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.

Protein Colloidal Aggregation Project

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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.

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

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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.

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.

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

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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.

Protein aggregation studied by forward light scattering and light transmission analysis

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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).

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

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Dasgupta, Anushka

Many studies have suggested that oxidative stress plays an important role in the pathophysiology of both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Yet, the mechanism by which oxidative stress leads to tissue damage in these disorders is unclear. Recent work from our laboratory has revealed that protein carbonylation, a major oxidative modification caused by severe and/or chronic oxidative stress conditions, is elevated in MS and EAE. Furthermore, protein carbonylation has been shown to alter protein structure leading to misfolding/aggregation. These findings prompted me to hypothesize that carbonylated proteins, formed as a consequence of oxidative stress and/or decreased proteasomal activity, promote protein aggregation to mediate neuronal apoptosis in vitro and in EAE. To test this novel hypothesis, I first characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of myelin-oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE in C57BL/6 mice [Chapter 2]. The results show that carbonylated proteins accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. I discovered not only that there is a temporal correlation between protein carbonylation and apoptosis but also that carbonyl levels are significantly higher in apoptotic cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are also present during the course of EAE, which seems to be due to reduced autophagy. In chapter 3, I show that when gluthathione levels are reduced to those in EAE spinal cord, both neuron-like PC12 (nPC12) cells and primary neuronal cultures accumulate carbonylated proteins and undergo cell death (both by necrosis and apoptosis). Immunocytochemical and biochemical studies also revealed a temporal

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

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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.

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

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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.

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

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...

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

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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

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

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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

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

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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.

Role of Prion Protein Aggregation in Neurotoxicity

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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.

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

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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.

The effect of pH profiles in methanogenic aggregates on the kinetics of acetate conversion

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Beer, de D.; Huisman, J.W.; Heuvel, van den J.C.; Ottengraf, S.P.P.

1993-01-01

Due to the conversion of acetic acid into the weaker carbonic acid and CH4, the pH inside methanogenic aggregates is higher than in the bulk liq. The pH profiles in aggregates were measured with pH microelectrodes. These profiles strongly det. the macro-kinetics of the aggregate, by their influence

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

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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.

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

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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.

Aggregation propensity of critical regions of the protein Tau

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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.

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

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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

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

International Nuclear Information System (INIS)

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

Al cation induces aggregation of serum proteins.

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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.

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

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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.

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

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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.

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

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

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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.

Automation of aggregate characterization using laser profiling and digital image analysis

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Kim, Hyoungkwan

2002-08-01

Particle morphological properties such as size, shape, angularity, and texture are key properties that are frequently used to characterize aggregates. The characteristics of aggregates are crucial to the strength, durability, and serviceability of the structure in which they are used. Thus, it is important to select aggregates that have proper characteristics for each specific application. Use of improper aggregate can cause rapid deterioration or even failure of the structure. The current standard aggregate test methods are generally labor-intensive, time-consuming, and subject to human errors. Moreover, important properties of aggregates may not be captured by the standard methods due to a lack of an objective way of quantifying critical aggregate properties. Increased quality expectations of products along with recent technological advances in information technology are motivating new developments to provide fast and accurate aggregate characterization. The resulting information can enable a real time quality control of aggregate production as well as lead to better design and construction methods of portland cement concrete and hot mix asphalt. This dissertation presents a system to measure various morphological characteristics of construction aggregates effectively. Automatic measurement of various particle properties is of great interest because it has the potential to solve such problems in manual measurements as subjectivity, labor intensity, and slow speed. The main efforts of this research are placed on three-dimensional (3D) laser profiling, particle segmentation algorithms, particle measurement algorithms, and generalized particle descriptors. First, true 3D data of aggregate particles obtained by laser profiling are transformed into digital images. Second, a segmentation algorithm and a particle measurement algorithm are developed to separate particles and process each particle data individually with the aid of various kinds of digital image

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

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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.

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

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

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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.

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.

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

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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.

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

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

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.

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

International Nuclear Information System (INIS)

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

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

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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.

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.

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

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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

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)

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

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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.

Random Subspace Aggregation for Cancer Prediction with Gene Expression Profiles

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Liying Yang

2016-01-01

Full Text Available Background. Precisely predicting cancer is crucial for cancer treatment. Gene expression profiles make it possible to analyze patterns between genes and cancers on the genome-wide scale. Gene expression data analysis, however, is confronted with enormous challenges for its characteristics, such as high dimensionality, small sample size, and low Signal-to-Noise Ratio. Results. This paper proposes a method, termed RS_SVM, to predict gene expression profiles via aggregating SVM trained on random subspaces. After choosing gene features through statistical analysis, RS_SVM randomly selects feature subsets to yield random subspaces and training SVM classifiers accordingly and then aggregates SVM classifiers to capture the advantage of ensemble learning. Experiments on eight real gene expression datasets are performed to validate the RS_SVM method. Experimental results show that RS_SVM achieved better classification accuracy and generