain FL to aggregate in the SBR.

  20. Alkali-aggregate reactivity (AAR) facts book.

    Science.gov (United States)

    2013-03-01

    This document provides detailed information on alkali-aggregate reactivity (AAR). It primarily discusses alkali-silica reaction (ASR), covering the chemistry, symptoms, test methods, prevention, specifications, diagnosis and prognosis, and mitigation...

  1. Inflammation Induces TDP-43 Mislocalization and Aggregation.

    Directory of Open Access Journals (Sweden)

    Ana Sofia Correia

    Full Text Available TAR DNA-binding protein 43 (TDP-43 is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS and frontotemporal lobar degeneration (FTLD. Here we report that lipopolysaccharide (LPS-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43(A315T transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.

  2. Controlling the aggregation and gelation of ß-lactoglobulin by the addition of its peptides

    NARCIS (Netherlands)

    Kosters, H.A.

    2012-01-01

    In this thesis the effects of peptides, or protein hydrolysates on the heat-induced aggregation and gelation of (concentrated) protein systems were studied. First, it was investigated if specific peptides could influence the heat-induced denaturation and aggregation of intact proteins solutions,

  3. Chaperone activity of human small heat shock protein-GST fusion proteins.

    Science.gov (United States)

    Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

    2017-07-01

    Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

  4. Platelet aggregation inhibitors in primary and secondary prevention of ischemic stroke

    Directory of Open Access Journals (Sweden)

    von der Schulenburg, Johann-Matthias

    2006-04-01

    Full Text Available Background: The ischaemic stroke (IS is one of the most frequent cause of death in Germany. Besides of non-drug many drug-based interventions are used in primary or secondary prevention of IS, among them the thrombocyte aggregation inhibitors (TAI. Objectives: The evaluation addresses the questions on medical efficacy and cost-effectiveness of the TAI administration in the prevention of IS as compared to the management of risk factors alone as well as to the use of anticoagulant drugs. Methods: The literature search for articles published after 1997 was conducted in December 2003 in the most important medical and economic databases. The medical analysis was performed on the basis of the most up-to date meta-analyses of randomised controlled trials (RCT as well as of new published RCT. The data from the studies for stroke, bleeding complications as well as for the combined endpoint "severe vascular events" (SVE: death or stroke or myocardial infarction were summarised in meta-analyses.In order to include grey literature contact has been taken up with the pharmaceutical manufacturers of TAI. Results are presented in a descriptive way. Results: The medical analysis included data from 184 RCT (vs. placebo and from 22 RCT (vs. anticoagulant drugs. The absolute reduction of IS (4.8% vs. 6.6%; p<0,00001 and SVE (10.0% vs. 12.4%; p<0,00001 were definitely higher than the absolute increase of bleeding complications (1.6% vs. 0.9%; p<0,00001, but relatively similar to this absolute increase in a subpopulation with a low risk for SVE. With regard to the stroke prevention, evidence of efficacy could be yielded for acetylsalicil acid (ASA, dipyridamole, cilostazol, ridogrel and the combination ASA with dipyridamole. ASA is less effective than anticoagulants in the prevention of ischaemic stroke in atrial fibrillation, however, it causes fewer bleeding complications.Low dosed ASA can be considered cost-effective in secondary prevention of ischemic stroke

  5. Isolation, characterization, and aggregation of a structured bacterial matrix precursor.

    Science.gov (United States)

    Chai, Liraz; Romero, Diego; Kayatekin, Can; Akabayov, Barak; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2013-06-14

    Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

  6. Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

    Science.gov (United States)

    Tokunaga, Yuhei; Matsumoto, Mitsuharu; Tokunaga, Masao; Arakawa, Tsutomu; Sugimoto, Yasushi

    2013-01-01

    Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine-rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His-tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low-pH harsh conditions, however, His-HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid-hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full-length His-HP when incubated with 10–20% 2,2,2-trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage. PMID:24038709

  7. Synthetic food additive dye "Tartrazine" triggers amorphous aggregation in cationic myoglobin.

    Science.gov (United States)

    Al-Shabib, Nasser Abdulatif; Khan, Javed Masood; Khan, Mohd Shahnawaz; Ali, Mohd Sajid; Al-Senaidy, Abdulrahman M; Alsenaidy, Mohammad A; Husain, Fohad Mabood; Al-Lohedan, Hamad A

    2017-05-01

    Protein aggregation, a characteristic of several neurodegenerative diseases, displays vast conformational diversity from amorphous to amyloid-like aggregates. In this study, we have explored the interaction of tartrazine with myoglobin protein at two different pHs (7.4 and 2.0). We have utilized various spectroscopic techniques (turbidity, Rayleigh light scattering (RLS), intrinsic fluorescence, Congo Red and far-UV CD) along with microscopy techniques i.e. atomic force microscopy (AFM) and transmission electron microscopy (TEM) to characterize the tartrazine-induced aggregation in myoglobin. The results showed that higher concentrations of tartrazine (2.0-10.0mM) induced amorphous aggregation in myoglobin at pH 2.0 via electrostatic interactions. However, tartrazine was not able to induce aggregation in myoglobin at pH 7.4; because of strong electrostatic repulsion between myoglobin and tartrazine at this pH. The tartrazine-induced amorphous aggregation process is kinetically very fast, and aggregation occurred without the formation of a nucleus. These results proposed that the electrostatic interaction is responsible for tartrazine-induced amorphous aggregation. This study may help in the understanding of mechanistic insight of aggregation by tartrazine. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Calcium binding to beta-2-microglobulin at physiological pH drives the occurrence of conformational changes which cause the protein to precipitate into amorphous forms that subsequently transform into amyloid aggregates.

    Directory of Open Access Journals (Sweden)

    Sukhdeep Kumar

    Full Text Available Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD varieties. We establish (a that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT, resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.

  9. Study on the interaction between oxolinic acid aggregates and protein and its analytical application

    International Nuclear Information System (INIS)

    Wu Xia; Zheng Jinhua; Ding Honghong; Ran Dehuan; Xu Wei; Song Yuanyuan; Yang Jinghe

    2007-01-01

    It was found that oxolinic acid (OA) at high concentration can self-assemble into nano- to micro- meter scale OA aggregates in Tris-HCl (pH 7.48) buffer solution. The nanoparticles of OA were adopted as fluorescence probes in the quantitative analysis of proteins. Under optimum conditions, the fluorescence quenching extent of nanometer scale OA aggregates was in proportion to the concentration of albumins in the range of 3.0 x 10 -8 to 3.0 x 10 -5 g mL -1 for bovine serum albumin (BSA) and 8.0 x 10 -8 to 8.0 x 10 -6 g mL -1 for human serum albumin (HSA). The detection limits (S/N = 3) were 3.4 x 10 -9 g mL -1 for BSA, and 2.6 x 10 -8 g mL -1 for HSA, respectively. Samples were satisfactorily determined. The interaction mechanism of the system was studied using fluorescence, UV-vis, resonance light scattering (RLS) and transmission electron microscope (TEM) technology, etc., indicating that the nonluminescent complex was formed between serum albumin molecular and OA, to disaggregate the self-association of OA, which resulted in the dominated static fluorescence quenching in the system

  10. The Role of the Heat Shock Protein B8 (HSPB8 in Motoneuron Diseases

    Directory of Open Access Journals (Sweden)

    Paola Rusmini

    2017-06-01

    Full Text Available Amyotrophic lateral sclerosis (ALS and spinal and bulbar muscular atrophy (SBMA are two motoneuron diseases (MNDs characterized by aberrant protein behavior in affected cells. In familial ALS (fALS and in SBMA specific gene mutations lead to the production of neurotoxic proteins or peptides prone to misfold, which then accumulate in form of aggregates. Notably, some of these proteins accumulate into aggregates also in sporadic ALS (sALS even if not mutated. To prevent proteotoxic stresses detrimental to cells, misfolded and/or aggregated proteins must be rapidly removed by the protein quality control (PQC system. The small heat shock protein B8 (HSPB8 is a chaperone induced by harmful events, like proteasome inhibition. HSPB8 is expressed both in motoneuron and muscle cells, which are both targets of misfolded protein toxicity in MNDs. In ALS mice models, in presence of the mutant proteins, HSPB8 is upregulated both in spinal cord and muscle. HSPB8 interacts with the HSP70 co-chaperone BAG3 and enhances the degradation of misfolded proteins linked to sALS, or causative of fALS and of SBMA. HSPB8 acts by facilitating autophagy, thereby preventing misfolded protein accumulation in affected cells. BAG3 and BAG1 compete for HSP70-bound clients and target them for disposal to the autophagy or proteasome, respectively. Enhancing the selective targeting of misfolded proteins by HSPB8-BAG3-HSP70 to autophagy may also decrease their delivery to the proteasome by the BAG1-HSP70 complex, thereby limiting possible proteasome overwhelming. Thus, approaches aimed at potentiating HSPB8-BAG3 may contribute to the maintenance of proteostasis and may delay MNDs progression.

  11. Cross-seeding of prions by aggregated α-synuclein leads to transmissible spongiform encephalopathy.

    Directory of Open Access Journals (Sweden)

    Elizaveta Katorcha

    2017-08-01

    Full Text Available Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's, prion and other diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features that were once thought to be unique to only one neurodegenerative disorder. However, the origin for the overlap remains unclear. One possibility is that diseases with mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by aggregated states of unrelated proteins. In the current study we examined whether prion replication can be induced by cross-seeding by α-synuclein or Aβ peptide. We found that α-synuclein aggregates formed in cultured cells or in vitro display cross-seeding activity and trigger misfolding of the prion protein (PrPC in serial Protein Misfolding Cyclic Amplification reactions, producing self-replicating PrP states characterized by a short C-terminal proteinase K (PK-resistant region referred to as PrPres. Non-fibrillar α-synuclein or fibrillar Aβ failed to cross-seed misfolding of PrPC. Remarkably, PrPres triggered by aggregated α-synuclein in vitro propagated in animals and, upon serial transmission, produced PrPSc and clinical prion disease characterized by spongiosis and astrocytic gliosis. The current study demonstrates that aggregated α-synuclein is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-replicating states that can lead to transmissible prion diseases upon serial passaging in wild type animals. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies.

  12. Trifluoroethanol modulates α-synuclein amyloid-like aggregate formation, stability and dissolution

    DEFF Research Database (Denmark)

    Di Carlo, Maria Giovanna; Vetri, Valeria; Buscarino, Gianpiero

    2016-01-01

    The conversion of proteins into amyloid fibrils and other amyloid-like aggregates is closely connected to the onset of a series of age-related pathologies. Upon changes in environmental conditions, amyloid-like aggregates may also undergo disassembly into oligomeric aggregates, the latter being r...

  13. Purification and Characterization of BmooAi: A New Toxin from Bothrops moojeni Snake Venom That Inhibits Platelet Aggregation

    Directory of Open Access Journals (Sweden)

    Mayara Ribeiro de Queiroz

    2014-01-01

    Full Text Available In this paper, we describe the purification/characterization of BmooAi, a new toxin from Bothrops moojeni that inhibits platelet aggregation. The purification of BmooAi was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacel column, molecular exclusion on a Sephadex G-75 column, and reverse-phase HPLC chromatography on a C2/C18 column. BmooAi was homogeneous by SDS-PAGE and shown to be a single-chain protein of 15,000 Da. BmooAi was analysed by MALDI-TOF Spectrometry and revealed two major components with molecular masses 7824.4 and 7409.2 as well as a trace of protein with a molecular mass of 15,237.4 Da. Sequencing of BmooAi by Edman degradation showed two amino acid sequences: IRDFDPLTNAPENTA and ETEEGAEEGTQ, which revealed no homology to any known toxin from snake venom. BmooAi showed a rather specific inhibitory effect on platelet aggregation induced by collagen, adenosine diphosphate, or epinephrine in human platelet-rich plasma in a dose-dependent manner, whereas it had little or no effect on platelet aggregation induced by ristocetin. The effect on platelet aggregation induced by BmooAi remained active even when heated to 100°C. BmooAi could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

  14. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-06-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

  15. Extracting Tenebrio molitor protein while preventing browning: effect of pH and NaCl on protein yield

    NARCIS (Netherlands)

    Yi, L.; Boekel, van T.; Lakemond, C.M.M.

    2017-01-01

    The potential of insects as an alternative protein source for food applications was investigated by studying the effect of pH and NaCl on extraction yield of water-soluble proteins from Tenebrio molitor, while preventing browning due to polyphenol oxidation. Minimum protein solubility (29.6%) was at

  16. Temperature dependent rapid annealing effect induces amorphous aggregation of human serum albumin.

    Science.gov (United States)

    Ishtikhar, Mohd; Ali, Mohd Sajid; Atta, Ayman M; Al-Lohedan, Hammad; Badr, Gamal; Khan, Rizwan Hasan

    2016-01-01

    This study represents an analysis of the thermal aggregation of human serum albumin (HSA) induced by novel rosin modified compounds. The aggregation process causes conformational alterations in the secondary and tertiary structures of the proteins. The conversion of globular protein to amorphous aggregates was carried out by spectroscopic, calorimetric and microscopic techniques to investigate the factors that are responsible for the structural, conformational and morphological alteration in the protein. Our outcome results show that the aggregation of HSA was dependent on the hydrophobicity, charge and temperature, because the formation of amorphous aggregates occurs in the presence of a novel cationic rosin compound, quaternary amine of rosin diethylaminoethyl ester (QRMAE), at 40°C and pH 7.4 (but at 25°C on similar pH value, there was no evidence of aggregate formation). In addition, the parent compound of QRMAE, i.e., abietic acid, and other derivatives such as nonionic rosin compounds [(RMPEG-750) and (RMA-MPEG-750)] do not shows the aggregating property. This work provides precise and necessary information that aid in the understanding the effects of rosin derivative compounds on HSA. This study also restrains important information for athletes, health providers, pharmaceutical companies, industries, and soft drink-processing companies. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Star Polymers Reduce Islet Amyloid Polypeptide Toxicity via Accelerated Amyloid Aggregation.

    Science.gov (United States)

    Pilkington, Emily H; Lai, May; Ge, Xinwei; Stanley, William J; Wang, Bo; Wang, Miaoyi; Kakinen, Aleksandr; Sani, Marc-Antonie; Whittaker, Michael R; Gurzov, Esteban N; Ding, Feng; Quinn, John F; Davis, Thomas P; Ke, Pu Chun

    2017-12-11

    Protein aggregation into amyloid fibrils is a ubiquitous phenomenon across the spectrum of neurodegenerative disorders and type 2 diabetes. A common strategy against amyloidogenesis is to minimize the populations of toxic oligomers and protofibrils by inhibiting protein aggregation with small molecules or nanoparticles. However, melanin synthesis in nature is realized by accelerated protein fibrillation to circumvent accumulation of toxic intermediates. Accordingly, we designed and demonstrated the use of star-shaped poly(2-hydroxyethyl acrylate) (PHEA) nanostructures for promoting aggregation while ameliorating the toxicity of human islet amyloid polypeptide (IAPP), the peptide involved in glycemic control and the pathology of type 2 diabetes. The binding of PHEA elevated the β-sheet content in IAPP aggregates while rendering a new morphology of "stelliform" amyloids originating from the polymers. Atomistic molecular dynamics simulations revealed that the PHEA arms served as rodlike scaffolds for IAPP binding and subsequently accelerated IAPP aggregation by increased local peptide concentration. The tertiary structure of the star nanoparticles was found to be essential for driving the specific interactions required to impel the accelerated IAPP aggregation. This study sheds new light on the structure-toxicity relationship of IAPP and points to the potential of exploiting star polymers as a new class of therapeutic agents against amyloidogenesis.

  18. Effect of homogenisation in formation of thermally induced aggregates in a non- and low- fat milk model system with microparticulated whey proteins

    DEFF Research Database (Denmark)

    Celigueta Torres, Isabel; Nieto, Gema; Nylander, Tommy

    2017-01-01

    in the formation of protein aggregates were studied by polyacrylamide gel electrophoresis and the final complexes visualised by darkfield microscopy. Homogenisation of non-fat milk systems led to partial adsorption of caseins onto microparticles, independently of the type of microparticle. On the contrary...... are responsible for the formation of heat-induced aggregates that influence the texture and sensory characteristics of the final product. The formation of heat-induced complexes was studied in non- and low-fat milk model systems, where microparticulated whey protein (MWP) was used as fat replacer. Five MWP types......, homogenisation of low-fat milk resulted in preferential adsorption of caseins onto fat globules, rather than onto microparticles. Further heating of the milk, led to the formation of heat induced complexes with different sizes and characteristics depending on the type of MWP and the presence or not of fat...

  19. Combined aspirin and cilostazol treatment is associated with reduced platelet aggregation and prevention of exercise-induced platelet activation.

    Science.gov (United States)

    Cleanthis, M; Bhattacharya, V; Smout, J; Ashour, H; Stansby, G

    2009-05-01

    Cilostazol has proven efficacy in increasing walking distance in claudicants, but it has not been demonstrated to be more effective than placebo in secondary cardiovascular prevention. The direct effect of exercise on platelet function remains less well defined. We have investigated the effect of combination treatment with aspirin and cilostazol on platelet activity in claudicants subjected to repeated treadmill exercise. Nineteen claudicants completed a double-blind, randomised, controlled, cross-over trial. Each subject received a 2-week course of aspirin (75mg) and placebo and aspirin and cilostazol (100mg twice daily). Following each 2-week treatment period, patients participated in a standardised treadmill test (3.2kmh(-1), 10 degrees incline) walking to maximal claudication distance. The exercise was repeated thrice in total, and blood was sampled before and after exercise. Platelet activation was measured using free platelet counting aggregation, flow cytometry for surface markers of platelet activation and soluble P-selectin assay. Compared to aspirin and placebo, combination treatment with aspirin and cilostazol was associated with reduced arachidonic-acid-induced platelet aggregation (pWilcoxon signed-rank test). Aspirin and placebo treatment were associated with elevated P-selectin expression, platelet-monocyte aggregation and reduced CD42b expression (pWilcoxon signed-rank test) post-exercise. No difference was seen in spontaneous platelet aggregation whilst soluble P-selectin was reduced post-exercise with combination treatment with aspirin and cilostazol (pWilcoxon signed-rank test). Combination treatment with aspirin and cilostazol results in suppression of platelet activation and reduces the effect of exercise on platelets. The benefit seen may be a result of cilostazol enhancing the inhibitory effect of aspirin on the cyclo-oxygenase pathway.

  20. Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.

    Science.gov (United States)

    Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

    2018-06-18

    The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development. Copyright © 2018. Published by Elsevier Inc.

  1. Effect of free cysteine on the denaturation and aggregation of holo α-lactalbumin

    DEFF Research Database (Denmark)

    Nielsen, Line R.; Lund, Marianne N.; Davies, Michael J.

    2018-01-01

    α-Lactalbumin (α-LA) is a key commercial whey protein for nutritional purposes. The holo protein (calcium saturated) is considered the most heat stable whey protein, capable of refolding from unfolded states under many conditions. This is due to the absence of free thiols (cysteine residues......) that are typically involved in thermal aggregation and thiol–disulphide exchange reactions of other whey proteins. Heating (0–120 min at 90 °C, pH 7.0) holo α-LA generates free thiols through thermal cleavage of disulphide bonds, resulting in aggregates comprising unfolded α-LA species. The addition of free cysteine...... promotes the formation of soluble aggregates, effectively decreasing the holding time required to reach a particular aggregate size in a dose-dependent manner (0.35–1.4 mM cysteine). Excess cysteine (≥14 mM) causes a destabilisation of α-LA, shown by decreased denaturation temperature and gel formation...

  2. Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri.

    Science.gov (United States)

    Mackenzie, Donald A; Jeffers, Faye; Parker, Mary L; Vibert-Vallet, Amandine; Bongaerts, Roy J; Roos, Stefan; Walter, Jens; Juge, Nathalie

    2010-11-01

    Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867(i)), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins

  3. Modeling In-Network Aggregation in VANETs

    NARCIS (Netherlands)

    Dietzel, Stefan; Kargl, Frank; Heijenk, Geert; Schaub, Florian

    2011-01-01

    The multitude of applications envisioned for vehicular ad hoc networks requires efficient communication and dissemination mechanisms to prevent network congestion. In-network data aggregation promises to reduce bandwidth requirements and enable scalability in large vehicular networks. However, most

  4. Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

    Science.gov (United States)

    Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A; Gratton, Enrico; Jones, Mark R

    2015-05-27

    DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.

  5. Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response.

    Directory of Open Access Journals (Sweden)

    Adi Ben Yehuda

    Full Text Available Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

  6. FLZ Attenuates α-Synuclein-Induced Neurotoxicity by Activating Heat Shock Protein 70.

    Science.gov (United States)

    Bao, Xiu-Qi; Wang, Xiao-Liang; Zhang, Dan

    2017-01-01

    Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. The pathology of PD is caused by progressive degeneration of dopaminergic neurons and is characterized by the presence of intracellular inclusions known as Lewy bodies, composed mainly of α-synuclein. Heat shock proteins (HSPs) are crucial in protein quality control in cells. HSP70 in particular prevents the aggregation of protein aggregation, such as α-synuclein, providing a degree of protection against PD. The compound FLZ has been shown to protect several PD models in previous studies and was reported as an HSP inducer to protect against MPP + -induced neurotoxicity, but the mechanism remains unclear. In this study, we investigated the effects of FLZ-mediated HSP70 induction in α-synuclein transgenic mice and cells. FLZ treatment alleviated motor dysfunction and improved dopaminergic neuronal function in α-synuclein transgenic mice. HSP70 protein expression and transcriptional activity were increased by FLZ treatment, eliciting a reduction of α-synuclein aggregation and associated toxicity. The inhibition of HSP70 by quercetin or HSP70 siRNA markedly attenuated the neuroprotective effects of FLZ, confirming that FLZ exerted a neuroprotective effect through HSP70. We revealed that FLZ directly bound to and increased the expression of Hip, a cochaperone of HSP70, which in turn enhanced HSP70 activity. In conclusion, we defined a critical role for HSP70 and its cochaperones activated by FLZ in preventing neurodegeneration and proposed that targeting the HSP70 system may represent a potential therapy for α-synuclein-related diseases, such as PD.

  7. Aggregation of flexible polyelectrolytes: Phase diagram and dynamics.

    Science.gov (United States)

    Tom, Anvy Moly; Rajesh, R; Vemparala, Satyavani

    2017-10-14

    Similarly charged polymers in solution, known as polyelectrolytes, are known to form aggregated structures in the presence of oppositely charged counterions. Understanding the dependence of the equilibrium phases and the dynamics of the process of aggregation on parameters such as backbone flexibility and charge density of such polymers is crucial for insights into various biological processes which involve biological polyelectrolytes such as protein, DNA, etc. Here, we use large-scale coarse-grained molecular dynamics simulations to obtain the phase diagram of the aggregated structures of flexible charged polymers and characterize the morphology of the aggregates as well as the aggregation dynamics, in the presence of trivalent counterions. Three different phases are observed depending on the charge density: no aggregation, a finite bundle phase where multiple small aggregates coexist with a large aggregate and a fully phase separated phase. We show that the flexibility of the polymer backbone causes strong entanglement between charged polymers leading to additional time scales in the aggregation process. Such slowing down of the aggregation dynamics results in the exponent, characterizing the power law decay of the number of aggregates with time, to be dependent on the charge density of the polymers. These results are contrary to those obtained for rigid polyelectrolytes, emphasizing the role of backbone flexibility.

  8. Structural insights into mechanisms for inhibiting amyloid β42 aggregation by non-catechol-type flavonoids.

    Science.gov (United States)

    Hanaki, Mizuho; Murakami, Kazuma; Akagi, Ken-ichi; Irie, Kazuhiro

    2016-01-15

    The prevention of 42-mer amyloid β-protein (Aβ42) aggregation is promising for the treatment of Alzheimer's disease. We previously described the site-specific inhibitory mechanism for Aβ42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aβ42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aβ42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The (1)H-(15)N SOFAST-HMQC NMR of Aβ42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular β-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aβ42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aβ42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aβ42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aβ42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aβ42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aβ42 aggregation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model.

    Science.gov (United States)

    Kakkar, Vaishali; Månsson, Cecilia; de Mattos, Eduardo P; Bergink, Steven; van der Zwaag, Marianne; van Waarde, Maria A W H; Kloosterhuis, Niels J; Melki, Ronald; van Cruchten, Remco T P; Al-Karadaghi, Salam; Arosio, Paolo; Dobson, Christopher M; Knowles, Tuomas P J; Bates, Gillian P; van Deursen, Jan M; Linse, Sara; van de Sluis, Bart; Emanuelsson, Cecilia; Kampinga, Harm H

    2016-04-21

    Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

    Science.gov (United States)

    Bengali, Aditya N; Tessier, Peter M

    2009-10-01

    "Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these

  11. Multivalent scaffolds induce galectin-3 aggregation into nanoparticles

    Directory of Open Access Journals (Sweden)

    Candace K. Goodman

    2014-07-01

    Full Text Available Galectin-3 meditates cell surface glycoprotein clustering, cross linking, and lattice formation. In cancer biology, galectin-3 has been reported to play a role in aggregation processes that lead to tumor embolization and survival. Here, we show that lactose-functionalized dendrimers interact with galectin-3 in a multivalent fashion to form aggregates. The glycodendrimer–galectin aggregates were characterized by dynamic light scattering and fluorescence microscopy methodologies and were found to be discrete particles that increased in size as the dendrimer generation was increased. These results show that nucleated aggregation of galectin-3 can be regulated by the nucleating polymer and provide insights that improve the general understanding of the binding and function of sugar-binding proteins.

  12. Proteomic Data From Human Cell Cultures Refine Mechanisms of Chaperone-Mediated Protein homeostasis

    OpenAIRE

    Finka, Andrija; Goloubinoff, Andrija Finka and Pierre

    2013-01-01

    In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here “the chaperome,” which can prevent formation of potentially harmful misfolded protein conformers and use the...

  13. Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb protein switches its activity from auto-aggregation to biofilm formation

    Directory of Open Access Journals (Sweden)

    Marija Miljković

    2016-09-01

    Full Text Available AggLb is the largest (318.6 kDa aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix (ECM, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody, an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.

  14. Stable, metastable, and kinetically trapped amyloid aggregate phases.

    Science.gov (United States)

    Miti, Tatiana; Mulaj, Mentor; Schmit, Jeremy D; Muschol, Martin

    2015-01-12

    Self-assembly of proteins into amyloid fibrils plays a key role in a multitude of human disorders that range from Alzheimer's disease to type II diabetes. Compact oligomeric species, observed early during amyloid formation, are reported as the molecular entities responsible for the toxic effects of amyloid self-assembly. However, the relation between early-stage oligomeric aggregates and late-stage rigid fibrils, which are the hallmark structure of amyloid plaques, has remained unclear. We show that these different structures occupy well-defined regions in a peculiar phase diagram. Lysozyme amyloid oligomers and their curvilinear fibrils only form after they cross a salt and protein concentration-dependent threshold. We also determine a boundary for the onset of amyloid oligomer precipitation. The oligomeric aggregates are structurally distinct from rigid fibrils and are metastable against nucleation and growth of rigid fibrils. These experimentally determined boundaries match well with colloidal model predictions that account for salt-modulated charge repulsion. The model also incorporates the metastable and kinetic character of oligomer phases. Similarities and differences of amyloid oligomer assembly to metastable liquid-liquid phase separation of proteins and to surfactant aggregation are discussed.

  15. Competitive Protein Adsorption - Multilayer Adsorption and Surface Induced Protein Aggregation

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2009-01-01

    In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces...... is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions...... of high concentration with investigation of single protein adsorption and interdependent adsorption between two specific proteins enables us to map protein adsorption sequences during competitive protein adsorption. Our study shows that proteins can adsorb in a multilayer fashion onto the polymer surfaces...

  16. Signature of an aggregation-prone conformation of tau

    Science.gov (United States)

    Eschmann, Neil A.; Georgieva, Elka R.; Ganguly, Pritam; Borbat, Peter P.; Rappaport, Maxime D.; Akdogan, Yasar; Freed, Jack H.; Shea, Joan-Emma; Han, Songi

    2017-03-01

    The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6*, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6(*) regions dramatically extend to distances commensurate with extended β-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended β-strand conformational state of PHF6(*) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.

  17. The role of alginate in Azotobacter vinelandii aggregation in submerged culture

    Directory of Open Access Journals (Sweden)

    Edith Coronado

    2008-01-01

    Full Text Available The culture of strain LA21, a non-mucoid strain of Azotobacter vinelandii derivative of ATCC 9046, revealed that alginate is not necessary for aggregate formation. In fact, the non-mucoid strain LA21 developed aggregates significantly larger than those of the mucoid strain (ATCC 9046, which suggests that alginate has a detrimental effect on the aggregate size, due to its properties as a surface active agent. Treating the aggregates with a protease caused a decrease in the equivalent diameter of the structures, suggesting the participation of extracellular proteins in the aggregation. Key words: Aggregation; Azotobacter vinelandii; alginate; mutant strain; mucoid.

  18. DETERMINATION OF THE THERMODYNAMICS OF β-LACTOGLOBULIN AGGREGATION USING ULTRA VIOLET LIGHT SCATTERING SPECTROSCOPY

    OpenAIRE

    Belton, Daniel; Austerberry, James

    2018-01-01

    The problem of protein aggregation is widely studied across a number of disciplines, where understanding the behaviour of the protein monomer, and its behaviour with co-solutes is imperative in order to devise solutions to the problem. Here we present a method for measuring the kinetics of protein aggregation based on ultra violet light scattering spectroscopy (UVLSS) across a range of NaCl conditions. Through measurement of wavelength dependant scattering and using the model protein β-lactog...

  19. Protein-peptide interactions in mixtures of whey peptides and whey proteins

    NARCIS (Netherlands)

    Creusot, N.; Gruppen, H.

    2007-01-01

    The effects of several conditions on the amounts and compositions of aggregates formed in mixtures of whey protein hydrolysate, made with Bacillus licheniformis protease, and whey protein isolate were investigated using response surface methodology. Next, the peptides present in the aggregates were

  20. The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains.

    Science.gov (United States)

    Shattuck, Jenifer E; Waechter, Aubrey C; Ross, Eric D

    2017-07-04

    Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN + ], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.

  1. The Role of Metal Binding in the Amyotrophic Lateral Sclerosis-Related Aggregation of Copper-Zinc Superoxide Dismutase

    Directory of Open Access Journals (Sweden)

    Ivana Sirangelo

    2017-08-01

    Full Text Available Protein misfolding and conformational changes are common hallmarks in many neurodegenerative diseases involving formation and deposition of toxic protein aggregates. Although many players are involved in the in vivo protein aggregation, physiological factors such as labile metal ions within the cellular environment are likely to play a key role. In this review, we elucidate the role of metal binding in the aggregation process of copper-zinc superoxide dismutase (SOD1 associated to amyotrophic lateral sclerosis (ALS. SOD1 is an extremely stable Cu-Zn metalloprotein in which metal binding is crucial for folding, enzymatic activity and maintenance of the native conformation. Indeed, demetalation in SOD1 is known to induce misfolding and aggregation in physiological conditions in vitro suggesting that metal binding could play a key role in the pathological aggregation of SOD1. In addition, this study includes recent advances on the role of aberrant metal coordination in promoting SOD1 aggregation, highlighting the influence of metal ion homeostasis in pathologic aggregation processes.

  2. Prions and prion-like proteins.

    Science.gov (United States)

    Fraser, Paul E

    2014-07-18

    Prions are self-replicating protein aggregates and are the primary causative factor in a number of neurological diseases in mammals. The prion protein (PrP) undergoes a conformational transformation leading to aggregation into an infectious cellular pathogen. Prion-like protein spreading and transmission of aggregates between cells have also been demonstrated for other proteins associated with Alzheimer disease and Parkinson disease. This protein-only phenomenon may therefore have broader implications in neurodegenerative disorders. The minireviews in this thematic series highlight the recent advances in prion biology and the roles these unique proteins play in disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Proceedings of the specialist research meeting on 'abnormal protein aggregation and the folding diseases, and their protection and repair system' [1-2 November 2012, Kumatori, Osaka (Japan)

    International Nuclear Information System (INIS)

    Sadakane, Yutaka; Fujii, Noriko

    2013-03-01

    For this meeting, we invited leading biochemistry, structural biology, physical chemistry, analytical chemistry, molecular biology, radiation biology, and enzymology researchers. During the meeting, it was reported that the triggers of abnormal protein aggregation include racemization and isomerization of aspartyl residues, deamination of appraisingly residues. The process and the protection of amyloid fibril formation, and the alteration of the higher order structure of abnormal aggregated protein were also covered. Furthermore, the new analytical methods for the detection of the abnormal protein were presented. The topics of this meeting were widespread and the discussion was exciting. This special issue was the proceedings of the meeting with collection of 11 papers. The one of the presented papers are indexed individually. (J.P.N.)

  4. Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Smirnov Vladimir N

    2002-10-01

    Full Text Available Abstract Background Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown. Results We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation. Conclusions The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

  5. Sequence dependent aggregation of peptides and fibril formation

    Science.gov (United States)

    Hung, Nguyen Ba; Le, Duy-Manh; Hoang, Trinh X.

    2017-09-01

    Deciphering the links between amino acid sequence and amyloid fibril formation is key for understanding protein misfolding diseases. Here we use Monte Carlo simulations to study the aggregation of short peptides in a coarse-grained model with hydrophobic-polar (HP) amino acid sequences and correlated side chain orientations for hydrophobic contacts. A significant heterogeneity is observed in the aggregate structures and in the thermodynamics of aggregation for systems of different HP sequences and different numbers of peptides. Fibril-like ordered aggregates are found for several sequences that contain the common HPH pattern, while other sequences may form helix bundles or disordered aggregates. A wide variation of the aggregation transition temperatures among sequences, even among those of the same hydrophobic fraction, indicates that not all sequences undergo aggregation at a presumable physiological temperature. The transition is found to be the most cooperative for sequences forming fibril-like structures. For a fibril-prone sequence, it is shown that fibril formation follows the nucleation and growth mechanism. Interestingly, a binary mixture of peptides of an aggregation-prone and a non-aggregation-prone sequence shows the association and conversion of the latter to the fibrillar structure. Our study highlights the role of a sequence in selecting fibril-like aggregates and also the impact of a structural template on fibril formation by peptides of unrelated sequences.

  6. Metabolic states following accumulation of intracellular aggregates: implications for neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    Alexei Vazquez

    Full Text Available The formation of intracellular aggregates is a common etiology of several neurodegenerative diseases. Mitochondrial defects and oxidative stress has been pointed as the major mechanistic links between the accumulation of intracellular aggregates and cell death. In this work we propose a "metabolic cell death by overcrowding" as an alternative hypothesis. Using a model of neuron metabolism, we predict that as the concentration of protein aggregates increases the neurons transit through three different metabolic phases. The first phase (0-6 mM corresponds with the normal neuron state, where the neuronal activity is sustained by the oxidative phosphorylation of lactate. The second phase (6-8.6 mM is characterized by a mixed utilization of lactate and glucose as energy substrates and a switch from ammonia uptake to ammonia release by neurons. In the third phase (8.6-9.3 mM neurons are predicted to support their energy demands from glycolysis and an alternative pathway for energy generation, involving reactions from serine synthesis, one carbon metabolism and the glycine cleavage system. The model also predicts a decrease in the maximum neuronal capacity for energy generation with increasing the concentration of protein aggregates. Ultimately this maximum capacity becomes zero when the protein aggregates reach a concentration of about 9.3 mM, predicting the cessation of neuronal activity.

  7. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are

  8. Effects of sucrose on rFVIIa aggregation and methionine oxidation

    DEFF Research Database (Denmark)

    Soenderkaer, Susanne; Carpenter, John F; van de Weert, Marco

    2004-01-01

    The aim of this study was to characterize the effects of sucrose on the stability of recombinant factor VIIa (rFVIIa), with special emphasis on aggregation and methionine oxidation, as well as to investigate the impact of various environmental conditions on the rFVIIa conformation. The stability...... of rFVIIa was studied at pH 5. Aggregation was monitored using size exclusion high-performance liquid chromatography (SE-HPLC), whereas formation of methionine oxidation products was measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Fourier transform infrared (FTIR...... the protein's surface, which shifts the protein molecular population away from expanded aggregation competent species and toward the compact native state, is thought to account for these observations. rFVIIa is sensitive to methionine oxidation; two mono-oxidized and one di-oxidized product were formed upon...

  9. Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis.

    Directory of Open Access Journals (Sweden)

    Jody Groenendyk

    Full Text Available Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function.We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER homeostasis, transient activation of the unfolded protein response (UPR pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA is sufficient to prevent cardiac fibrosis, and improved exercise tolerance.We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function.

  10. Disulfide scrambling in superoxide dismutase 1 reduces its cytotoxic effect in cultured cells and promotes protein aggregation.

    Directory of Open Access Journals (Sweden)

    Lina Leinartaitė

    Full Text Available Mutations in the gene coding for superoxide dismutase 1 (SOD1 are associated with familiar forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS. These mutations are believed to result in a "gain of toxic function", leading to neuronal degeneration. The exact mechanism is still unknown, but misfolding/aggregation events are generally acknowledged as important pathological events in this process. Recently, we observed that demetallated apoSOD1, with cysteine 6 and 111 substituted for alanine, is toxic to cultured neuroblastoma cells. This toxicity depended on an intact, high affinity Zn(2+ site. It was therefor contradictory to discover that wild-type apoSOD1 was not toxic, despite of its high affinity for Zn(2+. This inconsistency was hypothesized to originate from erroneous disulfide formation involving C6 and C111. Using high resolution non-reducing SDS-PAGE, we have in this study demonstrated that the inability of wild-type apoSOD1 to cause cell death stems from formation of non-native intra-molecular disulfides. Moreover, monomeric apoSOD1 variants capable of such disulfide scrambling aggregated into ThT positive oligomers under physiological conditions without agitation. The oligomers were stabilized by inter-molecular disulfides and morphologically resembled what has in other neurodegenerative diseases been termed protofibrils. Disulfide scrambling thus appears to be an important event for misfolding and aggregation of SOD1, but may also be significant for protein function involving cysteines, e.g. mitochondrial import and copper loading.

  11. Noncovalent PEGylation: different effects of dansyl-, L-tryptophan-, phenylbutylamino-, benzyl- and cholesteryl-PEGs on the aggregation of salmon calcitonin and lysozyme.

    Science.gov (United States)

    Mueller, Claudia; Capelle, Martinus A H; Seyrek, Emek; Martel, Sophie; Carrupt, Pierre-Alain; Arvinte, Tudor; Borchard, Gerrit

    2012-06-01

    Protein aggregation is a major instability that can occur during all stages of protein drug production and development. Protein aggregates may compromise the safety and efficacy of the final protein formulation. In this paper, various new excipients [phenylbutylamino-, benzyl-, and cholesteryl-polyethylene glycols (PEGs)] and their use for the reduction of aggregation of salmon calcitonin (sCT) and hen egg-white lysozyme (HEWL) by noncovalent PEGylation are presented. The ability to suppress aggregation of sCT in various buffer systems at a 1:1 molar ratio was assessed by following changes in protein conformation and aggregation state over time. The results are compared with that of dansyl- and L-tryptophan (Trp)-PEGs described in earlier publications. Furthermore, the influence of the different PEG-based excipients on the aggregation of HEWL was measured. HEWL aggregation was completely suppressed in the presence of cholesteryl-PEGs (2 and 5 kDa), whereas deterioration was observed using benzyl-methoxy polyethylene glycols (mPEGs; 2 and 5 kDa). Phenylbutylamino- and Trp-mPEG (2 kDa), as well as dansyl-PEGs of different molecular weight prolonged the lag phase of aggregation and reduced the aggregation velocity of HEWL. Copyright © 2012 Wiley Periodicals, Inc.

  12. Kinetic partitioning between aggregation and vesicle permeabilization by modified ADan

    DEFF Research Database (Denmark)

    Nesgaard, Lise W.; Vad, Brian; Christiansen, Gunna

    2009-01-01

    The neurodegenerative illness Familial Danish Dementia (FDD) is linked to formation and aggregation of the 34-residue ADan peptide, whose cytotoxicity may be mediated by membrane interactions. Here we characterize the derived peptide SerADan, in which the two cysteines found in ADan have been....... Aggregation is prevented at neutral/acidic pH and low ionic strength by anionic lipid vesicles. These vesicles are permeabilized by monomeric SerADan assembling on the membrane to form stable beta-sheet structures which are different from the solution aggregates. In contrast, solution ageing of SerADan first...

  13. Lipotoxicity induces hepatic protein inclusions through TBK1-mediated p62/SQSTM1 phosphorylation.

    Science.gov (United States)

    Cho, Chun-Seok; Park, Hwan-Woo; Ho, Allison; Semple, Ian A; Kim, Boyoung; Jang, Insook; Park, Haeli; Reilly, Shannon; Saltiel, Alan R; Lee, Jun Hee

    2017-12-18

    Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause non-alcoholic steatohepatitis (NASH). NASH in turn is associated with the accumulation of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). The formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here we show that upon saturated fatty acid-induced lipotoxicity, Tank-binding protein kinase 1 (TBK1) is activated and phosphorylates p62. The TBK1-mediated p62 phosphorylation is important for lipotoxicity-induced aggregation of ubiquitinated proteins and the formation of large protein inclusions in hepatocytes. In addition, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in the lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of the ubiquitin-p62 aggregates, not only in cultured hepatocytes, but also in mouse models of obesity and NASH. These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. This article is protected by copyright. All rights reserved. © 2017 by the American Association for the Study of Liver Diseases.

  14. α-Synuclein aggregation, seeding and inhibition by scyllo-inositol

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, Tarek [Biological Sciences, Sunnybrook Research Institute (Canada); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, M4N 3M5, ON (Canada); McLaurin, JoAnne, E-mail: jmclaurin@sri.utoronto.ca [Biological Sciences, Sunnybrook Research Institute (Canada); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, M4N 3M5, ON (Canada)

    2016-01-15

    Recent literature demonstrates the accelerated aggregation of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease (PD), by the presence of preformed fibrillar conformers in vitro. Furthermore, these preformed fibrillar seeds are suggested to accelerate pathological induction in vivo when injected into the brains of mice. Variation in the results of in vivo studies is proposed to be caused by α-synuclein conformational variants. To investigate the impact of amino acid sequence on seeding efficiency, human and mouse α-synuclein seeds, which vary at 7 amino acid residues, were generated and cross-seeding kinetics studied. Using transmission electron microscopy (TEM), we confirmed that mouse α-synuclein aggregated more rapidly than human α-synuclein. Subsequently, we determined that seeding of human and mouse α-synuclein was more rapid in the presence of seeds generated from the same species. In addition, an established amyloid inhibitor, scyllo-inositol, was examined for potential inhibitory effects on α-synuclein aggregation. TEM analysis of protein:inhibitor assays demonstrated that scyllo-inositol inhibits the aggregation of α-synuclein, suggesting the therapeutic potential of the small molecule in PD. - Highlights: • Mouse α-syn fibrillizes in a significantly shorter timeframe than human α-syn. • Seeding of monomers is more efficient when seeds originate from the same species. • scyllo-Inositol has anti-aggregation effects on mouse and human α-syn.

  15. Expression of organophosphorus-degradation gene ( opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

    Science.gov (United States)

    Li, Qiong; Tang, Qing; Xu, Xudong; Gao, Hong

    2010-11-01

    Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene ( opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, P psbA . From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.

  16. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

    International Nuclear Information System (INIS)

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-01-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

  17. Possible Function of Molecular Chaperones in Diseases Caused by Propagating Amyloid Aggregates

    Directory of Open Access Journals (Sweden)

    Vladimir F. Lazarev

    2017-05-01

    Full Text Available The vast majority of neurodegenerative pathologies stem from the formation of toxic oligomers and aggregates composed of wrongly folded proteins. These protein complexes can be released from pathogenic cells and enthralled by other cells, causing the formation of new aggregates in a prion-like manner. By this mechanism, migrating complexes can transmit a disorder to distant regions of the brain and promote gradually transmitting degenerative processes. Molecular chaperones can counteract the toxicity of misfolded proteins. In this review, we discuss recent data on the possible cytoprotective functions of chaperones in horizontally transmitting neurological disorders.

  18. Atomic force microscopy and Raman scattering spectroscopy studies on heat-induced fibrous aggregates of β-lactoglobulin

    OpenAIRE

    Ikeda, Shinya

    2003-01-01

    Nanometer-thick fibrous aggregates of β-lactoglobulin alone and its mixture with other globular proteins were formed by heating aqueous solutions at pH 2 with maintaining an effective level of electrostatic repulsion among denatured protein molecules. In atomic force microscopy (AFM) images, these fibrous aggregates appeared to be fairly uniform in width and height and composed of strings of globular elements. Fibrous aggregates formed in β-lactoglobulin individual systems were only slightly ...

  19. On the nature of the Cu-rich aggregates in brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sullivan, Brendan; Robison, Gregory; Osborn, Jenna; Kay, Martin; Thompson, Peter; Davis, Katherine; Zakharova, Taisiya; Antipova, Olga; Pushkar, Yulia

    2017-04-01

    Fulfilling a bevy of biological roles, copper is an essential metal for healthy brain function. Cu dyshomeostasis has been demonstrated to be involved in some neurological conditions including Menkes and Alzheimer’s diseases. We have previously reported localized Cu-rich aggregates in astrocytes of the subventricular zone (SVZ) in rodent brains with Cu concentrations in the hundreds of millimolar. Metallothionein, a cysteine-rich protein critical to metal homeostasis and known to participate in a variety of neuroprotective and neuroregenerative processes, was proposed as a binding protein. Here, we present an analysis of metallothionein(1,2) knockout (MTKO) mice and age-matched controls using X-ray fluorescence microscopy. In large structures such as the corpus callosum, cortex, and striatum, there is no significant difference in Cu, Fe, or Zn concentrations in MTKO mice compared to age-matched controls. In the astrocyte-rich subventricular zone where Cu-rich aggregates reside, approximately 1/3 as many Cu-rich aggregates persist in MTKO mice resulting in a decrease in periventricular Cu concentration. Aggregates in both wild-type and MTKO mice show XANES spectra characteristic of CuxSy multimetallic clusters and have similar [S]/[Cu] ratios. Consistent with assignment as a CuxSy multimetallic cluster, the astrocyte-rich SVZ of both MTKO and wild-type mice exhibit autofluorescent bodies, though MTKO mice exhibit fewer. Furthermore, XRF imaging of Au-labeled lysosomes and ubiquitin demonstrates a lack of co-localization with Cu-rich aggregates suggesting they are not involved in a degradation pathway. Overall, these data suggest that Cu in aggregates is bound by either metallothionein-3 or a yet unknown protein similar to metallothionein.

  20. Modeling and simulation of aggregation of membrane protein LAT with molecular variability in the number of binding sites for cytosolic Grb2-SOS1-Grb2.

    Directory of Open Access Journals (Sweden)

    Ambarish Nag

    Full Text Available The linker for activation of T cells (LAT, the linker for activation of B cells (LAB, and the linker for activation of X cells (LAX form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate. We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

  1. Electrostatic interactions drive native-like aggregation of human alanine:glyoxylate aminostransferase.

    Science.gov (United States)

    Dindo, Mirco; Conter, Carolina; Cellini, Barbara

    2017-11-01

    Protein aggregate formation is the basis of several misfolding diseases, including those displaying loss-of-function pathogenesis. Although aggregation is often attributed to the population of intermediates exposing hydrophobic surfaces, the contribution of electrostatic forces has recently gained attention. Here, we combined computational and in vitro studies to investigate the aggregation process of human peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme involved in glyoxylate detoxification. We demonstrated that AGT is susceptible to electrostatic aggregation due to its peculiar surface charge anisotropy and that PLP binding counteracts the self-association process. The two polymorphic mutations P11L and I340M exert opposite effects. The P11L substitution enhances the aggregation tendency, probably by increasing surface charge anisotropy, while I340M plays a stabilizing role. In light of these results, we examined the effects of the most common missense mutations leading to primary hyperoxaluria type I (PH1), a rare genetic disorder associated with abnormal calcium oxalate precipitation in the urinary tract. All of them endow AGT with a strong electrostatic aggregation propensity. Moreover, we predicted that pathogenic mutations of surface residues could alter charge distribution, thus inducing aggregation under physiological conditions. A global model describing the AGT aggregation process is provided. Overall, the results indicate that the contribution of electrostatic interactions in determining the fate of proteins and the effect of amino acid substitutions should not be underestimated and provide the basis for the development of new therapeutic strategies for PH1 aimed at increasing AGT stability. © 2017 Federation of European Biochemical Societies.

  2. Physiological serum copper concentrations found in malignancies cause unfolding induced aggregation of human serum albumin in vitro.

    Science.gov (United States)

    Rizvi, Asim; Furkan, Mohd; Naseem, Imrana

    2017-12-15

    Malignancies are characterized by several drastic metabolic changes, one of which is a progressive rise in the levels of serum copper. This rise in serum copper is documented across all malignancies and across malignancies in several species. This study aims to explore in vitro the effect of increased copper levels on the structure of the blood protein human serum albumin. Exposure of human serum albumin to physiologically relevant copper concentrations for 21 days resulted in structural modifications in the protein which were evident by changes in the intrinsic florescence. A loss of the predominantly alpha helical structure of human serum albumin was recorded along with a tendency to form protein aggregates. This aggregation was characterized by Thioflavin T and Congo Red assays. Rayleigh light scattering and turbidity assays confirmed aggregation. The aggregates were visually confirmed using transmission electron microscopy. This is the first report implicating increased copper levels as a cause of aggregation of blood proteins in malignancies. The physiological and biochemical implications of this phenomenon are discussed. Copyright © 2017. Published by Elsevier Inc.

  3. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    Directory of Open Access Journals (Sweden)

    Sophie A Comyn

    2016-07-01

    Full Text Available Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

  4. Protein and exercise in the prevention of sarcopenia and aging.

    Science.gov (United States)

    Naseeb, Manal A; Volpe, Stella L

    2017-04-01

    Aging is associated with a progressive decline in skeletal muscle mass and strength. The decline, known as sarcopenia, could lead to physical disability, poor quality of life, and death. In addition, the older population usually experiences age-related muscle changes that affect muscle mass, muscular strength, and functional abilities. The purpose of this review is to describe the role of protein and exercise in slowing the progression of sarcopenia. It will also discuss whether age-related changes can be attenuated by dietary protein and exercise in the older population. This review will also cover one of the possible mechanisms of how dietary protein and exercise are involved in sarcopenia prevention, as well as the available measurement tools. Based on the findings of this review, the adequate amount of protein required for older men and women needs to be revised and likely be higher. Moreover, studies are required to explore some inconclusive findings concerning sarcopenia in the older population. Further research is required to investigate the following: (1) the safety and effectiveness concerning the consumption of 1.4 g of protein/kg of body weight (or more) in this vulnerable population; (2) the effectiveness of amino acid supplementation in reducing progression of sarcopenia over time through longitudinal studies; (3) the preferred source and timing of protein for the older population to maintain muscular strength and attenuate sarcopenia; (4) exercise interventions, especially those of longer duration, in the attenuation of sarcopenia; (5) other types of exercise and their effects on age-related muscle changes; (6) the mechanism of how protein and exercise prevent muscle loss with aging; and (7) determine the best method to diagnose sarcopenia. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Association of Cerebral Amyloid-β Aggregation With Cognitive Functioning in Persons Without Dementia

    DEFF Research Database (Denmark)

    Jansen, Willemijn J; Ossenkoppele, Rik; Tijms, Betty M

    2018-01-01

    Importance: Cerebral amyloid-β aggregation is an early event in Alzheimer disease (AD). Understanding the association between amyloid aggregation and cognitive manifestation in persons without dementia is important for a better understanding of the course of AD and for the design of prevention tr...

  6. Gold nanoparticles for the bare-eye based and spectrophotometric detection of proteins, polynucleotides and DNA

    International Nuclear Information System (INIS)

    Lepoitevin, Mathilde; Lemouel, Marie; Bechelany, Mikhael; Janot, Jean-Marc; Balme, Sebastien

    2015-01-01

    We have explored the potential of using gold nanoparticles (Au-NPs) in optical and bare-eye discrimination of (a) proteins (such as bovine serum albumin and lysozyme), (b) homo-polynucleotides (such as poly-adenylic acid, poly-cytidylic acid, poly-uridylic acid and poly-inosinic acid), and (c) long chain DNA (from salmon, herring and thym). Such biomacromolecules can be detected and discriminated due to their ability to prevent the formation of blue aggregates from red (non-aggregated) citrate capped Au-NPs on addition of NaCl. The effect of these biomacromolecules on the aggregation was investigated by colorimetry and UV–vis spectrometry. The results show that the two proteins can be differentiated by colorimetry, and also salmon ssDNA and dsDNA. The Au-NPs can also discriminate the dsDNAs of salmon and herring. We conclude that the use of Au-NPs represent a viable candidate to future methods of DNA analysis on the basis of visual testing, particularly in the area of food analysis. (author)

  7. SHAPE CHARACTERIZATION OF CONCRETE AGGREGATE

    Directory of Open Access Journals (Sweden)

    Jing Hu

    2011-05-01

    Full Text Available As a composite material, the performance of concrete materials can be expected to depend on the properties of the interfaces between its two major components, aggregate and cement paste. The microstructure at the interfacial transition zone (ITZ is assumed to be different from the bulk material. In general, properties of conventional concrete have been found favoured by optimum packing density of the aggregate. Particle size is a common denominator in such studies. Size segregation in the ITZ among the binder particles in the fresh state, observed in simulation studies by concurrent algorithm-based SPACE system, additionally governs density as well as physical bonding capacity inside these shell-like zones around aggregate particles. These characteristics have been demonstrated qualitatively pertaining also after maturation of the concrete. Such properties of the ITZs have direct impact on composite properties. Despite experimental approaches revealed effects of aggregate grain shape on different features of material structure (among which density, and as a consequence on mechanical properties, it is still an underrated factor in laboratory studies, probably due to the general feeling that a suitable methodology for shape characterization is not available. A scientific argument hindering progress is the interconnected nature of size and shape. Presently, a practical problem preventing shape effects to be emphasized is the limitation of most computer simulation systems in concrete technology to spherical particles. New developments at Delft University of Technology will make it possible in the near future to generate jammed states, or other high-density fresh particle mixtures of non-spherical particles, which thereupon can be subjected to hydration algorithms. This paper will sketch the outlines of a methodological approach for shape assessment of loose (non-embedded aggregate grains, and demonstrate its use for two types of aggregate, allowing

  8. Acupuncture promotes mTOR-independent autophagic clearance of aggregation-prone proteins in mouse brain.

    Science.gov (United States)

    Tian, Tian; Sun, Yanhong; Wu, Huangan; Pei, Jian; Zhang, Jing; Zhang, Yi; Wang, Lu; Li, Bin; Wang, Lihua; Shi, Jiye; Hu, Jun; Fan, Chunhai

    2016-01-21

    Acupuncture has historically been practiced to treat medical disorders by mechanically stimulating specific acupoints with fine needles. Despite its well-documented efficacy, its biological basis remains largely elusive. In this study, we found that mechanical stimulation at the acupoint of Yanglingquan (GB34) promoted the autophagic clearance of α-synuclein (α-syn), a well known aggregation-prone protein closely related to Parkinson's disease (PD), in the substantia nigra par compacta (SNpc) of the brain in a PD mouse model. We found the protein clearance arose from the activation of the autophagy-lysosome pathway (ALP) in a mammalian target of rapamycin (mTOR)-independent approach. Further, we observed the recovery in the activity of dopaminergic neurons in SNpc, and improvement in the motor function at the behavior level of PD mice. Whereas acupuncture and rapamycin, a chemical mTOR inhibitor, show comparable α-syn clearance and therapeutic effects in the PD mouse model, the latter adopts a distinctly different, mTOR-dependent, autophagy induction process. Due to this fundamental difference, acupuncture may circumvent adverse effects of the rapamycin treatment. The newly discovered connection between acupuncture and autophagy not only provides a new route to understanding the molecular mechanism of acupuncture but also sheds new light on cost-effective and safe therapy of neurodegenerative diseases.

  9. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations.

    Science.gov (United States)

    Matveev, Vladimir V

    2010-06-09

    According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity."One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity."Josiah Willard Gibbs (1839-1903).

  10. SANS study of interaction of silica nanoparticles with BSA protein and their resultant structure

    International Nuclear Information System (INIS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2014-01-01

    Small angle neutron scattering (SANS) has been carried out to study the interaction of anionic silica nanoparticles (88 Å) with globular protein Bovine Serum Albumin (BSA) (M.W. 66.4 kD) in aqueous solution. The measurements have been carried out on fixed concentration (1 wt %) of Ludox silica nanoparticles with varying concentration of BSA (0–5 wt %) at pH7. Results show that silica nanoparticles and BSA coexist as individual entities at low concentration of BSA where electrostatic repulsive interactions between them prevent their aggregation. However, as the concentration of BSA increases (≥ 0.5 wt %), it induces the attractive depletion interaction among nanoparticles leading to finally their aggregation at higher BSA concentration (2 wt %). The aggregates are found to be governed by the diffusion limited aggregation (DLA) morphology of fractal nature having fractal dimension about 2.4

  11. Structure activity relationship study of curcumin analogues toward the amyloid-beta aggregation inhibitor.

    Science.gov (United States)

    Endo, Hitoshi; Nikaido, Yuri; Nakadate, Mamiko; Ise, Satomi; Konno, Hiroyuki

    2014-12-15

    Inhibition of the amyloid β aggregation process could possibly prevent the onset of Alzheimer's disease. In this article, we report a structure-activity relationship study of curcumin analogues for anti amyloid β aggregation activity. Compound 7, the ideal amyloid β aggregation inhibitor in vitro among synthesized curcumin analogues, has not only potent anti amyloid β aggregation effects, but also water solubility more than 160 times that of curcumin. In addition, new approaches to improve water solubility of curcumin-type compounds are proposed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. A molecular key for building hyphae aggregates: the role of the newly identified Streptomyces protein HyaS.

    Science.gov (United States)

    Koebsch, Ilona; Overbeck, Jens; Piepmeyer, Sophie; Meschke, Holger; Schrempf, Hildgund

    2009-05-01

    Streptomycetes produce many metabolites with medical and biotechnological applications. During fermentations, their hyphae build aggregates, a process in which the newly identified protein HyaS plays an important role. The corresponding hyaS gene is present within all investigated Streptomyces species. Reporter fusions indicate that transcription of hyaS occurs within substrate hyphae of the Streptomyces lividans wild type (WT). The HyaS protein is dominantly associated with the substrate hyphae. The WT strain forms cylindrically shaped clumps of densely packed substrate hyphae, often fusing to higher aggregates (pellets), which remain stably associated during shaking. Investigations by electron microscopy suggest that HyaS induces tight fusion-like contacts among substrate hyphae. In contrast, the pellets of the designed hyaS disruption mutant ΔH are irregular in shape, contain frequently outgrowing bunches of hyphae, and fuse less frequently. ΔH complemented with a plasmid carrying hyaS resembles the WT phenotype. Biochemical studies indicate that the C-terminal region of HyaS has amine oxidase activity. Investigations of ΔH transformants, each carrying a specifically mutated gene, lead to the conclusion that the in situ oxidase activity correlates with the pellet-inducing role of HyaS, and depends on the presence of certain histidine residues. Furthermore, the level of undecylprodigiosin, a red pigment with antibiotic activity, is influenced by the engineered hyaS subtype within a strain. These data present the first molecular basis for future manipulation of pellets, and concomitant production of secondary metabolites during biotechnological processes. © 2009 The Authors. Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.

  13. Descriptive parameters of the erythrocyte aggregation phenomenon using a laser transmission optical chip

    Science.gov (United States)

    Toderi, Martín A.; Castellini, Horacio V.; Riquelme, Bibiana D.

    2017-01-01

    The study of red blood cell (RBC) aggregation is of great interest because of its implications for human health. Altered RBC aggregation can lead to microcirculatory problems as in vascular pathologies, such as hypertension and diabetes, due to a decrease in the erythrocyte surface electric charge and an increase in the ligands present in plasma. The process of erythrocyte aggregation was studied in stasis situation (free shear stresses), using an optical chip based on the laser transmission technique. Kinetic curves of erythrocyte aggregation under different conditions were obtained, allowing evaluation and characterization of this process. Two main characteristics of blood that influence erythrocyte aggregation were analyzed: the erythrocyte surface anionic charge (EAC) after digestion with the enzyme trypsin and plasmatic protein concentration in suspension medium using plasma dissolutions in physiological saline with human albumin. A theoretical approach was evaluated to obtain aggregation and disaggregation ratios by syllectograms data fitting. Sensible parameters (Amp100, t) regarding a reduced erythrocyte EAC were determined, and other parameters (AI, M-Index) resulted that are representative of a variation in the plasmatic protein content of the suspension medium. These results are very useful for further applications in biomedicine.

  14. Guidance to Achieve Accurate Aggregate Quantitation in Biopharmaceuticals by SV-AUC.

    Science.gov (United States)

    Arthur, Kelly K; Kendrick, Brent S; Gabrielson, John P

    2015-01-01

    The levels and types of aggregates present in protein biopharmaceuticals must be assessed during all stages of product development, manufacturing, and storage of the finished product. Routine monitoring of aggregate levels in biopharmaceuticals is typically achieved by size exclusion chromatography (SEC) due to its high precision, speed, robustness, and simplicity to operate. However, SEC is error prone and requires careful method development to ensure accuracy of reported aggregate levels. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an orthogonal technique that can be used to measure protein aggregation without many of the potential inaccuracies of SEC. In this chapter, we discuss applications of SV-AUC during biopharmaceutical development and how characteristics of the technique make it better suited for some applications than others. We then discuss the elements of a comprehensive analytical control strategy for SV-AUC. Successful implementation of these analytical control elements ensures that SV-AUC provides continued value over the long time frames necessary to bring biopharmaceuticals to market. © 2015 Elsevier Inc. All rights reserved.

  15. Optimization on Fc for Improvement of Stability and Aggregation Resistance.

    Science.gov (United States)

    Chen, Xiaobo; Zeng, Fang; Huang, Tao; Cheng, Liang; Liu, Huan; Gong, Rui

    2016-01-01

    Fc-based therapeutics including therapeutic full-size monoclonal antibodies (mAbs) and Fcfusion proteins represent fastest-growing market in biopharmaceutical industrial. However, one major challenge during development of Fc-based therapeutics is how to maintain their efficacy in clinic use. Many factors may lead to failure in final marketing. For example, the stability and aggregation resistance might not be high enough for bearing the disadvantages during fermentation, purification, formulation, storage, shipment and other steps in manufacture and sale. Low stability and high aggregation tendency lead to decreased bioactivity and increased risk of immunogenicity resulting in serious side effect. Because Fc is one of the major parts in monoclonal antibodies and Fc-fusion proteins, engineering of Fc to increase its stability and reduce or eliminate aggregation due to incorrect association are of great importance and could further extend the potential of Fc-based therapeutics. Lots of studies focus on Fc optimization for better physical and chemical characteristics and function by structured-based computer-aid rational design, high-throughput screening expression system selection and other methods. The identification of optimized Fc mutants increases the clinic potential of currently existed therapeutics mAbs and Fc-fusion proteins, and accelerates the development of new Fc-based therapeutics. Here we provide an overview of the related field, and discuss recent advances and future directions in optimization of Fc-based therapeutics with modified stability and aggregation resistance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL

    International Nuclear Information System (INIS)

    Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E.

    2005-01-01

    Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein

  17. Early Response of Protein Quality Control in Gills Is Associated with Survival of Hypertonic Shock in Mozambique tilapia

    Science.gov (United States)

    Tang, Cheng-Hao; Lee, Tsung-Han

    2013-01-01

    The protein quality control (PQC) mechanism is essential for cell function and viability. PQC with proper biological function depends on molecular chaperones and proteases. The hypertonicity-induced protein damage and responses of PQC mechanism in aquatic organisms, however, are poorly understood. In this study, we examine the short-term effects of different hypertonic shocks on the levels of heat shock proteins (HSPs, e.g., HSP70 and HSP90), ubiquitin-conjugated proteins and protein aggregation in gills of the Mozambique tilapia (Oreochromis mossambicus). Following transfer from fresh water (FW) to 20‰ hypertonicity, all examined individuals survived to the end of experiment. Moreover, the levels of branchial HSPs and ubiquitin-conjugated proteins significantly increased at 3 and 24 h post-transfer, respectively. Up-regulation of HSPs and ubiquitin-conjugated proteins was sufficient to prevent the accumulation of aggregated proteins. However, the survival rate of tilapia dramatically declined at 5 h and all fish died within 7 h after direct transfer to 30‰ hypertonicity. We presumed that this result was due to the failed activation of gill PQC system, which resulted in elevating the levels of aggregated proteins at 3 and 4 h. Furthermore, in aggregated protein fractions, the amounts of gill Na+/K+-ATPase (NKA) remained relatively low when fish were transferred to 20‰ hypertonicity, whereas abundant NKA was found at 4 h post-transfer to 30‰ hypertonicity. This study demonstrated that the response of PQC in gills is earlier than observable changes in localization of ion-secreting transport proteins upon hypertonic challenge. To our knowledge, this is the first study to investigate the regulation of PQC mechanism in fish and characterize its important role in euryhaline teleost survival in response to hypertonic stress. PMID:23690986

  18. Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.

    Science.gov (United States)

    Manickam, Nagaraj; Sun, Xiuhua; Hakala, Kevin W; Weintraub, Susan T; Essex, David W

    2008-07-01

    Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

  19. Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases

    DEFF Research Database (Denmark)

    Salahuddin, Parveen; Siddiqi, Mohammad Khursheed; Khan, Sanaullah

    2016-01-01

    ’s disease (PD), Alzheimer’s disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other......In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post......-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially...

  20. Formation of non-toxic Aβ fibrils by small heat shock protein under heat-stress conditions

    Energy Technology Data Exchange (ETDEWEB)

    Sakono, Masafumi [Bioengineering Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); PRESTO, JST, Saitama (Japan); Utsumi, Arata [Bioengineering Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588 (Japan); Zako, Tamotsu, E-mail: zako@riken.jp [Bioengineering Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Abe, Tetsuya; Yohda, Masafumi [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588 (Japan); Maeda, Mizuo [Bioengineering Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2013-01-25

    Highlights: ► We examined effect of the quaternary structure of yeast sHsp on Aβ aggregation. ► Aβ aggregation was inhibited by the oligomeric form of sHsp, but not by dimeric sHsp. ► The fibrillar amyloids consisted of both Aβ and dimeric sHsp. ► They exhibited different inner structure and cytotoxicity from authentic Aβ amyloids. ► These results suggest the formation of new type fibrillar Aβ amyloid by sHsp. -- Abstract: Small heat shock protein (sHsp) is a molecular chaperone with a conserved alpha-crystallin domain that can prevent protein aggregation. It has been shown that sHsps exist as oligomers (12–40 mer) and their dissociation into small dimers or oligomers is functionally important. Since several sHsps are upregulated and co-localized with amyloid-β (Aβ) in senile plaques of patients with Alzheimer’s disease (AD), sHsps are thought to be involved in AD. Previous studies have also shown that sHsp can preventaggregation in vitro. However, it remains unclear how the quaternary structure of sHsp influences Aβ aggregation. In this study, we report for the first time the effect of the quaternary structure of sHsp on Aβ aggregation using sHsp from the fission yeast Schizosaccharomyces pombe (SpHsp16.0) showing a clear temperature-dependent structural transition between an oligomer (30 °C) and dimer (50 °C) state. Aβ aggregation was inhibited by the oligomeric form of SpHsp16.0. In contrast, amyloid fibrils were formed in the presence of dimeric SpHsp16.0. Interestingly, these amyloid fibrils consisted of both Aβ and SpHsp16.0 and showed a low ThT intensity and low cytotoxicity due to their low binding affinity to the cell surface. These results suggest the formation of novel fibrillar Aβ amyloid with different characteristics from that of the authentic Aβ amyloid fibrils formed in the absence of sHsp. Our results also suggest the potential protective role of sHsp in AD under stress conditions.

  1. Aggregate complexes of HIV-1 induced by multimeric antibodies.

    Science.gov (United States)

    Stieh, Daniel J; King, Deborah F; Klein, Katja; Liu, Pinghuang; Shen, Xiaoying; Hwang, Kwan Ki; Ferrari, Guido; Montefiori, David C; Haynes, Barton; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Michael, Nelson L; Robb, Merlin L; Kim, Jerome H; Denny, Thomas N; Tomaras, Georgia D; Shattock, Robin J

    2014-10-02

    Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry. The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA. These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.

  2. SUMOylation Is an Inhibitory Constraint that Regulates the Prion-like Aggregation and Activity of CPEB3

    Directory of Open Access Journals (Sweden)

    Bettina Drisaldi

    2015-06-01

    Full Text Available Protein synthesis is crucial for the maintenance of long-term-memory-related synaptic plasticity. The prion-like cytoplasmic polyadenylation element-binding protein 3 (CPEB3 regulates the translation of several mRNAs important for long-term synaptic plasticity in the hippocampus. Here, we provide evidence that the prion-like aggregation and activity of CPEB3 is controlled by SUMOylation. In the basal state, CPEB3 is a repressor and is soluble. Under these circumstances, CPEB3 is SUMOylated in hippocampal neurons both in vitro and in vivo. Following neuronal stimulation, CPEB3 is converted into an active form that promotes the translation of target mRNAs, and this is associated with a decrease of SUMOylation and an increase of aggregation. A chimeric CPEB3 protein fused to SUMO cannot form aggregates and cannot activate the translation of target mRNAs. These findings suggest a model whereby SUMO regulates translation of mRNAs and structural synaptic plasticity by modulating the aggregation of the prion-like protein CPEB3.

  3. Aggregation kinetics and structure of cryoimmunoglobulins clusters

    CERN Document Server

    De Spirito, M; Bassi, F A; Di Stasio, E; Giardina, B; Arcovito, G

    2002-01-01

    Cryoimmunoglobulins are pathological antibodies characterized by a temperature-dependent reversible insolubility. Rheumatoid factors (RF) are immunoglobulins possessing anti-immunoglobulin activity and usually consist of an IgM antibody that recognizes IgG as antigen. These proteins are present in sera of patients affected by a large variety of different pathologies, such as HCV infection, neoplastic and autoimmune diseases. Aggregation and precipitation of cryoimmunoglobulins, leading to vasculiti, are physical phenomena behind such pathologies. A deep knowledge of the physico-chemical mechanisms regulating such phenomena plays a fundamental role in biological and clinical applications. In this work, a preliminary investigation of the aggregation kinetics and of the final macro- molecular structure of the aggregates is presented. Through static light scattering techniques, the gyration radius R/sub g/ and the fractal dimension D/sub m/ of the growing clusters have been determined. However, while the initial ...

  4. Early stages in the biogenesis of eukaryotic β-barrel proteins.

    Science.gov (United States)

    Jores, Tobias; Rapaport, Doron

    2017-09-01

    The endosymbiotic organelles mitochondria and chloroplasts harbour, similarly to their prokaryotic progenitors, β-barrel proteins in their outer membrane. These proteins are encoded on nuclear DNA, translated on cytosolic ribosomes and imported into their target organelles by a dedicated machinery. Recent studies have provided insights into the import into the organelles and the membrane insertion of these proteins. Although the cytosolic stages of their biogenesis are less well defined, it is speculated that upon their synthesis, chaperones prevent β-barrel proteins from aggregation and keep them in an import-competent conformation. In this Review, we summarize the current knowledge about the biogenesis of β-barrel proteins, focusing on the early stages from the translation on cytosolic ribosomes to the recognition on the surface of the organelle. © 2017 Federation of European Biochemical Societies.

  5. Noncovalent pegylation by dansyl-poly(ethylene glycol)s as a new means against aggregation of salmon calcitonin.

    Science.gov (United States)

    Mueller, Claudia; Capelle, Martinus A H; Arvinte, Tudor; Seyrek, Emek; Borchard, Gerrit

    2011-05-01

    During all stages of protein drug development, aggregation is one of the most often encountered problems. Covalent conjugation of poly(ethylene glycol) (PEG), also called PEGylation, to proteins has been shown to reduce aggregation of proteins. In this paper, new excipients based on PEG are presented that are able to reduce aggregation of salmon calcitonin (sCT). Several PEG polymers consisting of a hydrophobic dansyl-headgroup attached to PEGs of different molecular weights have been synthesized and characterized physicochemically. After addition of dansyl-methoxypoly(ethylene glycol) (mPEG) 2 kDa to a 40 times molar excess of sCT resulted in an increase in dansyl-fluorescence and a decrease in 90° light scatter suggesting possible interactions. The aggregation of sCT in different buffer systems in presence or absence of the different dansyl-PEGs was measured by changes in Nile red fluorescence and turbidity. Dansyl-mPEG 2 kDa in a 1:1 molar ratio to sCT strongly reduced aggregation. Reduction of sCT aggregation was also measured for the bivalent dansyl-PEG 3 kDa in a 1:1 molar ratio. Dansyl-mPEG 5 kDa deteriorated sCT aggregation. Potential cytotoxicity and hemolysis were investigated. This paper shows that dansyl-PEGs are efficacious in reducing aggregation of sCT. Copyright © 2010 Wiley-Liss, Inc.

  6. Molecular simulations of lipid-mediated protein-protein interactions

    NARCIS (Netherlands)

    de Meyer, F.J.M.; Venturoli, M.; Smit, B.

    2008-01-01

    Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the

  7. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations

    Directory of Open Access Journals (Sweden)

    Matveev Vladimir V

    2010-06-01

    Full Text Available Abstract According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates. Cell function is considered as a transition between two states (two states model, the resting state and state of activity (this applies to the cell as a whole and to its individual structures. In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins, and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity. "One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity." Josiah Willard Gibbs (1839-1903

  8. Interaction between holo transferrin and HSA-PPIX complex in the presence of lomefloxacin: An evaluation of PPIX aggregation in protein-protein interactions

    Science.gov (United States)

    Sattar, Zohreh; Iranfar, Hediye; Asoodeh, Ahmad; Saberi, Mohammad Reza; Mazhari, Mahboobeh; Chamani, Jamshidkhan

    2012-11-01

    Human serum albumin (HSA) and holo transferrin (TF) are two serum carrier proteins that are able to interact with each other, thereby altering their binding behavior toward their ligands. During the course of this study, the interaction between HSA-PPIX and TF, in the presence and absence of lomefloxacin (LMF), was for the first time investigated using different spectroscopic and molecular modeling techniques. Fluorescence spectroscopy experiments were performed in order to study conformational changes of proteins. The RLS technique was utilized to investigate the effect of LMF on J-aggregation of PPIX, which is the first report of its kind. Our findings present clear-cut evidence for the alteration of interactions between HSA and TF in the presence of PPIX and changes in drug-binding to HSA and HSA-PPIX complex upon interaction with TF. Moreover, molecular modeling studies suggested that the binding site for LMF became switched in the presence of PPIX, and that LMF bound to the site IIA of HSA. The obtained results should give new insight into research in this field and may cast some light on the dynamics of drugs in biological systems.

  9. Methylglyoxal-induced modification causes aggregation of myoglobin

    Science.gov (United States)

    Banerjee, Sauradipta; Maity, Subhajit; Chakraborti, Abhay Sankar

    2016-02-01

    Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. We have investigated the in vitro effect of MG (200 μM) on the monomeric heme protein myoglobin (Mb) (100 μM) in a time-dependent manner (7 to 18 days incubation at 25 °C). MG induces significant structural alterations of the heme protein, including heme loss, changes in tryptophan fluorescence, decrease of α-helicity with increased β-sheet content etc. These changes occur gradually with increased period of incubation. Incubation of Mb with MG for 7 days results in formation of the AGE adducts: carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87 and carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133. On increasing the period of incubation up to 14 days, additional AGEs namely, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139 have been detected. MG also induces aggregation of Mb, which is clearly evident with longer period of incubation (18 days), and appears to have amyloid nature. MG-derived AGEs may thus have an important role as the precursors of protein aggregation, which, in turn, may be associated with physiological complications.

  10. Effects of vegetation restoration on the aggregate stability and distribution of aggregate-associated organic carbon in a typical karst gorge region

    Science.gov (United States)

    Tang, F. K.; Cui, M.; Lu, Q.; Liu, Y. G.; Guo, H. Y.; Zhou, J. X.

    2015-08-01

    Changes in soil utilization significantly affect aggregate stability and aggregate-associated soil organic carbon (SOC). A field investigation and indoor analysis were conducted in order to study the soil aggregate stability and organic carbon distribution in the water-stable aggregates (WSA) of the bare land (BL), grassland (GL), shrubland (SL), and woodland (WL) in a typical karst gorge region. The results indicated that the BL, GL, SL, and WL were dominated by particles with sizes > 5 mm under dry sieving treatment, and that the soil aggregate contents of various sizes decreased as the particle size decreased. In addition, the BL, GL, SL, and WL were predominantly comprised of WSA sieving treatment, and that the WSA contents initially increased, then decreased, and then increased again as the particle size decreased. Furthermore, at a soil depth of 0-60 cm, the mean weight diameter (MWD), geometrical mean diameter (GMD), and fractal dimensions (D) of the dry aggregates and water-stable aggregates in the different types of land were ranked, in descending order, as WL > GL > SL > BL. The contents of WSA > 0.25 mm, MWD and GMD increased significantly, in that order, and the percentage of aggregate destruction (PAD) and fractal dimensions decreased significantly as the soil aggregate stability improved. The results of this study indicated that, as the SOC contents increased after vegetation restoration, the average SOC content of WL was 2.35, 1.37, and 1.26 times greater than that in the BL, GL, and SL, respectively. The total SOC and SOC associated in WSA of various sizes were the highest at a soil depth of 0-20 cm. In addition, the SOC contents of the WSA increased as the soil aggregate sizes decreased. The SOC contents of the WSA aggregates aggregate SOC contents. The woodland and grassland facilitated WSA stability and SOC protection, thus, promoting the natural restoration of vegetation by reducing artificial disturbances could effectively restore the ecology

  11. Covalent α-synuclein dimers: chemico-physical and aggregation properties.

    Directory of Open Access Journals (Sweden)

    Micaela Pivato

    Full Text Available The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson's disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1-104 and 29-140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results

  12. Far-infrared radiation protects viability in a cell model of Spinocerebellar Ataxia by preventing polyQ protein accumulation and improving mitochondrial function.

    Science.gov (United States)

    Chang, Jui-Chih; Wu, Shey-Lin; Hoel, Fredrik; Cheng, Yu-Shan; Liu, Ko-Hung; Hsieh, Mingli; Hoel, August; Tronstad, Karl Johan; Yan, Kuo-Chia; Hsieh, Ching-Liang; Lin, Wei-Yong; Kuo, Shou-Jen; Su, Shih-Li; Liu, Chin-San

    2016-07-29

    Far infrared radiation (FIR) is currently investigated as a potential therapeutic strategy in various diseases though the mechanism is unknown. Presently, we tested if FIR mediates beneficial effects in a cell model of the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3). SCA3 is caused by a mutation leading to an abnormal polyglutamine expansion (PolyQ) in ataxin-3 protein. The consequent aggregation of mutant ataxin-3 results in disruption of vital cell functions. In this study, neuroblastoma cells (SK-N-SH) was transduced to express either non-pathogenic ataxin-3-26Q or pathogenic ataxin-3-78Q proteins. The cells expressing ataxin-3-78Q demonstrated decreased viability, and increased sensitivity to metabolic stress in the presence rotenone, an inhibitor of mitochondrial respiration. FIR exposure was found to protect against these effects. Moreover, FIR improved mitochondrial respiratory function, which was significantly compromised in ataxin-3-78Q and ataxin-3-26Q expressing cells. This was accompanied by decreased levels of mitochondrial fragmentation in FIR treated cells, as observed by fluorescence microscopy and protein expression analysis. Finally, the expression profile LC3-II, Beclin-1 and p62 suggested that FIR prevent the autophagy inhibiting effects observed in ataxin-3-78Q expressing cells. In summary, our results suggest that FIR have rescuing effects in cells expressing mutated pathogenic ataxin-3, through recovery of mitochondrial function and autophagy.

  13. NMR of alpha-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation

    NARCIS (Netherlands)

    Fernández, Claudio O.; Hoyer, Wolfgang; Zweckstetter, Markus; Jares-Erijman, Elizabeth A.; Subramaniam, Vinod; Griesinger, Christian; Jovin, Thomas M.

    2004-01-01

    The aggregation of α-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of α-synuclein

  14. Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Lebrun, Marielle [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Thelen, Nicolas; Thiry, Marc [University of Liege (ULg), GIGA-Neurosciences, Laboratory of Cellular and Tissular Biology, Liege (Belgium); Riva, Laura; Ote, Isabelle; Condé, Claude; Vandevenne, Patricia [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Di Valentin, Emmanuel [University of Liege (ULg), GIGA-Viral Vectors Platform, Liege (Belgium); Bontems, Sébastien [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Sadzot-Delvaux, Catherine, E-mail: csadzot@ulg.ac.be [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium)

    2014-04-15

    The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.

  15. Consequential secondary structure alterations and aggregation during prolonged casein glycation.

    Science.gov (United States)

    Jindal, Supriya; Naeem, Aabgeena

    2013-05-01

    Non-enzymatic glycosylation (glycation) of casein is a process used not just to ameliorate the quality of dairy products but also to increase the shelf life of canned foods, including baby milk supplements. Incubation of κ-casein with reducing sugars for 15 days at physiological temperature showed the formation of a molten globule state at day 9 and 12 during fructation and glucation respectively. This state exhibits substantial secondary structure and maximum ANS binding. Later on, glycation resulted in the formation of aggregates at day 12 in presence of fructose and day 15 in presence of glucose. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and FTIR. These aggregates showed altered tryptophan environment, decrease ANS binding relative to molten globule state and increase in Thioflavin T fluorescence. Aggregates were also accompanied by the accumulation of AGEs, indicative of structural damage to the protein and formation of potentially harmful species at the physiological level. Fructose was more reactive than glucose and thus caused early and significant changes in the protein. From our studies, we conclude that controlling the extent of the Maillard reaction in the food industry is essential to counter its negative effects and expand its safety spectrum.

  16. Changes in blood aggregation with differences in molecular weight and degree of deacetylation of chitosan

    International Nuclear Information System (INIS)

    Hattori, Hidemi; Ishihara, Masayuki

    2015-01-01

    Because the molecular weight (Mw) and degree of deacetylation (DDA) of chitosan can vary depending on the purification method, the identification of appropriate chitosan structures is important for developing more effective hemostatic agents. In this study, the influence of varying Mw and DDA of chitosan on blood aggregation was characterized by 10 types of chitosan with different Mw and DDA, including oligomers. The highest aggregation of whole blood, washed erythrocytes and platelets in platelet-rich plasma (PRP) were observed in chitosan with Mw of 8.6 kDa or more and with DDA of 75 to 88%. However, chitosan with too high Mw (247 kDa) inhibited the aggregation of whole blood, washed erythrocytes and PRP at high chitosan concentration. At certain concentrations, chitosan with 75–85% DDA and 50–190 kDa and chitosan with 87.6% DDA and 247 kDa both aggregated proteins in PRP. Chitosan oligomer did not affect blood aggregation. The results suggested that the aggregation by chitosan depended on the interaction of positively charged chitosan with negatively charged erythrocytes, platelets and plasma protein. It seemed that a suitable balance of positive charge in chitosan and negative charge in hemocytes and some kinds of proteins was important. To develop a hemostatic with effective blood aggregation, the chitosan should not be limited to a single Mw and a single DDA but may be a mixed chitosan with wide range of Mw (8.6–247 kDa) and DDA of 75 to 88%. (paper)

  17. Tracking hypoxic signaling within encapsulated cell aggregates.

    Science.gov (United States)

    Skiles, Matthew L; Sahai, Suchit; Blanchette, James O

    2011-12-16

    , is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 10(10) pfu/mL. Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion. Western blot analysis of encapsulated cells in 20% and 1% oxygen also

  18. Polyglycerol coatings of glass vials for protein resistance.

    Science.gov (United States)

    Höger, Kerstin; Becherer, Tobias; Qiang, Wei; Haag, Rainer; Friess, Wolfgang; Küchler, Sarah

    2013-11-01

    Proteins are surface active molecules which undergo non-specific adsorption when getting in contact with surfaces such as the primary packaging material. This process is critical as it may cause a loss of protein content or protein aggregation. To prevent unspecific adsorption, protein repellent coatings are of high interest. We describe the coating of industrial relevant borosilicate glass vials with linear methoxylated polyglycerol, hyperbranched polyglycerol, and hyperbranched methoxylated polyglycerol. All coatings provide excellent protein repellent effects. The hyperbranched, non-methoxylated coating performed best. The protein repellent properties were maintained also after applying industrial relevant sterilization methods (≥200 °C). Marginal differences in antibody stability between formulations stored in bare glass vials and coated vials were detected after 3 months storage; the protein repellent effect remained largely stable. Here, we describe a new material suitable for the coating of primary packaging material of proteins which significantly reduces the protein adsorption and thus could present an interesting new possibility for biomedical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Heterologous aggregates promote de novo prion appearance via more than one mechanism.

    Directory of Open Access Journals (Sweden)

    Fatih Arslan

    2015-01-01

    Full Text Available Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q/asparagine (N-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN

  20. Aloe emodin, an anthroquinone from Aloe vera acts as an anti aggregatory agent to the thermally aggregated hemoglobin

    Science.gov (United States)

    Furkan, Mohammad; Alam, Md Tauqir; Rizvi, Asim; Khan, Kashan; Ali, Abad; Shamsuzzaman; Naeem, Aabgeena

    2017-05-01

    Aggregation of proteins is a physiological process which contributes to the pathophysiology of several maladies including diabetes mellitus, Huntington's and Alzheimer's disease. In this study we have reported that aloe emodin (AE), an anthroquinone, which is one of the active components of the Aloe vera plant, acts as an inhibitor of hemoglobin (Hb) aggregation. Hb was thermally aggregated at 60 °C for four days as evident by increased thioflavin T and ANS fluorescence, shifted congo red absorbance, appearance of β sheet structure, increase in turbidity and presence of oligomeric aggregates. Increasing concentration of AE partially reverses the aggregation of the model heme protein (hemoglobin). The maximum effect of AE was observed at 100 μM followed by saturation at 125 μM. The results were confirmed by UV-visible spectrometry, intrinsic fluorescence, ThT, ANS, congo red assay as well as transmission electron microscopy (TEM). These results were also supported by fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) which shows the disappearance of β sheet structure and appearance of α helices. This study will serve as baseline for translatory research and the development of AE based therapeutics for diseases attributed to protein aggregation.

  1. Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

    Science.gov (United States)

    Rizzo, Joseph M; Shi, Shuai; Li, Yunsong; Semple, Andrew; Esposito, Jessica J; Yu, Shenjiang; Richardson, Daisy; Antochshuk, Valentyn; Shameem, Mohammed

    2015-05-01

    In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  2. Generation of Soluble Advanced Glycation End Products Receptor (sRAGE)-Binding Ligands during Extensive Heat Treatment of Whey Protein/Lactose Mixtures Is Dependent on Glycation and Aggregation

    NARCIS (Netherlands)

    Liu, Fahui; Teodorowicz, Gosia; Wichers, Harry J.; Boekel, van Tiny; Hettinga, Kasper A.

    2016-01-01

    Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE

  3. Protein Self-Assembly and Protein-Induced DNA Morphologies

    Science.gov (United States)

    Mawhinney, Matthew T.

    The ability of biomolecules to associate into various structural configurations has a substantial impact on human physiology. The synthesis of protein polypeptide chains using the information encoded by DNA is mediated through the use of regulatory proteins, known as transcription factors. Some transcription factors perform function by inducing local curvature in deoxyribonucleic acid (DNA) strands, the mechanisms of which are not entirely known. An important architectural protein, eleven zinc finger CTCF (11 ZF CTCF) is involved in genome organization and hypothesized to mediate DNA loop formation. Direct evidence for these CTCF-induced DNA loops has yet to be observed. In this thesis, the effect of 11 ZF CTCF on DNA morphology is examined using atomic force microscopy, a powerful technique for visualizing biomolecules with nanometer resolution. The presence of CTCF is revealed to induce a variety of morphologies deviating from the relaxed state of control DNA samples, including compact circular complexes, meshes, and networks. Images reveal quasi-circular DNA/CTCF complexes consistent with a single DNA molecule twice wrapped around the protein. The structures of DNA and proteins are highly important for operations in the cell. Structural irregularities may lead to a variety of issues, including more than twenty human pathologies resulting from aberrant protein misfolding into amyloid aggregates of elongated fibrils. Insulin deficiency and resistance characterizing type 2 diabetes often requires administration of insulin. Injectable and inhalable delivery methods have been documented to result in the deposition of amyloid fibrils. Oligomers, soluble multiprotein assemblies, are believed to play an important role in this process. Insulin aggregation under physiological conditions is not well understood and oligomers have not yet been fully characterized. In this thesis, in vitro insulin aggregation at acidic and neutral pH is explored using a variety of techniques

  4. Familial Aggregation of Insomnia.

    Science.gov (United States)

    Jarrin, Denise C; Morin, Charles M; Rochefort, Amélie; Ivers, Hans; Dauvilliers, Yves A; Savard, Josée; LeBlanc, Mélanie; Merette, Chantal

    2017-02-01

    There is little information about familial aggregation of insomnia; however, this type of information is important to (1) improve our understanding of insomnia risk factors and (2) to design more effective treatment and prevention programs. This study aimed to investigate evidence of familial aggregation of insomnia among first-degree relatives of probands with and without insomnia. Cases (n = 134) and controls (n = 145) enrolled in a larger epidemiological study were solicited to invite their first-degree relatives and spouses to complete a standardized sleep/insomnia survey. In total, 371 first-degree relatives (Mage = 51.9 years, SD = 18.0; 34.3% male) and 138 spouses (Mage = 55.5 years, SD = 12.2; 68.1% male) completed the survey assessing the nature, severity, and frequency of sleep disturbances. The dependent variable was insomnia in first-degree relatives and spouses. Familial aggregation was claimed if the risk of insomnia was significantly higher in the exposed (relatives of cases) compared to the unexposed cohort (relatives of controls). The risk of insomnia was also compared between spouses in the exposed (spouses of cases) and unexposed cohort (spouses of controls). The risk of insomnia in exposed and unexposed biological relatives was 18.6% and 10.4%, respectively, yielding a relative risk (RR) of 1.80 (p = .04) after controlling for age and sex. The risk of insomnia in exposed and unexposed spouses was 9.1% and 4.2%, respectively; however, corresponding RR of 2.13 (p = .28) did not differ significantly. Results demonstrate evidence of strong familial aggregation of insomnia. Additional research is warranted to further clarify and disentangle the relative contribution of genetic and environmental factors in insomnia. © Sleep Research Society 2016. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  5. Predicting protein aggregation during storage in lyophilized solids using solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS).

    Science.gov (United States)

    Moorthy, Balakrishnan S; Schultz, Steven G; Kim, Sherry G; Topp, Elizabeth M

    2014-06-02

    Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products.

  6. Hierarchical organization in aggregates of protein molecules

    DEFF Research Database (Denmark)

    Bohr, Henrik; Kyhle, Anders; Sørensen, Alexis Hammer

    1997-01-01

    of the solution and the density of protein are varied shows the existence of specific growth processes resulting in different branch-like structures. The resulting structures are strongly influenced by the shape of each protein molecule. Lysozyme and ribonuclease are found to form spherical structures...

  7. Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species.

    Science.gov (United States)

    Needham, Lisa-Maria; Weber, Judith; Fyfe, James W B; Kabia, Omaru M; Do, Dung T; Klimont, Ewa; Zhang, Yu; Rodrigues, Margarida; Dobson, Christopher M; Ghandi, Sonia; Bohndiek, Sarah E; Snaddon, Thomas N; Lee, Steven F

    2018-02-01

    Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H 2 O 2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H 2 O 2 . We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H 2 O 2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

  8. Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species

    Science.gov (United States)

    Needham, Lisa-Maria; Weber, Judith; Fyfe, James W. B.; Kabia, Omaru M.; Do, Dung T.; Klimont, Ewa; Zhang, Yu; Rodrigues, Margarida; Dobson, Christopher M.; Ghandi, Sonia; Bohndiek, Sarah E.; Snaddon, Thomas N.; Lee, Steven F.

    2018-02-01

    Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H2O2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H2O2. We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H2O2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

  9. Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats.

    Science.gov (United States)

    Gadi, Dounia; Bnouham, Mohamed; Aziz, Mohammed; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq; Legrand, Chantal; Lafeve, Françoise Fauvel; Mekhfi, Hassane

    2009-08-17

    Many cardiovascular diseases are associated with an increase in blood platelet activity. In Morocco, parsley (Petroselinum crispum, Apiaceae) is one of the medicinal herbs used to treat cardiovascular diseases such as arterial hypertension. In this study, crude aqueous extract (CAE) of parsley was evaluated for its anti-platelet activity in experimental animals on platelet aggregation in vitro and ex vivo; and on bleeding time in vivo. The in vitro aggregation was monitored after pre-incubation of platelets with CAE. The bleeding time and ex vivo aggregation were performed after oral treatment. CAE inhibited dose dependently platelet aggregation in vitro induced by thrombin, ADP, collagen and epinephrine. The oral administration of CAE (3g/kg) inhibited significantly (pparsley may be benefit in the normalization of platelet hyperactivation, in the nutritional prevention of cardiovascular diseases and are potentially interesting in the development of new prevention strategies.

  10. Interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low pH.

    Science.gov (United States)

    Ye, A; Streicher, C; Singh, H

    2011-12-01

    Whey protein beverages have been shown to be astringent at low pH. In the present study, the interactions between model whey proteins (β-lactoglobulin and lactoferrin) and human saliva in the pH range from 7 to 2 were investigated using particle size, turbidity, and ζ-potential measurements and sodium dodecyl sulfate-PAGE. The correlation between the sensory results of astringency and the physicochemical data was discussed. Strong interactions between β-lactoglobulin and salivary proteins led to an increase in the particle size and turbidity of mixtures of both unheated and heated β-lactoglobulin and human saliva at pH ∼3.4. However, the large particle size and high turbidity that occurred at pH 2.0 were the result of aggregation of human salivary proteins. The intense astringency in whey protein beverages may result from these increases in particle size and turbidity at these pH values and from the aggregation and precipitation of human salivary proteins alone at pH salivary proteins in the interaction is a key factor in the perception of astringency in whey protein beverages. At any pH, the increases in particle size and turbidity were much smaller in mixtures of lactoferrin and saliva, which suggests that aggregation and precipitation may not be the only mechanism linked to the perception of astringency in whey protein. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  11. Particle Aggregation During Fe(III) Bioreduction in Nontronite

    Science.gov (United States)

    Jaisi, D. P.; Dong, H.; Hi, Z.; Kim, J.

    2005-12-01

    This study was performed to evaluate the rate and mechanism of particle aggregation during bacterial Fe (III) reduction in different size fractions of nontronite and to investigate the role of different factors contributing to particle aggregation. To achieve this goal, microbial Fe(III) reduction experiments were performed with lactate as an electron donor, Fe(III) in nontronite as an electron acceptor, and AQDS as an electron shuttle in bicarbonate buffer using Shewanella putrefaceins CN32. These experiments were performed with and without Na- pyrophosphate as a dispersant in four size fractions of nontronite (0.12-0.22, 0.41-0.69, 0.73-0.96 and 1.42-1.8 mm). The rate of nontronite aggregation during the Fe(III) bioreduction was measured by analyzing particle size distribution using photon correlation spectroscopy (PCS) and SEM images analysis. Similarly, the changes in particle morphology during particle aggregation were determined by analyses of SEM images. Changes in particle surface charge were measured with electrophoretic mobility analyzer. The protein and carbohydrate fraction of EPS produced by cells during Fe(III) bioreduction was measured using Bradford and phenol-sulfuric acid extraction method, respectively. In the presence of the dispersant, the extent of Fe(III) bioreduction was 11.5-12.2% within the first 56 hours of the experiment. There was no measurable particle aggregation in control experiments. The PCS measurements showed that the increase in the effective diameter (95% percentile) was by a factor of 3.1 and 1.9 for particle size of 0.12-0.22 mm and 1.42-1.80 mm, respectively. The SEM image analyses also gave the similar magnitude of increase in particle size. In the absence of the dispersant, the extent of Fe(III) bioreduction was 13.4-14.5% in 56 hours of the experiment. The rate of aggregation was higher than that in the presence of the dispersant. The increase in the effective diameter (95% percentile) was by a factor of 13.6 and 4.1 for

  12. Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

    Science.gov (United States)

    Finka, Andrija; Goloubinoff, Pierre

    2013-09-01

    In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10% of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

  13. Enhanced Shear-induced Platelet Aggregation Due to Low-temperature Storage

    Science.gov (United States)

    2013-07-01

    Grewal PK, Wandall HH, Josefsson EC, Sorensen AL, Larson G, Marth JD, Hartwig JH, Hoffmeister KM. Dual roles for hepatic lectin receptors in the clearance ...PLT aggregation due to low temperature storage may be a beneficial strategy to prevent severe bleeding in trauma . P latelets (PLTs) are transfused to...prevent bleed- ing due to thrombocytopenia associated with hematologic malignancies or to manage severe blood loss during surgery or trauma . PLTs are

  14. The Heteroaryldihydropyrimidine Bay 38-7690 Induces Hepatitis B Virus Core Protein Aggregates Associated with Promyelocytic Leukemia Nuclear Bodies in Infected Cells.

    Science.gov (United States)

    Huber, Andrew D; Wolf, Jennifer J; Liu, Dandan; Gres, Anna T; Tang, Jing; Boschert, Kelsey N; Puray-Chavez, Maritza N; Pineda, Dallas L; Laughlin, Thomas G; Coonrod, Emily M; Yang, Qiongying; Ji, Juan; Kirby, Karen A; Wang, Zhengqiang; Sarafianos, Stefan G

    2018-04-25

    Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly in vitro have been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp in vitro and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level. IMPORTANCE Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and

  15. Kinetics of Single-Enzyme Reactions on Vesicles: Role of Substrate Aggregation

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2015-03-01

    Enzymatic reactions occurring in vivo on lipid membranes can be influenced by various factors including macromolecular crowding in general and substrate aggregation in particular. In academic studies, the role of these factors can experimentally be clarified by tracking single-enzyme kinetics occurring on individual lipid vesicles. To extend the conceptual basis for such experiments, we analyze herein the corresponding kinetics mathematically with emphasis on the role of substrate aggregation. In general, the aggregation may occur on different length scales. Small aggregates may e.g. contain a few proteins or peptides while large aggregates may be mesoscopic as in the case of lipid domains which can be formed in the membranes composed of different lipids. We present a kinetic model describing comprehensively the effect of aggregation of the former type on the dependence of the reaction rate on substrate membrane concentration. The results obtained with physically reasonable parameters indicate that the aggregation-related deviations from the conventional Michaelis-Menten kinetics may be appreciable. Special Issue Comments: This theoretical article is focused on single-enzyme reactions occurring in parallel with substrate aggregation on individual vesicles. This subject is related to a few Special Issue articles concerning enzyme dynamics6,7 and function8 and mathematical aspects of stochastic kinetics.9

  16. Exceptional heat stability of high protein content dispersions containing whey protein particles

    NARCIS (Netherlands)

    Saglam, D.; Venema, P.; Vries, de R.J.; Linden, van der E.

    2014-01-01

    Due to aggregation and/or gelation during thermal treatment, the amount of whey proteins that can be used in the formulation of high protein foods e.g. protein drinks, is limited. The aim of this study was to replace whey proteins with whey protein particles to increase the total protein content and

  17. An Aggregation Model for Energy Resources Management and Market Negotiations

    Directory of Open Access Journals (Sweden)

    Omid Abrishambaf

    2018-03-01

    Full Text Available Currently the use of distributed energy resources, especially renewable generation, and demand response programs are widely discussed in scientific contexts, since they are a reality in nowadays electricity markets and distribution networks. In order to benefit from these concepts, an efficient energy management system is needed to prevent energy wasting and increase profits. In this paper, an optimization based aggregation model is presented for distributed energy resources and demand response program management. This aggregation model allows different types of customers to participate in electricity market through several tariffs based demand response programs. The optimization algorithm is a mixed-integer linear problem, which focuses on minimizing operational costs of the aggregator. Moreover, the aggregation process has been done via K-Means clustering algorithm, which obtains the aggregated costs and energy of resources for remuneration. By this way, the aggregator is aware of energy available and minimum selling price in order to participate in the market with profit. A realistic low voltage distribution network has been proposed as a case study in order to test and validate the proposed methodology. This distribution network consists of 25 distributed generation units, including photovoltaic, wind and biomass generation, and 20 consumers, including residential, commercial, and industrial buildings.

  18. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Österlund, Tobias; Liu, Zihe

    2013-01-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

  19. Rydberg aggregates

    Science.gov (United States)

    Wüster, S.; Rost, J.-M.

    2018-02-01

    We review Rydberg aggregates, assemblies of a few Rydberg atoms exhibiting energy transport through collective eigenstates, considering isolated atoms or assemblies embedded within clouds of cold ground-state atoms. We classify Rydberg aggregates, and provide an overview of their possible applications as quantum simulators for phenomena from chemical or biological physics. Our main focus is on flexible Rydberg aggregates, in which atomic motion is an essential feature. In these, simultaneous control over Rydberg-Rydberg interactions, external trapping and electronic energies, allows Born-Oppenheimer surfaces for the motion of the entire aggregate to be tailored as desired. This is illustrated with theory proposals towards the demonstration of joint motion and excitation transport, conical intersections and non-adiabatic effects. Additional flexibility for quantum simulations is enabled by the use of dressed dipole-dipole interactions or the embedding of the aggregate in a cold gas or Bose-Einstein condensate environment. Finally we provide some guidance regarding the parameter regimes that are most suitable for the realization of either static or flexible Rydberg aggregates based on Li or Rb atoms. The current status of experimental progress towards enabling Rydberg aggregates is also reviewed.

  20. C9orf72 poly GA RAN-translated protein plays a key role in amyotrophic lateral sclerosis via aggregation and toxicity.

    Science.gov (United States)

    Lee, Youn-Bok; Baskaran, Pranetha; Gomez-Deza, Jorge; Chen, Han-Jou; Nishimura, Agnes L; Smith, Bradley N; Troakes, Claire; Adachi, Yoshitsugu; Stepto, Alan; Petrucelli, Leonard; Gallo, Jean-Marc; Hirth, Frank; Rogelj, Boris; Guthrie, Sarah; Shaw, Christopher E

    2017-12-15

    An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD. © The Author 2017. Published by Oxford University Press.

  1. Fibril Formation from Pea Protein and Sesequent Gel Formation

    NARCIS (Netherlands)

    Munialo, C.D.; Martin, A.H.; Linden, van der E.; Jongh, de H.H.J.

    2014-01-01

    The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20

  2. Fibril Formation from Pea Protein and Subsequent Gel Formation

    NARCIS (Netherlands)

    Munialo, XC.D.; Martin, A.H.; Linden, E. van der; Jongh, H.H.J de

    2014-01-01

    The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20

  3. Resistance of Sendai virus (HVJ) F-protein against gamma irradiation

    International Nuclear Information System (INIS)

    Hosokawa, Yasushi

    1980-01-01

    Sendai virus envelope proteins (hemagglutinin and F-protein), containing disulfide bonds in their molecules, were almost caused neither cleavage of protein molecules nor disappearance of antigenisitics by 10 5 R of γ-irradiation, but the liberated inner proteins (P, NP and M-protein) were severed into low molecules. By irradiation, hemagglutinin and F-protein of the virions were polymerized or aggregated together in such a way that the aggregate was not dissociated by SDS-2ME treatment on Slab gel electrophoresis, but in the liberated states hemagglutinin alone was aggregated and F-protein was separated with little loss of its anigenisity. From above results, the interaction between hemagglutinin and F-protein, and the utility of irradiation for isolation of F-protein were discussed. (author)

  4. Silencing the Odorant Binding Protein RferOBP1768 Reduces the Strong Preference of Palm Weevil for the Major Aggregation Pheromone Compound Ferrugineol

    Directory of Open Access Journals (Sweden)

    Binu Antony

    2018-03-01

    Full Text Available In insects, perception of the environment—food, mates, and prey—is mainly guided by chemical signals. The dynamic process of signal perception involves transport to odorant receptors (ORs by soluble secretory proteins, odorant binding proteins (OBPs, which form the first stage in the process of olfactory recognition and are analogous to lipocalin family proteins in vertebrates. Although OBPs involved in the transport of pheromones to ORs have been functionally identified in insects, there is to date no report for Coleoptera. Furthermore, there is a lack of information on olfactory perception and the molecular mechanism by which OBPs participate in the transport of aggregation pheromones. We focus on the red palm weevil (RPW Rhynchophorus ferrugineus, the most devastating quarantine pest of palm trees worldwide. In this work, we constructed libraries of all OBPs and selected antenna-specific and highly expressed OBPs for silencing through RNA interference. Aggregation pheromone compounds, 4-methyl-5-nonanol (ferrugineol and 4-methyl-5-nonanone (ferruginone, and a kairomone, ethyl acetate, were then sequentially presented to individual RPWs. The results showed that antenna-specific RferOBP1768 aids in the capture and transport of ferrugineol to ORs. Silencing of RferOBP1768, which is responsible for pheromone binding, significantly disrupted pheromone communication. Study of odorant perception in palm weevil is important because the availability of literature regarding the nature and role of olfactory signaling in this insect may reveal likely candidates representative of animal olfaction and, more generally, of molecular recognition. Knowledge of OBPs recognizing the specific pheromone ferrugineol will allow for designing biosensors for the detection of this key compound in weevil monitoring in date palm fields.

  5. The role of tears in preventing protein deposition on contact lenses

    NARCIS (Netherlands)

    Boot, N.; Kok, J.; Kijlstra, A.

    1989-01-01

    Recently the presence of a coating inhibitory factor was described in human tears which can prevent the binding of proteins to a solid phase. In these earlier studies depositions of lactoferrin and IgG onto plastic was studied. In the study described here, peroxidase conjugated albumin was used as a

  6. Fibril formation from pea protein and subsequent gel formation.

    Science.gov (United States)

    Munialo, Claire Darizu; Martin, Anneke H; van der Linden, Erik; de Jongh, Harmen H J

    2014-03-19

    The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20 h at pH 2.0. Following heating of pea proteins, it was observed that all of the proteins were hydrolyzed into peptides and that 50% of these peptides were assembled into fibrils. Changes on a structural level in pea proteins were studied using circular dichroism, transmission electron microscopy, and particle size analysis. During the fibril assembly process, an increase in aggregate size was observed, which coincided with an increase in thioflavin T binding, indicating the presence of β-sheet aggregates. Fibrils made using pea proteins were more branched and curly. Gel formation of preformed fibrils was induced by slow acidification from pH 7.0 to a final pH of around pH 5.0. The ability of pea protein-based fibrillar gels to fracture during an amplitude sweep was comparable to those of soy protein and whey protein-based fibrillar gels, although gels prepared from fibrils made using pea protein and soy protein were weaker than those of whey protein. The findings show that fibrils can be prepared from pea protein, which can be incorporated into protein-based fibrillar gels.

  7. Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease.

    Science.gov (United States)

    Harrington, Charles R; Storey, John M D; Clunas, Scott; Harrington, Kathleen A; Horsley, David; Ishaq, Ahtsham; Kemp, Steven J; Larch, Christopher P; Marshall, Colin; Nicoll, Sarah L; Rickard, Janet E; Simpson, Michael; Sinclair, James P; Storey, Lynda J; Wischik, Claude M

    2015-04-24

    Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μM) required to reverse behavioral deficits and pathology in Tau transgenic mice. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Directory of Open Access Journals (Sweden)

    Zeng YX

    2016-04-01

    stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington’s disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results. Keywords: Huntington’s disease, mutant Huntingtin, xyloketal derivatives, Caenorhabditis elegans, protein misfolding, molecular target

  9. On the characterization of intermediates in the isodesmic aggregation pathway of hen lysozyme at alkaline pH.

    Directory of Open Access Journals (Sweden)

    Vijay Kumar Ravi

    Full Text Available Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer's, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM-0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6 ± 0.6 × 10(-5 s(-1. Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.

  10. Characterizing harmful advanced glycation end-products (AGEs) and ribosylated aggregates of yellow mustard seed phytocystatin: Effects of different monosaccharides

    Science.gov (United States)

    Ahmed, Azaj; Shamsi, Anas; Bano, Bilqees

    2017-01-01

    Advanced glycation end products (AGEs) are at the core of variety of diseases ranging from diabetes to renal failure and hence gaining wide consideration. This study was aimed at characterizing the AGEs of phytocystatin isolated from mustard seeds (YMP) when incubated with different monosaccharides (glucose, ribose and mannose) using fluorescence, ultraviolet, circular dichroism (CD) spectroscopy and microscopy. Ribose was found to be the most potent glycating agent as evident by AGEs specific fluorescence and absorbance. YMP exists as a molten globule like structure on day 24 as depicted by high ANS fluorescence and altered intrinsic fluorescence. Glycated YMP as AGEs and ribose induced aggregates were observed at day 28 and 32 respectively. In our study we have also examined the anti-aggregative potential of polyphenol, resveratrol. Our results suggested the anti-aggregative behavior of resveratrol as it prevented the in vitro aggregation of YMP, although further studies are required to decode the mechanism by which resveratrol prevents the aggregation.

  11. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    2013-01-01

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  12. Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

    Directory of Open Access Journals (Sweden)

    Zhi-Xue Xu

    2016-01-01

    Full Text Available The abnormal fibrillation of human islet amyloid polypeptide (hIAPP has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−-epigallocatechin gallate (EGCG is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III/EGCG complex in molar ratio of 1 : 1 as Al(EGCG(H2O2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes.

  13. Mineral trioxide aggregate: part 2 - a review of the material aspects.

    Science.gov (United States)

    Malhotra, Neeraj; Agarwal, Antara; Mala, Kundabala

    2013-03-01

    The purpose of this two-part series is to review the composition, properties, and products of mineral trioxide aggregate (MTA) materials. PubMed and MedLine electronic databases were used to identify scientific papers from January 1991 to May 2010. Based on the selected inclusion criteria, citations were referenced from the scientific peer-reviewed dental literature. Mineral trioxide aggregate is a refined form of the parent compound, Portland cement (PC), and demonstrates a strong biocompatibility due to the high pH level and the material's ability to form hydroxyapatite. Mineral trioxide aggregate materials provide better microleakage protection than traditional endodontic materials as observed in findings from dye-leakage, fluid-filtration, protein-leakage, and bacterial penetration-leakage studies and has been recognized as a bioactive material. Various MTA commercial products are available, including gray mineral trioxide aggregate (GMTA), white mineral trioxide aggregate (WMTA), and mineral trioxide aggregate-Angelus (AMTA). Although these materials are indicated for various dental uses and applications, long-term in-vivo clinical studies are needed. Part 1 of this article highlighted and discussed the composition and characteristics of the material. Part 2 provides an overview of commercially available MTA materials.

  14. Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

    Science.gov (United States)

    Hill, Sarah J; Mordes, Daniel A; Cameron, Lisa A; Neuberg, Donna S; Landini, Serena; Eggan, Kevin; Livingston, David M

    2016-11-29

    Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

  15. Explaining the length threshold of polyglutamine aggregation

    International Nuclear Information System (INIS)

    De Los Rios, Paolo; Hafner, Marc; Pastore, Annalisa

    2012-01-01

    The existence of a length threshold, of about 35 residues, above which polyglutamine repeats can give rise to aggregation and to pathologies, is one of the hallmarks of polyglutamine neurodegenerative diseases such as Huntington’s disease. The reason why such a minimal length exists at all has remained one of the main open issues in research on the molecular origins of such classes of diseases. Following the seminal proposals of Perutz, most research has focused on the hunt for a special structure, attainable only above the minimal length, able to trigger aggregation. Such a structure has remained elusive and there is growing evidence that it might not exist at all. Here we review some basic polymer and statistical physics facts and show that the existence of a threshold is compatible with the modulation that the repeat length imposes on the association and dissociation rates of polyglutamine polypeptides to and from oligomers. In particular, their dramatically different functional dependence on the length rationalizes the very presence of a threshold and hints at the cellular processes that might be at play, in vivo, to prevent aggregation and the consequent onset of the disease. (paper)

  16. Explaining the length threshold of polyglutamine aggregation

    Science.gov (United States)

    De Los Rios, Paolo; Hafner, Marc; Pastore, Annalisa

    2012-06-01

    The existence of a length threshold, of about 35 residues, above which polyglutamine repeats can give rise to aggregation and to pathologies, is one of the hallmarks of polyglutamine neurodegenerative diseases such as Huntington’s disease. The reason why such a minimal length exists at all has remained one of the main open issues in research on the molecular origins of such classes of diseases. Following the seminal proposals of Perutz, most research has focused on the hunt for a special structure, attainable only above the minimal length, able to trigger aggregation. Such a structure has remained elusive and there is growing evidence that it might not exist at all. Here we review some basic polymer and statistical physics facts and show that the existence of a threshold is compatible with the modulation that the repeat length imposes on the association and dissociation rates of polyglutamine polypeptides to and from oligomers. In particular, their dramatically different functional dependence on the length rationalizes the very presence of a threshold and hints at the cellular processes that might be at play, in vivo, to prevent aggregation and the consequent onset of the disease.

  17. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus, Prevents Platelet Activation in Human Platelets

    Directory of Open Access Journals (Sweden)

    Ye-Ming Lee

    2012-01-01

    Full Text Available Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.. Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH● formation, and phospholipase C (PLCγ2, protein kinase C (PKC, mitogen-activated protein kinase (MAPK, and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

  18. The thermodynamics of protein aggregation reactions may underpin the enhanced metabolic efficiency associated with heterosis, some balancing selection, and the evolution of ploidy levels.

    Science.gov (United States)

    Ginn, B R

    2017-07-01

    Identifying the physical basis of heterosis (or "hybrid vigor") has remained elusive despite over a hundred years of research on the subject. The three main theories of heterosis are dominance theory, overdominance theory, and epistasis theory. Kacser and Burns (1981) identified the molecular basis of dominance, which has greatly enhanced our understanding of its importance to heterosis. This paper aims to explain how overdominance, and some features of epistasis, can similarly emerge from the molecular dynamics of proteins. Possessing multiple alleles at a gene locus results in the synthesis of different allozymes at reduced concentrations. This in turn reduces the rate at which each allozyme forms soluble oligomers, which are toxic and must be degraded, because allozymes co-aggregate at low efficiencies. The model developed in this paper can explain how heterozygosity impacts the metabolic efficiency of an organism. It can also explain why the viabilities of some inbred lines seem to decline rapidly at high inbreeding coefficients (F > 0.5), which may provide a physical basis for truncation selection for heterozygosity. Finally, the model has implications for the ploidy level of organisms. It can explain why polyploids are frequently found in environments where severe physical stresses promote the formation of soluble oligomers. The model can also explain why complex organisms, which need to synthesize aggregation-prone proteins that contain intrinsically unstructured regions (IURs) and multiple domains because they facilitate complex protein interaction networks (PINs), tend to be diploid while haploidy tends to be restricted to relatively simple organisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Nanoimaging to Prevent and Treat Alzheimer’s and Parkinson’s Diseases. Scientific/Technical report

    Energy Technology Data Exchange (ETDEWEB)

    Yuri L. Lyubchenko, PhD, DSc

    2012-12-20

    This project will develop innovative approaches to characterization of the very early stages of protein aggregation that eventually can be translated to the development of early diagnostic tools and efficient treatments for Alzheimer’s, Parkinson’s and Huntington’s diseases. Funding will be used to acquire nanoimaging technology for nanoscale imaging, manipulation and analysis of biomedical materials to develop treatments that will repair disabled proteins and cure diseases that result from protein malfunction, specifically Alzheimer’s and Parkinson’s diseases. Expected outcomes include tests for early diagnosis and therapeutic treatments for these devastating neurological diseases. To elucidate the mechanisms of protein misfolding, we will establish an extensive program of experimental studies using a broad arsenal of advanced nanoscale and traditional techniques that will be integrated with molecular-scale modeling of protein misfolding and the nucleation of aggregate structures. To identify intracellular machinery or/and multicomponent complexes critically involved in protein misfolding, we will characterize interactions between targeted proteins and specific intracellular components or metabolites that impact on protein conformational pathways leading to protein misfolding accompanied by formation of toxic aggregated morphologies. To design innovative nanotechnology tools for the control of intracellular protein misfolding and aggregation processes, we will develop a predictive molecular scale model for intracellular protein misfolding and the formation of toxic aggregates. Verified through experimental studies, the objective is to establish an enabling foundation for the engineering of novel molecular diagnostics and therapeutics for various cellular pathologies.

  20. How do eubacterial organisms manage aggregation-prone proteome? [v1; ref status: indexed, http://f1000r.es/3k3

    Directory of Open Access Journals (Sweden)

    Rishi Das Roy

    2014-06-01

    Full Text Available Eubacterial genomes vary considerably in their nucleotide composition. The percentage of genetic material constituted by guanosine and cytosine (GC nucleotides ranges from 20% to 70%.  It has been posited that GC-poor organisms are more dependent on protein folding machinery. Previous studies have ascribed this to the accumulation of mildly deleterious mutations in these organisms due to population bottlenecks. This phenomenon has been supported by protein folding simulations, which showed that proteins encoded by GC-poor organisms are more prone to aggregation than proteins encoded by GC-rich organisms. To test this proposition using a genome-wide approach, we classified different eubacterial proteomes in terms of their aggregation propensity and chaperone-dependence using multiple machine learning models. In contrast to the expected decrease in protein aggregation with an increase in GC richness, we found that the aggregation propensity of proteomes increases with GC content. A similar and even more significant correlation was obtained with the GroEL-dependence of proteomes: GC-poor proteomes have evolved to be less dependent on GroEL than GC-rich proteomes. We thus propose that a decrease in eubacterial GC content may have been selected in organisms facing proteostasis problems.

  1. Physical mechanism for biopolymers to aggregate and maintain in non-equilibrium states.

    Science.gov (United States)

    Ma, Wen-Jong; Hu, Chin-Kun

    2017-06-08

    Many human or animal diseases are related to aggregation of proteins. A viable biological organism should maintain in non-equilibrium states. How protein aggregate and why biological organisms can maintain in non-equilibrium states are not well understood. As a first step to understand such complex systems problems, we consider simple model systems containing polymer chains and solvent particles. The strength of the spring to connect two neighboring monomers in a polymer chain is controlled by a parameter s with s → ∞ for rigid-bond. The strengths of bending and torsion angle dependent interactions are controlled by a parameter s A with s A  → -∞ corresponding to no bending and torsion angle dependent interactions. We find that for very small s A , polymer chains tend to aggregate spontaneously and the trend is independent of the strength of spring. For strong springs, the speed distribution of monomers in the parallel (along the direction of the spring to connect two neighboring monomers) and perpendicular directions have different effective temperatures and such systems are in non-equilibrium states.

  2. Intra-chain organisation of hydrophobic residues controls inter-chain aggregation rates of amphiphilic polymers

    Science.gov (United States)

    Varilly, Patrick; Willard, Adam P.; Kirkegaard, Julius B.; Knowles, Tuomas P. J.; Chandler, David

    2017-04-01

    Aggregation of amphiphiles through the action of hydrophobic interactions is a common feature in soft condensed matter systems and is of particular importance in the context of biophysics as it underlies both the generation of functional biological machinery as well as the formation of pathological misassembled states of proteins. Here we explore the aggregation behaviour of amphiphilic polymers using lattice Monte Carlo calculations and show that the distribution of hydrophobic residues within the polymer sequence determines the facility with which dry/wet interfaces can be created and that such interfaces drive the aggregation process.

  3. Human stefin B normal and patho-physiological role: molecular and cellular aspects of amyloid-type aggregation of certain EPM1 mutants.

    Directory of Open Access Journals (Sweden)

    Mira ePolajnar

    2012-08-01

    Full Text Available Epilepsies are characterised by abnormal electrophysiological activity of the brain. Among various types of inherited epilepsies different epilepsy syndromes, among them progressive myoclonus epilepsies with features of ataxia and neurodegeneration, are counted. The progressive myoclonus epilepsy of type 1 (EPM1, also known as Unverricht-Lundborg disease presents with features of cerebellar atrophy and increased oxidative stress. It has been found that EPM1 is caused by mutations in human cystatin B gene (human stefin B. We first describe the role of protein aggregation in other neurodegenerative conditions. Protein aggregates appear intraneurally but are also excreted, such as is the case with senile plaques of amyloid- β (Aβ that accumulate in the brain parenchyma and vessel walls. A common characteristic of such diseases is the change of the protein conformation towards β secondary structure that accounts for the strong tendency of such proteins to aggregate and form amyloid fibrils. Second, we describe the patho-physiology of EPM1 and the normal and aberrant roles of stefin B in a mouse model of the disease. Furthermore, we discuss how the increased protein aggregation observed with some of the mutants of human stefin B may relate to the neurodegeneration that occurs in rare EPM1 patients. Our hypothesis (Ceru et al., 2005 states that some of the EPM1 mutants of human stefin B may undergo aggregation in neural cells, thus gaining additional toxic function (apart from loss of normal function. Our in vitro experiments thus far have confirmed that 4 mutants undergo increased aggregation relative to the wild-type protein. It has been shown that the R68X mutant forms amyloid-fibrils very rapidly, even at neutral pH and forms perinuclear inclusions, whereas the G4R mutant exhibits a prolonged lag phase, during which the toxic prefibrillar aggregates accumulate and are scattered more diffusely over the cytoplasm. Initial experiments on the G50E

  4. Chaperone-protease networks in mitochondrial protein homeostasis.

    Science.gov (United States)

    Voos, Wolfgang

    2013-02-01

    As essential organelles, mitochondria are intimately integrated into the metabolism of a eukaryotic cell. The maintenance of the functional integrity of the mitochondrial proteome, also termed protein homeostasis, is facing many challenges both under normal and pathological conditions. First, since mitochondria are derived from bacterial ancestor cells, the proteins in this endosymbiotic organelle have a mixed origin. Only a few proteins are encoded on the mitochondrial genome, most genes for mitochondrial proteins reside in the nuclear genome of the host cell. This distribution requires a complex biogenesis of mitochondrial proteins, which are mostly synthesized in the cytosol and need to be imported into the organelle. Mitochondrial protein biogenesis usually therefore comprises complex folding and assembly processes to reach an enzymatically active state. In addition, specific protein quality control (PQC) processes avoid an accumulation of damaged or surplus polypeptides. Mitochondrial protein homeostasis is based on endogenous enzymatic components comprising a diverse set of chaperones and proteases that form an interconnected functional network. This review describes the different types of mitochondrial proteins with chaperone functions and covers the current knowledge of their roles in protein biogenesis, folding, proteolytic removal and prevention of aggregation, the principal reactions of protein homeostasis. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation.

    Science.gov (United States)

    Jang, Ja-Young; Kim, Tae-Su; Cai, Jingmei; Kim, Jihyun; Kim, Youngeun; Shin, Kyungha; Kim, Kwang Sei; Park, Sung Kyeong; Lee, Sung-Pyo; Choi, Ehn-Kyoung; Rhee, Man Hee; Kim, Yun-Bae

    2013-12-01

    The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-dependent manner. Rats were orally administered with nattokinase for 1 week, and their carotid arteries were exposed. Arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Nattokinase delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 160 mg/kg. In addition, a high dose (500 mg/kg) of nattokinase fully prevented the occlusion, as achieved with aspirin (30 mg/kg). The results indicate that nattokinase extracted from fermented soybean inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is suggested that nattokinase could be a good candidate without adverse effects for the improvement of blood flow.

  6. S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding.

    Science.gov (United States)

    Nakamura, T; Lipton, S A

    2007-07-01

    Although activation of glutamate receptors is essential for normal brain function, excessive activity leads to a form of neurotoxicity known as excitotoxicity. Key mediators of excitotoxic damage include overactivation of N-methyl-D-aspartate (NMDA) receptors, resulting in excessive Ca(2+) influx with production of free radicals and other injurious pathways. Overproduction of free radical nitric oxide (NO) contributes to acute and chronic neurodegenerative disorders. NO can react with cysteine thiol groups to form S-nitrosothiols and thus change protein function. S-nitrosylation can result in neuroprotective or neurodestructive consequences depending on the protein involved. Many neurodegenerative diseases manifest conformational changes in proteins that result in misfolding and aggregation. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. Molecular chaperones - such as protein-disulfide isomerase, glucose-regulated protein 78, and heat-shock proteins - can provide neuroprotection by facilitating proper protein folding. Here, we review the effect of S-nitrosylation on protein function under excitotoxic conditions, and present evidence that NO contributes to degenerative conditions by S-nitrosylating-specific chaperones that would otherwise prevent accumulation of misfolded proteins and neuronal cell death. In contrast, we also review therapeutics that can abrogate excitotoxic damage by preventing excessive NMDA receptor activity, in part via S-nitrosylation of this receptor to curtail excessive activity.

  7. Mutant Huntingtin Gene-Dose Impacts on Aggregate Deposition, DARPP32 Expression and Neuroinflammation in HdhQ150 Mice

    Science.gov (United States)

    Young, Douglas; Mayer, Franziska; Vidotto, Nella; Schweizer, Tatjana; Berth, Ramon; Abramowski, Dorothee; Shimshek, Derya R.; van der Putten, P. Herman; Schmid, Peter

    2013-01-01

    Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model. PMID:24086450

  8. Hydrodynamic effects on β-amyloid (16-22) peptide aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Chiricotto, Mara; Sterpone, Fabio, E-mail: fabio.sterpone@ibpc.fr [Laboratoire de Biochimie Théorique, IBPC, CNRS UPR9080, University Paris Diderot, Sorbonne Paris Cité, 13 rue Pierre et Marie Curie, 75005 Paris (France); Melchionna, Simone [CNR-ISC, Institute for Complex System, Consiglio Nazionale delle Ricerche, Rome (Italy); Derreumaux, Philippe [Laboratoire de Biochimie Théorique, IBPC, CNRS UPR9080, University Paris Diderot, Sorbonne Paris Cité, 13 rue Pierre et Marie Curie, 75005 Paris (France); IUF, Institut Universitaire de France, Boulevard Saint Michel, 75005 Paris (France)

    2016-07-21

    Computer simulations based on simplified representations are routinely used to explore the early steps of amyloid aggregation. However, when protein models with implicit solvent are employed, these simulations miss the effect of solvent induced correlations on the aggregation kinetics and lifetimes of metastable states. In this work, we apply the multi-scale Lattice Boltzmann Molecular Dynamics technique (LBMD) to investigate the initial aggregation phases of the amyloid Aβ{sub 16−22} peptide. LBMD includes naturally hydrodynamic interactions (HIs) via a kinetic on-lattice representation of the fluid kinetics. The peptides are represented by the flexible OPEP coarse-grained force field. First, we have tuned the essential parameters that control the coupling between the molecular and fluid evolutions in order to reproduce the experimental diffusivity of elementary species. The method is then deployed to investigate the effect of HIs on the aggregation of 100 and 1000 Aβ{sub 16−22} peptides. We show that HIs clearly impact the aggregation process and the fluctuations of the oligomer sizes by favouring the fusion and exchange dynamics of oligomers between aggregates. HIs also guide the growth of the leading largest cluster. For the 100 Aβ{sub 16−22} peptide system, the simulation of ∼300 ns allowed us to observe the transition from ellipsoidal assemblies to an elongated and slightly twisted aggregate involving almost the totality of the peptides. For the 1000 Aβ{sub 16−22} peptides, a system of unprecedented size at quasi-atomistic resolution, we were able to explore a branched disordered fibril-like structure that has never been described by other computer simulations, but has been observed experimentally.

  9. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

    Science.gov (United States)

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E.; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R.; Ross, Ian; Parton, Robert G.; Böcking, Till; Gambin, Yann

    2016-01-01

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils. PMID:27892477

  10. Hypochlorous and peracetic acid induced oxidation of dairy proteins.

    Science.gov (United States)

    Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

    2011-02-09

    Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

  11. Marine Synechococcus Aggregation

    Science.gov (United States)

    Neuer, S.; Deng, W.; Cruz, B. N.; Monks, L.

    2016-02-01

    Cyanobacteria are considered to play an important role in the oceanic biological carbon pump, especially in oligotrophic regions. But as single cells are too small to sink, their carbon export has to be mediated by aggregate formation and possible consumption by zooplankton producing sinking fecal pellets. Here we report results on the aggregation of the ubiquitous marine pico-cyanobacterium Synechococcus as a model organism. We first investigated the mechanism behind such aggregation by studying the potential role of transparent exopolymeric particles (TEP) and the effects of nutrient (nitrogen or phosphorus) limitation on the TEP production and aggregate formation of these pico-cyanobacteria. We further studied the aggregation and subsequent settling in roller tanks and investigated the effects of the clays kaolinite and bentonite in a series of concentrations. Our results show that despite of the lowered growth rates, Synechococcus in nutrient limited cultures had larger cell-normalized TEP production, formed a greater volume of aggregates, and resulted in higher settling velocities compared to results from replete cultures. In addition, we found that despite their small size and lack of natural ballasting minerals, Synechococcus cells could still form aggregates and sink at measureable velocities in seawater. Clay minerals increased the number and reduced the size of aggregates, and their ballasting effects increased the sinking velocity and carbon export potential of aggregates. In comparison with the Synechococcus, we will also present results of the aggregation of the pico-cyanobacterium Prochlorococcus in roller tanks. These results contribute to our understanding in the physiology of marine Synechococcus as well as their role in the ecology and biogeochemistry in oligotrophic oceans.

  12. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding.

    Science.gov (United States)

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.

  13. Mechanisms of plastein formation, and prospective food and nutraceutical applications of the peptide aggregates

    Directory of Open Access Journals (Sweden)

    Min Gong

    2015-03-01

    Full Text Available Plastein is a protease-induced peptide aggregate with prospective application in enhancing the nutritional quality of proteins and debittering protein hydrolysates. These properties are yet to be applied in product development possibly due to economic considerations (production cost vs. product yields. This paper reviews currently proposed mechanisms of plastein formation including condensation, transpeptidation and physical interaction of aggregating peptides. Emerging findings indicate that plastein possesses bioactivities, thereby expanding its prospective application. The role of proteases in inducing peptide interaction in plastein remains unclear. Understanding the protease function will facilitate the development of efficient proteases and scalable industrial processes for plastein production.

  14. Antioxidant Treatment and Induction of Autophagy Cooperate to Reduce Desmin Aggregation in a Cellular Model of Desminopathy.

    Directory of Open Access Journals (Sweden)

    Eva Cabet

    Full Text Available Desminopathies, a subgroup of myofibrillar myopathies (MFMs, the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the desmin gene (DES, encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant DES. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes, antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.

  15. Paradoxical Acceleration of Dithiothreitol-Induced Aggregation of Insulin in the Presence of a Chaperone

    Directory of Open Access Journals (Sweden)

    Boris Kurganov

    2010-11-01

    Full Text Available The kinetics of dithiothreitol (DTT-induced aggregation of human recombinant insulin and the effect of α-crystallin, a representative of the family of small heat shock proteins, on the aggregation process have been studied using dynamic light scattering technique. Analysis of the distribution of the particles by size measured in the course of aggregation showed that the initial stage of the aggregation process was the stage of formation of the start aggregates with a hydrodynamic radius (Rh of about 90 nm. When studying the effect of α-crystallin on the rate of DTT-induced aggregation of insulin, it was demonstrated that low concentrations of α-crystallin dramatically accelerated the aggregation process, whereas high concentrations of α-crystallin suppressed insulin aggregation. In the present study, at the molar stoichiometric ratio (insulin:α-crystallin less than 1:0.5, a pronounced accelerating effect of α-crystallin was observed; whereas a ratio exceeding the value of 1:0.6 caused suppression of insulin aggregation. The mechanisms underlying the dual effect of α-crystallin have been proposed. It is assumed that heterogeneous nucleation occurring on the surface of the α-crystallin particle plays the key role in the paradoxical acceleration of insulin aggregation by α-crystallin that may provide an alternative biologically significant pathway of the aggregation process.

  16. Mammalian amyloidogenic proteins promote prion nucleation in yeast.

    Science.gov (United States)

    Chandramowlishwaran, Pavithra; Sun, Meng; Casey, Kristin L; Romanyuk, Andrey V; Grizel, Anastasiya V; Sopova, Julia V; Rubel, Aleksandr A; Nussbaum-Krammer, Carmen; Vorberg, Ina M; Chernoff, Yury O

    2018-03-02

    Fibrous cross-β aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-β (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Aggregation-induced emission: phenomenon, mechanism and applications.

    Science.gov (United States)

    Hong, Yuning; Lam, Jacky W Y; Tang, Ben Zhong

    2009-08-07

    It is textbook knowledge that chromophore aggregation generally quenches light emission. In this feature article, we give an account on how we observed an opposite phenomenon termed aggregation-induced emission (AIE) and identified the restriction of intramolecular rotation as a main cause for the AIE effect. Based on the mechanistic understanding, we developed a series of new fluorescent and phosphorescent AIE systems with emission colours covering the entire visible spectral region and luminescence quantum yields up to unity. We explored high-tech applications of the AIE luminogens as, for example, fluorescence sensors (for explosive, ion, pH, temperature, viscosity, pressure, etc.), biological probes (for protein, DNA, RNA, sugar, phospholipid, etc.), immunoassay markers, PAGE visualization agents, polarized light emitters, monitors for layer-by-layer assembly, reporters for micelle formation, multistimuli-responsive nanomaterials, and active layers in the fabrication of organic light-emitting diodes.

  18. Defining and systematic analyses of aggregation indices to evaluate degree of calcium oxalate crystal aggregation

    Science.gov (United States)

    Chaiyarit, Sakdithep; Thongboonkerd, Visith

    2017-12-01

    Crystal aggregation is one of the most crucial steps in kidney stone pathogenesis. However, previous studies of crystal aggregation were rarely done and quantitative analysis of aggregation degree was handicapped by a lack of the standard measurement. We thus performed an in vitro assay to generate aggregation of calcium oxalate monohydrate (COM) crystals with various concentrations (25-800 µg/ml) in saturated aggregation buffer. The crystal aggregates were analyzed by microscopic examination, UV-visible spectrophotometry, and GraphPad Prism6 software to define a total of 12 aggregation indices (including number of aggregates, aggregated mass index, optical density, aggregation coefficient, span, number of aggregates at plateau time-point, aggregated area index, aggregated diameter index, aggregated symmetry index, time constant, half-life, and rate constant). The data showed linear correlation between crystal concentration and almost all of these indices, except only for rate constant. Among these, number of aggregates provided the greatest regression coefficient (r=0.997; pr=0.993; pr=‑0.993; pr=0.991; p<0.001 for both). These five indices are thus recommended as the most appropriate indices for quantitative analysis of COM crystal aggregation in vitro.

  19. Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

    Science.gov (United States)

    Reiz, Bela; Li, Liang

    2010-09-01

    Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

  20. Sustainable aggregates production : green applications for aggregate by-products.

    Science.gov (United States)

    2015-06-01

    Increased emphasis in the construction industry on sustainability and recycling requires production of : aggregate gradations with lower dust (cleaner aggregates) and smaller maximum sizeshence, increased : amount of quarry by-products (QBs). QBs ...

  1. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    International Nuclear Information System (INIS)

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E.

    1987-01-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of [ 3 H]serotonin, or alter the dose-responsive binding of 125 I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF

  2. Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils.

    Science.gov (United States)

    Tsujimura, Atsushi; Taguchi, Katsutoshi; Watanabe, Yoshihisa; Tatebe, Harutsugu; Tokuda, Takahiko; Mizuno, Toshiki; Tanaka, Masaki

    2015-01-01

    The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinson's disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils. Copyright © 2015. Published by Elsevier Inc.

  3. Aggregate stability and soil degradation in the tropics

    International Nuclear Information System (INIS)

    Mbagwu, J.S.C.

    2004-01-01

    Aggregate stability is a measure of the structural stability of soils. Factors that influence aggregate stability are important in evaluating the ease with which soils erode by water and/or wind, the potential of soils to crust and/or seal, soil permeability, quasi-steady state infiltration rates and seedling emergence and in predicting the capacity of soils to sustain long-term crop production. Aggregate stability of soils can be measured by the wet-sieving or raindrop techniques. A reduction in soil aggregate stability implies an increase in soil degradation. Hence aggregate stability and soil degradation are interwoven. The measures used can either be preventive or remedial. Preventive practices minimize the chances of soil degradation occurring or the magnitude or severity of the damage when the degradation manifests. These include in Nigeria, (i) manuring and mulching, (ii) planted fallows and cover crops, (iii) sustainable farming systems, (iv) adequate rotations, (v) home gardens or compound farms, (vi) alley cropping and related agro forestry systems, and (vii) chemical fertilizers which are mainly remedial measures. Because of alterations in soil properties that affect particular land uses, soils may degrade for one crop (maize rather sorghum). As long as some land use is possible soil degradation is not always an absolute concept. Decline in agricultural productivity should be evaluated in terms of inputs such as fertilizer use, water management and tillage methods. We can alleviate some types of soil degradation by use of micronutrients, inorganic fertilizers and organic residues. Soil that responds to management practices cannot be said to be degraded. Since crop growth depends on weather, degraded soils may be more sensitive to harsh weather (e.g. drought, temperature) than undegraded soils. A soil is degraded if its productivity falls below the economic threshold even under favourable weather conditions or with judicious inputs. All human

  4. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    Science.gov (United States)

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  5. Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

    Directory of Open Access Journals (Sweden)

    Hiroshi Yamaguchi

    2014-02-01

    Full Text Available Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  6. Proteins in Soy Might Have a Higher Role in Cancer Prevention than Previously Expected: Soybean Protein Fractions Are More Effective MMP-9 Inhibitors Than Non-Protein Fractions, Even in Cooked Seeds

    Directory of Open Access Journals (Sweden)

    Ana Lima

    2017-02-01

    Full Text Available The search for anticancer MMP-9 inhibitors (MMPIs in food products has become a major goal for research. MMPIs in soy have been related only to saponins and isoflavones, but recently, low specific protein fractions in soybeans were shown to reduce MMP-9 activity as well. The present work aimed at comparing the MMPI potential of protein fractions (P and non-protein fractions (NP isolated from soybean seeds, before and after soaking and cooking, mimicking dietary exposures. Reverse and substrate zymography, as well as a fluoregenic DQ gelatin assay were used to evaluate MMP-9 activities. Colon cancer cell migration and proliferation was also tested in HT29 cells. Regarding MMP-9 inhibition, proteins in soy presented IC50 values 100 times lower than non-protein extracts, and remained active after cooking, suggesting that proteins may be more effective MMP-9 inhibitors than non-protein compounds. Using the determined IC50 concentrations, NP fractions were able to induce higher inhibitions of HT29 cell migration and proliferation, but not through MMP-9 inhibition, whilst protein fractions were shown to specifically inhibit MMP-9 activity. Overall, our results show that protein fractions in soybeans might have a higher role in soy-related cancer prevention as MMPIs than previously expected. Being nontoxic and active at lower concentrations, the discovery of these heat-resistant specific MMPI proteins in soy can be of significant importance for cancer preventive diets, particularly considering the increasing use of soy proteins in food products and the controversy around isoflavones amongst consumers.

  7. ALS-linked mutant SOD1 proteins promote Aβ aggregates in ALS through direct interaction with Aβ.

    Science.gov (United States)

    Jang, Ja-Young; Cho, Hyungmin; Park, Hye-Yoon; Rhim, Hyangshuk; Kang, Seongman

    2017-11-04

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons. Aggregation of ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) is a hallmark of a subset of familial ALS (fALS). Recently, intracellular amyloid-β (Aβ) is detected in motor neurons of both sporadic and familial ALS. We have previously shown that intracellular Aβ specifically interacts with G93A, an ALS-linked SOD1 mutant. However, little is known about the pathological and biological effect of this interaction in neurons. In this study, we have demonstrated that the Aβ-binding region is exposed on the SOD1 surface through the conformational changes due to misfolding of SOD1. Interestingly, we found that the intracellular aggregation of Aβ is enhanced through the direct interaction of Aβ with the Aβ-binding region exposed to misfolded SOD1. Ultimately, increased Aβ aggregation by this interaction promotes neuronal cell death. Consistent with this result, Aβ aggregates was three-fold higher in the brains of G93A transgenic mice than those of non Tg. Our study provides the first direct evidence that Aβ, an AD-linked factor, is associated to the pathogenesis of ALS and provides molecular clues to understand common aggregation mechanisms in the pathogenesis of neurodegenerative diseases. Furthermore, it will provide new insights into the development of therapeutic approaches for ALS. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. From Preventive to Permissive Checks

    DEFF Research Database (Denmark)

    Sharp, Paul Richard; Weisdorf, Jacob Louis

    The Malthusian "preventive check" mechanism has been well documented for pre-industrial England through evidence for a negativcorrelation between the marriage rate and the price of wheat. Other literature, however, speculates that the correlation was in fact positive from the early nineteenth cen...... shows that this result is not in fact inconsistent with a stylized Malthusian mechanism, and can be understood within the context of an increasing dominance of shocks to aggregate demand rather than to aggregate supply...

  9. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

    Science.gov (United States)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    2006-05-01

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

  10. Influence of Aggregate Wettability with Different Lithology Aggregates on Concrete Drying Shrinkage

    Directory of Open Access Journals (Sweden)

    Yuanchen Guo

    2015-01-01

    Full Text Available The correlation of the wettability of different lithology aggregates and the drying shrinkage of concrete materials is studied, and some influential factors such as wettability and wetting angle are analyzed. A mercury porosimeter is used to measure the porosities of different lithology aggregates accurately, and the pore size ranges that significantly affect the drying shrinkage of different lithology aggregate concretes are confirmed. The pore distribution curve of the different coarse aggregates is also measured through a statistical method, and the contact angle of different coarse aggregates and concrete is calculated according to the linear fitting relationship. Research shows that concrete strength is determined by aggregate strength. Aggregate wettability is not directly correlated with concrete strength, but wettability significantly affects concrete drying shrinkage. In all types’ pores, the greatest impacts on wettability are capillary pores and gel pores, especially for the pores of the size locating 2.5–50 nm and 50–100 nm two ranges.

  11. Beyond the role of dietary protein and amino acids in the prevention of diet-induced obesity.

    Science.gov (United States)

    Petzke, Klaus J; Freudenberg, Anne; Klaus, Susanne

    2014-01-20

    High-protein diets have been shown to prevent the development of diet-induced obesity and can improve associated metabolic disorders in mice. Dietary leucine supplementation can partially mimic this effect. However, the molecular mechanisms triggering these preventive effects remain to be satisfactorily explained. Here we review studies showing a connection between high protein or total amino nitrogen intake and obligatory water intake. High amino nitrogen intake may possibly lower lipid storage, and prevent insulin resistance. Suggestions are made for further systematical studies to explore the relationship between water consumption, satiety, and energy expenditure. Moreover, these examinations should better distinguish between leucine-specific and unspecific effects. Research in this field can provide important information to justify dietary recommendations and strategies in promoting long-term weight loss and may help to reduce health problems associated with the comorbidities of obesity.

  12. RELATIONSHIPS BETWEEN SOIL MICROBIAL BIOMASS, AGGREGATE STABILITY AND AGGREGATE ASSOCIATED-C: A MECHANISTIC APPROACH

    Directory of Open Access Journals (Sweden)

    Patrizia Guidi

    2014-01-01

    Full Text Available For the identification of C pools involved in soil aggregation, a physically-based aggregate fractionation was proposed, and  additional pretreatments were used in the measurement of the 1-2 mm aggregate stability in order to elucidate the relevance of the role of soil microorganisms with respect to the different aggregate breakdown mechanisms. The study was carried out on three clay loam Regosols, developed on calcareous shales, known history of organic cultivation.Our results showed that the soil C pool controlling the process of stabilisation of aggregates was related to the microbial community. We identified the resistance to fast wetting as the major mechanism of aggregate stability driven by microorganims. The plausible hypothesis is that organic farming promotes fungi growth, improving water repellency of soil aggregates by fungal hydrophobic substances. By contrast, we failed in the identification of C pools controlling the formation of aggregates, probably because of the disturbance of mechanical tillage which contributes to the breakdown of soil aggregates.The physically-based aggregate fractionation proposed in this study resulted useful in the  mechanistically understanding of the role of microorganisms in soil aggregation and it might be suggested for studying the impact of management on C pools, aggregates properties and their relationships in agricultural soils.

  13. Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

    Science.gov (United States)

    2011-01-01

    Background Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules ("salt-in" strategy) or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy). Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs. Results The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and may help the structure preventing aggregation and/or loss of function in hypersaline environments. Conclusions Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules. PMID:22192175

  14. Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

    Directory of Open Access Journals (Sweden)

    Siglioccolo Alessandro

    2011-12-01

    Full Text Available Abstract Background Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules ("salt-in" strategy or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy. Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs. Results The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and may help the structure preventing aggregation and/or loss of function in hypersaline environments. Conclusions Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules.

  15. Proteomic screening for amyloid proteins.

    Directory of Open Access Journals (Sweden)

    Anton A Nizhnikov

    Full Text Available Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

  16. Glycerol-Induced Aggregation of the Oligomeric L-Asparaginase II from E. coli Monitored with ATR-FTIR

    Directory of Open Access Journals (Sweden)

    Koba Adeishvili

    2001-06-01

    Full Text Available Abstract: In this paper attenuated total reflectance Fourier transform infrared spectroscopy has been employed for the study of the structural composition of aggregates of the oligomeric L-asparaginase II from E.coli formed in the presence of glycerol after the induction of refolding of the protein. Apart from the perfect coincidence of the secondary structure composition of EcA2 as determined by FTIR and crystallography [1], it has also been shown that secondary structure of protein in asparaginase deposits is similar to that of the native conformation: 20.7% extended, 22.3% disordered, 31.4% helix and 25.6% turn/bend/β sheet. Certain structural similarities in the range of experimental error was observed for all three protein deposits presented in this paper, indicating a common structural basis for the composition of this types of aggregates. It is concluded that in the constitution of such precipitates, a partially folded (molten globule like state(s is involved, and its stabilisation is a key factor leading to the aggregation. The results presented in this paper might serve to be a good explanation and an excellent basis for the fundamental theory of protein (oligomers precipitation by osmotic substances.

  17. Spherical aggregates composed of gold nanoparticles

    International Nuclear Information System (INIS)

    Chen, C-C; Kuo, P-L; Cheng, Y-C

    2009-01-01

    Alkylated triethylenetetramine (C12E3) was synthesized and used as both a reductant in the preparation of gold nanoparticles by the reduction of HAuCl 4 and a stabilizer in the subsequent self-assembly of the gold nanoparticles. In acidic aqueous solution, spherical aggregates (with a diameter of about 202 ± 22 nm) of gold nanoparticles (with the mean diameter of ∼18.7 nm) were formed. The anion-induced ammonium adsorption of the alkylated amines on the gold nanoparticles was considered to provide the electrostatic repulsion and steric hindrance between the gold nanoparticles, which constituted the barrier that prevented the individual particles from coagulating. However, as the amino groups became deprotonated with increasing pH, the ammonium adsorption was weakened, and the amino groups were desorbed from the gold surface, resulting in discrete gold particles. The results indicate that the morphology of the reduced gold nanoparticles is controllable through pH-'tunable' aggregation under the mediation of the amino groups of alkylated amine to create spherical microstructures.

  18. Uniform deposition of water-soluble CdS quantum dots on TiO2 nanotube arrays by cyclic voltammetric electrodeposition: Effectively prevent aggregation and enhance visible-light photocatalytic activity

    International Nuclear Information System (INIS)

    Zhang, Xiaojiao; Lin, Shiwei; Liao, Jianjun; Pan, Nengqian; Li, Danhong; Cao, Xiankun; Li, Jianbao

    2013-01-01

    Highlights: • Water-soluble CdS QDs were deposited on the TNTAs by DC electrodeposition, CV electrodeposition, and SILAR. • The CV method could effectively prevent the aggregation and uniformly deposit CdS QDs onto the TNTAs. • The CTAB/CdS/TNTAs prepared by the CV method exhibited superior photoelectrical properties and photocatalytic activity. -- Abstract: Water-soluble CdS quantum dots (QDs) covered with cationic surfactant-cetyltrimethylammonium bromide (CTAB) were deposited on the highly ordered TiO 2 nanotube arrays (TNTAs) by various methods, such as direct current (DC) electrodeposition, cyclic voltammetric (CV) electrodeposition, and successive ionic layer adsorption reaction (SILAR). The morphology measurements show that CTAB capping could well control the QD size and the CV method could effectively prevent the nanoparticle aggregation and uniformly deposit QDs onto TNTAs. Among all the deposition methods studied, the sample prepared by the CV method possesses superior photoelectrical properties and photocatalytic activity. A maximum photoconversion efficiency of 2.81% is achieved for the CdS/TNTAs prepared by CV electrodeposition, which exhibits about 17 times enhancement over the efficiency of the sample prepared by DC electrodeposition. And the photocatalytic degradation of methyl orange under visible-light irradiation demonstrates that the rate constant of the sample prepared by the CV method is almost seven times of that of the untreated TNTAs. Moreover, the underlying mechanism for the improving properties has been discussed

  19. RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

    Science.gov (United States)

    Lim, C J; Spiegelman, G B; Weeks, G

    2001-08-15

    Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

  20. Heat Shock Proteins: A Review of the Molecular Chaperones for Plant Immunity.

    Science.gov (United States)

    Park, Chang-Jin; Seo, Young-Su

    2015-12-01

    As sessile organisms, plants are exposed to persistently changing stresses and have to be able to interpret and respond to them. The stresses, drought, salinity, chemicals, cold and hot temperatures, and various pathogen attacks have interconnected effects on plants, resulting in the disruption of protein homeostasis. Maintenance of proteins in their functional native conformations and preventing aggregation of non-native proteins are important for cell survival under stress. Heat shock proteins (HSPs) functioning as molecular chaperones are the key components responsible for protein folding, assembly, translocation, and degradation under stress conditions and in many normal cellular processes. Plants respond to pathogen invasion using two different innate immune responses mediated by pattern recognition receptors (PRRs) or resistance (R) proteins. HSPs play an indispensable role as molecular chaperones in the quality control of plasma membrane-resident PRRs and intracellular R proteins against potential invaders. Here, we specifically discuss the functional involvement of cytosolic and endoplasmic reticulum (ER) HSPs/chaperones in plant immunity to obtain an integrated understanding of the immune responses in plant cells.

  1. Heat Shock Proteins: A Review of the Molecular Chaperones for Plant Immunity

    Directory of Open Access Journals (Sweden)

    Chang-Jin Park

    2015-12-01

    Full Text Available As sessile organisms, plants are exposed to persistently changing stresses and have to be able to interpret and respond to them. The stresses, drought, salinity, chemicals, cold and hot temperatures, and various pathogen attacks have interconnected effects on plants, resulting in the disruption of protein homeostasis. Maintenance of proteins in their functional native conformations and preventing aggregation of non-native proteins are important for cell survival under stress. Heat shock proteins (HSPs functioning as molecular chaperones are the key components responsible for protein folding, assembly, translocation, and degradation under stress conditions and in many normal cellular processes. Plants respond to pathogen invasion using two different innate immune responses mediated by pattern recognition receptors (PRRs or resistance (R proteins. HSPs play an indispensable role as molecular chaperones in the quality control of plasma membrane-resident PRRs and intracellular R proteins against potential invaders. Here, we specifically discuss the functional involvement of cytosolic and endoplasmic reticulum (ER HSPs/chaperones in plant immunity to obtain an integrated understanding of the immune responses in plant cells.

  2. Occurrence State and Molecular Structure Analysis of Extracellular Proteins with Implications on the Dewaterability of Waste-Activated Sludge.

    Science.gov (United States)

    Wu, Boran; Ni, Bing-Jie; Horvat, Kristine; Song, Liyan; Chai, Xiaoli; Dai, Xiaohu; Mahajan, Devinder

    2017-08-15

    The occurrence state and molecular structure of extracellular proteins were analyzed to reveal the influencing factors on the water-holding capacities of protein-like substances in waste-activated sludge (WAS). The gelation process of extracellular proteins verified that advanced oxidation processes (AOPs) for WAS dewaterability improvement eliminated the water affinity of extracellular proteins and prevented these macromolecules from forming stable colloidal aggregates. Isobaric tags for relative and absolute quantitation proteomics identified that most of the extracellular proteins were originally derived from the intracellular part and the proteins originally located in the extracellular part were mainly membrane-associated. The main mechanism of extracellular protein transformation during AOPs could be represented by the damage of the membrane or related external encapsulating structure and the release of intracellular substances. For the selected representative extracellular proteins, the strong correlation (R 2 > 0.97, p proteins on the interstitial water removal from WAS.

  3. Aggregate versus Individual-Level Sexual Behavior Assessment: How Much Detail Is Needed to Accurately Estimate HIV/STI Risk?

    Science.gov (United States)

    Pinkerton, Steven D.; Galletly, Carol L.; McAuliffe, Timothy L.; DiFranceisco, Wayne; Raymond, H. Fisher; Chesson, Harrell W.

    2010-01-01

    The sexual behaviors of HIV/sexually transmitted infection (STI) prevention intervention participants can be assessed on a partner-by-partner basis: in aggregate (i.e., total numbers of sex acts, collapsed across partners) or using a combination of these two methods (e.g., assessing five partners in detail and any remaining partners in aggregate).…

  4. Human surfactant protein A2 gene mutations impair dimmer/trimer assembly leading to deficiency in protein sialylation and secretion.

    Directory of Open Access Journals (Sweden)

    Yi Song

    Full Text Available Surfactant protein A2 (SP-A2 plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA, a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2.

  5. ROCK and PRK-2 Mediate the Inhibitory Effect of Y-27632 on Polyglutamine Aggregation

    Science.gov (United States)

    Shao, Jieya; Welch, William J.; Diamond, Marc I.

    2009-01-01

    Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, H-1077, HA-1152), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal. PMID:18423405

  6. Nanoparticle Surface Specific Adsorption of Zein and Its Self-assembled Behavior of Nanocubes Formation in Relation to On-Off SERS: Understanding Morphology Control of Protein Aggregates.

    Science.gov (United States)

    Navdeep; Banipal, Tarlok Singh; Kaur, Gurinder; Bakshi, Mandeep Singh

    2016-01-27

    Zein, an industrially important protein, is characterized in terms of its food and pharmaceutical coating applications by using surface enhanced Raman spectroscopy (SERS) on Au, Ag, and PbS nanoparticles (NPs). Its specific surface adsorption behavior on Ag NPs produced self-assembled zein nanocubes which demonstrated on and off SERS activity. Both SERS characterization as well as nanocube formation of zein helped us to understand the complex protein aggregation behavior in shape controlled morphologies, a process with significant ramifications in protein crystallization to achieve ordered morphologies. Interestingly, nanocube formation was promoted in the presence of Ag rather than Au or PbS NPs under in situ synthesis and discussed in terms of specific adsorption. Zein fingerprinting was much more clear and enhanced on Au surface in comparison to Ag while PbS did not demonstrate SERS due to its semiconducting nature.

  7. The influence of N-terminal acetylation on micelle-induced conformational changes and aggregation of α-Synuclein.

    Directory of Open Access Journals (Sweden)

    David Ruzafa

    Full Text Available The biological function of α-Synuclein has been related to binding to lipids and membranes but these interactions can also mediate α-Synuclein aggregation, which is associated to Parkinson's disease and other neuropathologies. In brain tissue α-Synuclein is constitutively N-acetylated, a modification that plays an important role in its conformational propensity, lipid and membrane binding, and aggregation propensity. We studied the interactions of the lipid-mimetic SDS with N-acetylated and non-acetylated α-Synuclein, as well as their early-onset Parkinson's disease variants A30P, E46K and A53T. At low SDS/protein ratios α-Synuclein forms oligomeric complexes with SDS micelles with relatively low α-helical structure. These micellar oligomers can efficiently nucleate aggregation of monomeric α-Synuclein, with successive formation of oligomers, protofibrils, curly fibrils and mature amyloid fibrils. N-acetylation reduces considerably the rate of aggregation of WT α-Synuclein. However, in presence of any of the early-onset Parkinson's disease mutations the protective effect of N-acetylation against micelle-induced aggregation becomes impaired. At higher SDS/protein ratios, N-acetylation favors another conformational transition, in which a second type of α-helix-rich, non-aggregating oligomers become stabilized. Once again, the Parkinson's disease mutations disconnect the influence of N-acetylation in promoting this transition. These results suggest a cooperative link between the N-terminus and the region of the mutations that may be important for α-Synuclein function.

  8. Foam stabilization by solid particle aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Guignot, S.; Faure, S. [CEA Marcoule, Lab. des Procedes Avances de Decontamination, 30 (France); Pitois, O. [UniversiteParis-Est Marne-La-Valle, Lab. Physique des Materiaux Divises et des Interfaces (LPMDI), 77 - Marne la Vallee (France)

    2008-07-01

    irreversible plugs. To establish the respective role of aggregates size and concentration, a series of experiments is currently performed consisting in studying the flow of foaming solution through glass-beds of various pore sizes containing the aggregates. The behaviour of foams containing sonicated particles appears to be more surprising since they do not display any yield stress and their sizes prevent them from being efficient plugs. Study of the flow of suspensions on the Plateau Border scale is currently performed to elucidate this behaviour. (authors)

  9. MHBCDA: Mobility and Heterogeneity aware Bandwidth Efficient Cluster based Data Aggregation for Wireless Sensor Network

    DEFF Research Database (Denmark)

    Dnyaneshwar, Mantri; Prasad, Neeli R.; Prasad, Ramjee

    2013-01-01

    Wireless Sensor Network (WSN) offers a variety of novel applications for mobile targets. It generates the large amount of redundant sensing data. The data aggregation techniques are extensively used to reduce the energy consumption and increase the network lifetime, although it has the side effect...... efficient. It exploits correlation of data packets generated by varying the packet generation rate. It prevents transmission of redundant data packets by improving energy consumption by 4.11% and prolongs the network life by 34.45% as compared with state-of-the-art solutions.......-based Data Aggregation (MHBCDA) algorithm for the randomly distributed nodes. It considers the mobile sink based packet aggregation for the heterogeneous WSN. It uses predefined region for the aggregation at cluster head to minimize computation and communication cost. The MHBCDA is energy and bandwidth...

  10. From preventive to permissive checks

    DEFF Research Database (Denmark)

    Weisdorf, Jacob Louis; Sharp, Paul Richard

    2009-01-01

    The Malthusian "preventive check" mechanism has been well documented for pre-industrial England through evidence for a negative correlation between the marriage rate and the price of wheat. Other literature, however, speculates that the correlation was in fact positive from the early nineteenth c...... model shows that this result is not in fact inconsistent with a stylised Malthusian mechanism, and can be understood within the context of an increasing dominance of shocks to aggregate demand rather than to aggregate supply....

  11. The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease.

    Science.gov (United States)

    Stürner, Elisabeth; Behl, Christian

    2017-01-01

    In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC) system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy). One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 ( BCL-2-associated athanogene 3 ). Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer's disease (tau-protein), Huntington's disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate.

  12. Ligand-promoted protein folding by biased kinetic partitioning.

    Science.gov (United States)

    Hingorani, Karan S; Metcalf, Matthew C; Deming, Derrick T; Garman, Scott C; Powers, Evan T; Gierasch, Lila M

    2017-04-01

    Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

  13. Effect of aggregate graining compositions on skid resistance of Exposed Aggregate Concrete pavement

    Science.gov (United States)

    Wasilewska, Marta; Gardziejczyk, Wladysław; Gierasimiuk, Pawel

    2018-05-01

    The paper presents the evaluation of skid resistance of EAC (Exposed Aggregate Concrete) pavements which differ in aggregate graining compositions. The tests were carried out on concrete mixes with a maximum aggregate size of 8 mm. Three types of coarse aggregates were selected depending on their resistance to polishing which was determined on the basis of the PSV (Polished Stone Value). Basalt (PSV 48), gabbro (PSV 50) and trachybasalt (PSV 52) aggregates were chosen. For each type of aggregate three graining compositions were designed, which differed in the content of coarse aggregate > 4mm. Their content for each series was as follows: A - 38%, B - 50% and C - 68%. Evaluation of the skid resistance has been performed using the FAP (Friction After Polishing) test equipment also known as the Wehner/Schulze machine. Laboratory method enables to compare the skid resistance of different types of wearing course under specified conditions simulating polishing processes. In addition, macrotexture measurements were made on the surface of each specimen using the Elatexure laser profile. Analysis of variance showed that at significance level α = 0.05, aggregate graining compositions as well as the PSV have a significant influence on the obtained values of the friction coefficient μm of the tested EAC pavements. The highest values of the μm have been obtained for EAC with the lowest amount of coarse aggregates (compositions A). In these cases the resistance to polishing of the aggregate does not significantly affect the friction coefficients. This is related to the large areas of cement mortar between the exposed coarse grains. Based on the analysis of microscope images, it was observed that the coarse aggregates were not sufficiently exposed. It has been proved that PSV significantly affected the coefficient of friction in the case of compositions B and C. This is caused by large areas of exposed coarse aggregate. The best parameters were achieved for the EAC pavements

  14. Barcoding heat shock proteins to human diseases: looking beyond the heat shock response.

    Science.gov (United States)

    Kakkar, Vaishali; Meister-Broekema, Melanie; Minoia, Melania; Carra, Serena; Kampinga, Harm H

    2014-04-01

    There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR)--and thus generally restoring the disturbed protein homeostasis associated with such diseases--has often been suggested as a therapeutic strategy. However, most data on activating the HSR or its downstream targets in mouse models of diseases associated with aggregate formation have been rather disappointing. The human chaperonome consists of many more heat shock proteins (HSPs) that are not regulated by the HSR, however, and researchers are now focusing on these as potential therapeutic targets. In this Review, we summarize the existing literature on a set of aggregation diseases and propose that each of them can be characterized or 'barcoded' by a different set of HSPs that can rescue specific types of aggregation. Some of these 'non-canonical' HSPs have demonstrated effectiveness in vivo, in mouse models of protein-aggregation disease. Interestingly, several of these HSPs also cause diseases when mutated--so-called chaperonopathies--which are also discussed in this Review.

  15. Effect of the LHCII pigment-protein complex aggregation on photovoltaic properties of sensitized TiO2 solar cells.

    Science.gov (United States)

    Yang, Yiqun; Jankowiak, Ryszard; Lin, Chen; Pawlak, Krzysztof; Reus, Michael; Holzwarth, Alfred R; Li, Jun

    2014-10-14

    A modified dye-sensitized solar cell consisting of a thin TiO2 barrier layer sensitized with natural trimeric light-harvesting complex II (LHCII) from spinach was used as a biomimetic model to study the effects of LHCII aggregation on the photovoltaic properties. The aggregation of individual trimers induced molecular reorganization, which dramatically increased the photocurrent. The morphology of small- and large-size LHCII aggregates deposited on a surface was confirmed by atomic force microscopy. Enhanced LHCII immobilization was accomplished via electrostatic interaction with amine-functionalized photoanodes. The photocurrent responses of the assembled solar cells under illumination at three characteristic wavelength bands in the UV-Vis absorption spectra of LHCII solutions confirmed that a significant photocurrent was generated by LHCII photosensitizers. The enhanced photocurrent by large aggregated LHCII is shown to correlate with the quenching in the far-red fluorescence deriving from chlorophyll-chlorophyll charge transfer states that are effectively coupled with the TiO2 surface and thus inject electrons into the TiO2 conduction band. The large aggregated LHCII with more chlorophyll-chlorophyll charge transfer states is a much better sensitizer since it injects electrons more efficiently into the conduction band of TiO2 than the small aggregated LHCII mostly consisting of unquenched chlorophyll excited state. The assembled solar cells demonstrated remarkable stability in both aqueous buffer and acetonitrile electrolytes over 30 days.

  16. Concrete produced with recycled aggregates

    Directory of Open Access Journals (Sweden)

    J. J. L. Tenório

    Full Text Available This paper presents the analysis of the mechanical and durable properties of recycled aggregate concrete (RAC for using in concrete. The porosity of recycled coarse aggregates is known to influence the fresh and hardened concrete properties and these properties are related to the specific mass of the recycled coarse aggregates, which directly influences the mechanical properties of the concrete. The recycled aggregates were obtained from construction and demolition wastes (CDW, which were divided into recycled sand (fine and coarse aggregates. Besides this, a recycled coarse aggregate of a specific mass with a greater density was obtained by mixing the recycled aggregates of the CDW with the recycled aggregates of concrete wastes (CW. The concrete was produced in laboratory by combining three water-cement ratios, the ratios were used in agreement with NBR 6118 for structural concretes, with each recycled coarse aggregates and recycled sand or river sand, and the reference concrete was produced with natural aggregates. It was observed that recycled aggregates can be used in concrete with properties for structural concrete. In general, the use of recycled coarse aggregate in combination with recycled sand did not provide good results; but when the less porous was used, or the recycled coarse aggregate of a specific mass with a greater density, the properties of the concrete showed better results. Some RAC reached bigger strengths than the reference concrete.

  17. Protein-protein interactions as a strategy towards protein-specific drug design: the example of ataxin-1.

    Directory of Open Access Journals (Sweden)

    Cesira de Chiara

    Full Text Available A main challenge for structural biologists is to understand the mechanisms that discriminate between molecular interactions and determine function. Here, we show how partner recognition of the AXH domain of the transcriptional co-regulator ataxin-1 is fine-tuned by a subtle balance between self- and hetero-associations. Ataxin-1 is the protein responsible for the hereditary spinocerebellar ataxia type 1, a disease linked to protein aggregation and transcriptional dysregulation. Expansion of a polyglutamine tract is essential for ataxin-1 aggregation, but the sequence-wise distant AXH domain plays an important aggravating role in the process. The AXH domain is also a key element for non-aberrant function as it intervenes in interactions with multiple protein partners. Previous data have shown that AXH is dimeric in solution and forms a dimer of dimers when crystallized. By solving the structure of a complex of AXH with a peptide from the interacting transcriptional repressor CIC, we show that the dimer interface of AXH is displaced by the new interaction and that, when blocked by the CIC peptide AXH aggregation and misfolding are impaired. This is a unique example in which palindromic self- and hetero-interactions within a sequence with chameleon properties discriminate the partner. We propose a drug design strategy for the treatment of SCA1 that is based on the information gained from the AXH/CIC complex.

  18. iHAT: interactive Hierarchical Aggregation Table for Genetic Association Data

    Directory of Open Access Journals (Sweden)

    Heinrich Julian

    2012-05-01

    Full Text Available Abstract In the search for single-nucleotide polymorphisms which influence the observable phenotype, genome wide association studies have become an important technique for the identification of associations between genotype and phenotype of a diverse set of sequence-based data. We present a methodology for the visual assessment of single-nucleotide polymorphisms using interactive hierarchical aggregation techniques combined with methods known from traditional sequence browsers and cluster heatmaps. Our tool, the interactive Hierarchical Aggregation Table (iHAT, facilitates the visualization of multiple sequence alignments, associated metadata, and hierarchical clusterings. Different color maps and aggregation strategies as well as filtering options support the user in finding correlations between sequences and metadata. Similar to other visualizations such as parallel coordinates or heatmaps, iHAT relies on the human pattern-recognition ability for spotting patterns that might indicate correlation or anticorrelation. We demonstrate iHAT using artificial and real-world datasets for DNA and protein association studies as well as expression Quantitative Trait Locus data.

  19. Graph Aggregation

    NARCIS (Netherlands)

    Endriss, U.; Grandi, U.

    Graph aggregation is the process of computing a single output graph that constitutes a good compromise between several input graphs, each provided by a different source. One needs to perform graph aggregation in a wide variety of situations, e.g., when applying a voting rule (graphs as preference

  20. Failure of protein quality control in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kabashi, Edor; Durham, Heather D

    2006-01-01

    The protein chaperoning and ubiquitin-proteasome systems perform many homeostatic functions within cells involving protein folding, transport and degradation. Of paramount importance is ridding cells of mutant or post-translationally modified proteins that otherwise tend to aggregate into insoluble complexes and form inclusions. Such inclusions are characteristic of many neurodegenerative diseases and implicate protein misfolding and aggregation as common aspects of pathogenesis. In the most common familial form of ALS, mutations in SOD1 promote misfolding of the protein and target it for degradation by proteasomes. Although proteasomes can degrade the mutant proteins efficiently, altered solubility and aggregation of mutant SOD1 are features of the disease and occur most prominently in the most vulnerable cells and tissues. Indeed, lumbar spinal cord of mutant SOD1 transgenic mice show early reduction in their capacity for protein chaperoning and proteasome-mediated hydrolysis of substrates, and motor neurons are particularly vulnerable to aggregation of mutant SOD1. A high threshold for upregulating key pathways in response to the stress of added substrate load may contribute to this vulnerability. The broad spectrum neuroprotective capability and efficacy of some chaperone-based therapies in preclinical models makes these pathways attractive as targets for therapy in ALS, as well as other neurodegenerative diseases. A better understanding of the mechanisms governing the regulation of protein chaperones and UPS components would facilitate development of treatments that upregulate these pathways in a coordinated manner in neural tissue without long term toxicity.

  1. Mfa4, an Accessory Protein of Mfa1 Fimbriae, Modulates Fimbrial Biogenesis, Cell Auto-Aggregation, and Biofilm Formation in Porphyromonas gingivalis.

    Science.gov (United States)

    Ikai, Ryota; Hasegawa, Yoshiaki; Izumigawa, Masashi; Nagano, Keiji; Yoshida, Yasuo; Kitai, Noriyuki; Lamont, Richard J; Yoshimura, Fuminobu; Murakami, Yukitaka

    2015-01-01

    Porphyromonas gingivalis, a gram-negative obligate anaerobic bacterium, is considered to be a key pathogen in periodontal disease. The bacterium expresses Mfa1 fimbriae, which are composed of polymers of Mfa1. The minor accessory components Mfa3, Mfa4, and Mfa5 are incorporated into these fimbriae. In this study, we characterized Mfa4 using genetically modified strains. Deficiency in the mfa4 gene decreased, but did not eliminate, expression of Mfa1 fimbriae. However, Mfa3 and Mfa5 were not incorporated because of defects in posttranslational processing and leakage into the culture supernatant, respectively. Furthermore, the mfa4-deficient mutant had an increased tendency to auto-aggregate and form biofilms, reminiscent of a mutant completely lacking Mfa1. Notably, complementation of mfa4 restored expression of structurally intact and functional Mfa1 fimbriae. Taken together, these results indicate that the accessory proteins Mfa3, Mfa4, and Mfa5 are necessary for assembly of Mfa1 fimbriae and regulation of auto-aggregation and biofilm formation of P. gingivalis. In addition, we found that Mfa3 and Mfa4 are processed to maturity by the same RgpA/B protease that processes Mfa1 subunits prior to polymerization.

  2. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

    Science.gov (United States)

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

  3. Resistance wheel exercise from mid-life has minimal effect on sciatic nerves from old mice in which sarcopenia was prevented.

    Science.gov (United States)

    Krishnan, Vidya S; White, Zoe; Terrill, Jessica R; Hodgetts, Stuart I; Fitzgerald, Melinda; Shavlakadze, Tea; Harvey, Alan R; Grounds, Miranda D

    2017-10-01

    The ability of resistance exercise, initiated from mid-life, to prevent age-related changes in old sciatic nerves, was investigated in male and female C57BL/6J mice. Aging is associated with cellular changes in old sciatic nerves and also loss of skeletal muscle mass and function (sarcopenia). Mature adult mice aged 15 months (M) were subjected to increasing voluntary resistance wheel exercise (RWE) over a period of 8 M until 23 M of age. This prevented sarcopenia in the old 23 M aged male and female mice. Nerves of control sedentary (SED) males at 3, 15 and 23 M of age, showed a decrease in the myelinated axon numbers at 15 and 23 M, a decreased g-ratio and a significantly increased proportion of myelinated nerves containing electron-dense aggregates at 23 M. Myelinated axon and nerve diameter, and axonal area, were increased at 15 M compared with 3 and 23 M. Exercise increased myelinated nerve profiles containing aggregates at 23 M. S100 protein, detected with immunoblotting was increased in sciatic nerves of 23 M old SED females, but not males, compared with 15 M, with no effect of exercise. Other neuronal proteins showed no significant alterations with age, gender or exercise. Overall the RWE had no cellular impact on the aging nerves, apart from an increased number of old nerves containing aggregates. Thus the relationship between cellular changes in aging nerves, and their sustained capacity for stimulation of old skeletal muscles to help maintain healthy muscle mass in response to exercise remains unclear.

  4. Atomic force microscopy-based antibody recognition imaging of proteins in the pathological deposits in Pseudoexfoliation Syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Creasey, Rhiannon [School of Chemical and Physical Sciences, Flinders University of SA, GPO Box 2100, Adelaide, SA 5001 (Australia); Sharma, Shiwani [School of Medicine, Ophthalmology, Flinders University of SA, GPO Box 2100, Adelaide, SA 5001 (Australia); Gibson, Christopher T. [School of Chemical and Physical Sciences, Flinders University of SA, GPO Box 2100, Adelaide, SA 5001 (Australia); Craig, Jamie E. [School of Medicine, Ophthalmology, Flinders University of SA, GPO Box 2100, Adelaide, SA 5001 (Australia); Ebner, Andreas [Institute for Biophysics, Johannes Kepler Universitaet Linz, Altenbergerstr. 69, A-4040 Linz (Austria); Becker, Thomas [Nanochemistry Research Institute, Curtin University, GPO Box U1987, Perth, 6845 WA (Australia); Hinterdorfer, Peter [Institute for Biophysics, Johannes Kepler Universitaet Linz, Altenbergerstr. 69, A-4040 Linz (Austria); Voelcker, Nicolas H., E-mail: nico.voelcker@flinders.edu.au [School of Chemical and Physical Sciences, Flinders University of SA, GPO Box 2100, Adelaide, SA 5001 (Australia)

    2011-07-15

    The phenomenon of protein aggregation is of considerable interest to various disciplines, including the field of medicine. A range of disease pathologies are associated with this phenomenon. One of the ocular diseases hallmarked by protein aggregation is the Pseudoexfoliation (PEX) Syndrome. This condition is characterized by the deposition of insoluble proteinaceous material on the anterior human lens capsule. Genomic and proteomic analyses have revealed an association of specific genetic markers and various proteins, respectively, with PEX syndrome. However, the ultrastructure of the protein aggregates is poorly characterized. This study seeks to build capacity to determine the molecular nature of PEX aggregates on human lens capsules in their native state by AFM-based antibody recognition imaging. Lysyl oxidase-Like 1 (LOXL1), a protein identified as a component of PEX aggregates, is detected by an antibody-modified AFM probe. Topographical AFM images and antibody recognition images are obtained using three AFM-based techniques: TREC, phase and force-volume imaging. LOXL1 is found to be present on the lens capsule surface, and is localized around fibrous protein aggregates. Our evaluation shows that TREC imaging is best suited for human tissue imaging and holds significant potential for imaging of human disease tissues in their native state. -- Highlights: {yields} Atomic force microscopy techniques were applied to diseased human tissues. {yields} LOXL1 protein was detected on the small fibers of Pseudoexfoliation deposits. {yields} PicoTREC was the optimum technique for investigating protein aggregates.

  5. High pressure as a tool for investigating protein-ligand interactions

    International Nuclear Information System (INIS)

    Marchal, S; Lange, R; Tortora, P; Balny, C

    2004-01-01

    In recent years, the application of pressure on biological systems has gained increasing interest. Pressure-induced destabilization of electrostatic and hydrophobic interactions is currently exploited to study conformational protein stability and macromolecular assemblies of proteins. Due to links between severe human pathologies and ordered protein oligomerization into aggregates, which have become apparent, a better knowledge of the molecular and structural determinants that ensure the packing efficiency and stability of such complexes has taken on special importance. Here, we report the effect of pressure on the property of human ataxin-3 of aggregation. The results indicate the importance of its polyglutamine chain length in the stability and the tendency of the protein to form spheroids. Partial unfolding of the protein leading to solvent exposure of hydrophobic domains appears to be a prerequisite in the aggregation process of ataxin-3

  6. The Ixodes scapularis Salivary Protein, Salp15, Prevents the Association of HIV-1 gp120 and CD4

    OpenAIRE

    Juncadella, Ignacio J.; Garg, Renu; Bates, Tonya C.; Olivera, Elias R.; Anguita, Juan

    2007-01-01

    Ixodes scapularis salivary protein, Salp15, inhibits CD4+ T cell activation by binding to the most-extracellular domains of the CD4 molecule, potentially overlapping with the gp120-binding region. We now show that Salp15 inhibits the interaction of gp120 and CD4. Furthermore, Salp15 prevents syncytia formation between HL2/3 (a stable HeLa cell line expressing the envelope protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interactio...

  7. Distinct thermodynamic signatures of oligomer generation in the aggregation of the amyloid-β peptide

    Science.gov (United States)

    Cohen, Samuel I. A.; Cukalevski, Risto; Michaels, Thomas C. T.; Šarić, Andela; Törnquist, Mattias; Vendruscolo, Michele; Dobson, Christopher M.; Buell, Alexander K.; Knowles, Tuomas P. J.; Linse, Sara

    2018-05-01

    Mapping free-energy landscapes has proved to be a powerful tool for studying reaction mechanisms. Many complex biomolecular assembly processes, however, have remained challenging to access using this approach, including the aggregation of peptides and proteins into amyloid fibrils implicated in a range of disorders. Here, we generalize the strategy used to probe free-energy landscapes in protein folding to determine the activation energies and entropies that characterize each of the molecular steps in the aggregation of the amyloid-β peptide (Aβ42), which is associated with Alzheimer's disease. Our results reveal that interactions between monomeric Aβ42 and amyloid fibrils during fibril-dependent secondary nucleation fundamentally reverse the thermodynamic signature of this process relative to primary nucleation, even though both processes generate aggregates from soluble peptides. By mapping the energetic and entropic contributions along the reaction trajectories, we show that the catalytic efficiency of Aβ42 fibril surfaces results from the enthalpic stabilization of adsorbing peptides in conformations amenable to nucleation, resulting in a dramatic lowering of the activation energy for nucleation.

  8. In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Paolo Arosio

    Full Text Available Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL and light chain deposition diseases (LCDD. In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T(m at pH 7.4. The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4(-≫Cl(->H(2PO(4(-, confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.

  9. Spinal cord homogenates from SOD1 familial amyotrophic lateral sclerosis induce SOD1 aggregation in living cells.

    Directory of Open Access Journals (Sweden)

    Edward Pokrishevsky

    Full Text Available Mutant Cu/Zn superoxide dismutase (SOD1 can confer its misfolding on wild-type SOD1 in living cells; the propagation of misfolding can also be transmitted between cells in vitro. Recent studies identified fluorescently-tagged SOD1G85R as a promiscuous substrate that is highly prone to aggregate by a variety of templates, in vitro and in vivo. Here, we utilized several SOD1-GFP reporter proteins with G37R, G85R, or G93A mutations in SOD1. We observed that human spinal cord homogenates prepared from SOD1 familial ALS (FALS can induce significantly more intracellular reporter protein aggregation than spinal cord homogenates from sporadic ALS, Alzheimer's disease, multiple system atrophy or healthy control individuals. We also determined that the induction of reporter protein aggregation by SOD1-FALS tissue homogenates can be attenuated by incubating the cells with the SOD1 misfolding-specific antibody 3H1, or the small molecule 5-fluorouridine. Our study further implicates SOD1 as the seeding particle responsible for the spread of SOD1-FALS neurodegeneration from its initial onset site(s, and demonstrates two potential therapeutic strategies for SOD1-mediated disease. This work also comprises a medium-throughput cell-based platform of screening potential therapeutics to attenuate propagated aggregation of SOD1.

  10. Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.

    Science.gov (United States)

    Connell, Jodi L; Kim, Jiyeon; Shear, Jason B; Bard, Allen J; Whiteley, Marvin

    2014-12-23

    Microbes frequently live in nature as small, densely packed aggregates containing ∼10(1)-10(5) cells. These aggregates not only display distinct phenotypes, including resistance to antibiotics, but also, serve as building blocks for larger biofilm communities. Aggregates within these larger communities display nonrandom spatial organization, and recent evidence indicates that this spatial organization is critical for fitness. Studying single aggregates as well as spatially organized aggregates remains challenging because of the technical difficulties associated with manipulating small populations. Micro-3D printing is a lithographic technique capable of creating aggregates in situ by printing protein-based walls around individual cells or small populations. This 3D-printing strategy can organize bacteria in complex arrangements to investigate how spatial and environmental parameters influence social behaviors. Here, we combined micro-3D printing and scanning electrochemical microscopy (SECM) to probe quorum sensing (QS)-mediated communication in the bacterium Pseudomonas aeruginosa. Our results reveal that QS-dependent behaviors are observed within aggregates as small as 500 cells; however, aggregates larger than 2,000 bacteria are required to stimulate QS in neighboring aggregates positioned 8 μm away. These studies provide a powerful system to analyze the impact of spatial organization and aggregate size on microbial behaviors.

  11. Information Aggregation in Organizations

    OpenAIRE

    Schulte, Elisabeth

    2006-01-01

    This dissertation contributes to the analysis of information aggregation procedures within organizations. Facing uncertainty about the consequences of a collective decision, information has to be aggregated before making a choice. Two main questions are addressed. Firstly, how well is an organization suited for the aggregation of decision-relevant information? Secondly, how should an organization be designed in order to aggregate information efficiently? The main part deals with information a...

  12. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  13. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    Directory of Open Access Journals (Sweden)

    Ewan West

    2015-06-01

    Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ and the loss of synapses. Aggregation of the cellular prion protein (PrPC by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2 and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

  14. External electric field: An effective way to prevent aggregation of Mg atoms on γ-graphyne for high hydrogen storage capacity

    International Nuclear Information System (INIS)

    Liu, Ping-Ping; Zhang, Hong; Cheng, Xin-Lu; Tang, Yong-Jian

    2016-01-01

    Highlights: • Due to large pores in the sheet of γ-graphyne, it should be a potential materials for energy storage applications. Our calculations might motivate active experimental efforts in designing high-efficiency hydrogen storage media. • For the first time, we use an applied external electric field to prevent Mg atoms from clustering using density functional theory (DFT) calculations. • The results demonstrate that, for Mg-G after electric field (F = 0.05 V/nm) treatment, ten H_2 molecules per Mg atom can be adsorbed and the hydrogen storage capacities reach to 10.64 wt%, with the average binding energies of 0.28 eV/H_2. - Abstract: In this article, we investigate the hydrogen storage capacity of Mg-decorated γ-graphyne (Mg-G) based on DFT calculations. Our results indicate that an external electric field can effectively prevent Mg atoms aggregating on γ-graphyne sheet. The Mg-G, after electric field (F = 0.05 V/nm) treatment, can store up to ten H_2 molecules and the hydrogen storage capacity is 10.64 wt%, with the average adsorption energy of 0.28 eV/H_2. Our calculations demonstrate that Mg-G is a potential material for hydrogen storage with high capacity and might motivate active experimental efforts in designing hydrogen storage media.

  15. The biology of non-native proteins

    NARCIS (Netherlands)

    Herczenik, E.

    2007-01-01

    Protein misfolding diseases are linked by common principles of protein aggregation, plaque development and tissue damage. There is no adequate therapy for these highly debilitating diseases. This thesis aims to increase the understanding of protein misfolding diseases, which will hopefully lead to

  16. Graph Theory and Ion and Molecular Aggregation in Aqueous Solutions

    Science.gov (United States)

    Choi, Jun-Ho; Lee, Hochan; Choi, Hyung Ran; Cho, Minhaeng

    2018-04-01

    In molecular and cellular biology, dissolved ions and molecules have decisive effects on chemical and biological reactions, conformational stabilities, and functions of small to large biomolecules. Despite major efforts, the current state of understanding of the effects of specific ions, osmolytes, and bioprotecting sugars on the structure and dynamics of water H-bonding networks and proteins is not yet satisfactory. Recently, to gain deeper insight into this subject, we studied various aggregation processes of ions and molecules in high-concentration salt, osmolyte, and sugar solutions with time-resolved vibrational spectroscopy and molecular dynamics simulation methods. It turns out that ions (or solute molecules) have a strong propensity to self-assemble into large and polydisperse aggregates that affect both local and long-range water H-bonding structures. In particular, we have shown that graph-theoretical approaches can be used to elucidate morphological characteristics of large aggregates in various aqueous salt, osmolyte, and sugar solutions. When ion and molecular aggregates in such aqueous solutions are treated as graphs, a variety of graph-theoretical properties, such as graph spectrum, degree distribution, clustering coefficient, minimum path length, and graph entropy, can be directly calculated by considering an ensemble of configurations taken from molecular dynamics trajectories. Here we show percolating behavior exhibited by ion and molecular aggregates upon increase in solute concentration in high solute concentrations and discuss compelling evidence of the isomorphic relation between percolation transitions of ion and molecular aggregates and water H-bonding networks. We anticipate that the combination of graph theory and molecular dynamics simulation methods will be of exceptional use in achieving a deeper understanding of the fundamental physical chemistry of dissolution and in describing the interplay between the self-aggregation of solute

  17. Graph Theory and Ion and Molecular Aggregation in Aqueous Solutions.

    Science.gov (United States)

    Choi, Jun-Ho; Lee, Hochan; Choi, Hyung Ran; Cho, Minhaeng

    2018-04-20

    In molecular and cellular biology, dissolved ions and molecules have decisive effects on chemical and biological reactions, conformational stabilities, and functions of small to large biomolecules. Despite major efforts, the current state of understanding of the effects of specific ions, osmolytes, and bioprotecting sugars on the structure and dynamics of water H-bonding networks and proteins is not yet satisfactory. Recently, to gain deeper insight into this subject, we studied various aggregation processes of ions and molecules in high-concentration salt, osmolyte, and sugar solutions with time-resolved vibrational spectroscopy and molecular dynamics simulation methods. It turns out that ions (or solute molecules) have a strong propensity to self-assemble into large and polydisperse aggregates that affect both local and long-range water H-bonding structures. In particular, we have shown that graph-theoretical approaches can be used to elucidate morphological characteristics of large aggregates in various aqueous salt, osmolyte, and sugar solutions. When ion and molecular aggregates in such aqueous solutions are treated as graphs, a variety of graph-theoretical properties, such as graph spectrum, degree distribution, clustering coefficient, minimum path length, and graph entropy, can be directly calculated by considering an ensemble of configurations taken from molecular dynamics trajectories. Here we show percolating behavior exhibited by ion and molecular aggregates upon increase in solute concentration in high solute concentrations and discuss compelling evidence of the isomorphic relation between percolation transitions of ion and molecular aggregates and water H-bonding networks. We anticipate that the combination of graph theory and molecular dynamics simulation methods will be of exceptional use in achieving a deeper understanding of the fundamental physical chemistry of dissolution and in describing the interplay between the self-aggregation of solute

  18. A Novel Method to Quantify Soil Aggregate Stability by Measuring Aggregate Bond Energies

    Science.gov (United States)

    Efrat, Rachel; Rawlins, Barry G.; Quinton, John N.; Watts, Chris W.; Whitmore, Andy P.

    2016-04-01

    Soil aggregate stability is a key indicator of soil quality because it controls physical, biological and chemical functions important in cultivated soils. Micro-aggregates are responsible for the long term sequestration of carbon in soil, therefore determine soils role in the carbon cycle. It is thus vital that techniques to measure aggregate stability are accurate, consistent and reliable, in order to appropriately manage and monitor soil quality, and to develop our understanding and estimates of soil as a carbon store to appropriately incorporate in carbon cycle models. Practices used to assess the stability of aggregates vary in sample preparation, operational technique and unit of results. They use proxies and lack quantification. Conflicting results are therefore drawn between projects that do not provide methodological or resultant comparability. Typical modern stability tests suspend aggregates in water and monitor fragmentation upon exposure to an un-quantified amount of ultrasonic energy, utilising a laser granulometer to measure the change in mean weight diameter. In this project a novel approach has been developed based on that of Zhu et al., (2009), to accurately quantify the stability of aggregates by specifically measuring their bond energies. The bond energies are measured operating a combination of calorimetry and a high powered ultrasonic probe, with computable output function. Temperature change during sonication is monitored by an array of probes which enables calculation of the energy spent heating the system (Ph). Our novel technique suspends aggregates in heavy liquid lithium heteropolytungstate, as opposed to water, to avoid exposing aggregates to an immeasurable disruptive energy source, due to cavitation, collisions and clay swelling. Mean weight diameter is measured by a laser granulometer to monitor aggregate breakdown after successive periods of calculated ultrasonic energy input (Pi), until complete dispersion is achieved and bond

  19. The Sso7d protein of Sulfolobus solfataricus: in vitro relationship among different activities

    Directory of Open Access Journals (Sweden)

    Annamaria Guagliardi

    2002-01-01

    Full Text Available The physiological role of the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus is unknown. In vitro studies have shown that Sso7d promotes annealing of complementary DNA strands (Guagliardi et al. 1997, induces negative supercoiling (Lopez-Garcia et al. 1998, and chaperones the disassembly and renaturation of protein aggregates in an ATP hydrolysis-dependent manner (Guagliardi et al. 2000. In this study, we examined the relationships among the binding of Sso7d to double-stranded DNA, its interaction with protein aggregates, and its ATPase activity. Experiments with 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that exposed hydrophobic surfaces in Sso7d are responsible for interactions with protein aggregates and double-stranded DNA, whereas the site of ATPase activity has a non-hydrophobic character. The interactions of Sso7d with double-stranded DNA and with protein aggregates are mutually exclusive events, suggesting that the disassembly activity and the DNA-related activities of Sso7d may be competitive in vivo. In contrast, the hydrolysis of ATP by Sso7d is independent of the binding of Sso7d to double-stranded DNA or protein aggregates.

  20. Curcumin Attenuates Amyloid-β Aggregate Toxicity and Modulates Amyloid-β Aggregation Pathway.

    Science.gov (United States)

    Thapa, Arjun; Jett, Stephen D; Chi, Eva Y

    2016-01-20

    The abnormal misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet enriched insoluble deposits initiates a cascade of events leading to pathological processes and culminating in cognitive decline in Alzheimer's disease (AD). In particular, soluble oligomeric/prefibrillar Aβ have been shown to be potent neurotoxins. The naturally occurring polyphenol curcumin has been shown to exert a neuroprotective effect against age-related neurodegenerative diseases such as AD. However, its protective mechanism remains unclear. In this study, we investigated the effects of curcumin on the aggregation of Aβ40 as well as Aβ40 aggregate induced neurotoxicity. Our results show that the curcumin does not inhibit Aβ fibril formation, but rather enriches the population of "off-pathway" soluble oligomers and prefibrillar aggregates that were nontoxic. Curcumin also exerted a nonspecific neuroprotective effect, reducing toxicities induced by a range of Aβ conformers, including monomeric, oligomeric, prefibrillar, and fibrillar Aβ. The neuroprotective effect is possibly membrane-mediated, as curcumin reduced the extent of cell membrane permeabilization induced by Aβ aggregates. Taken together, our study shows that curcumin exerts its neuroprotective effect against Aβ induced toxicity through at least two concerted pathways, modifying the Aβ aggregation pathway toward the formation of nontoxic aggregates and ameliorating Aβ-induced toxicity possibly through a nonspecific pathway.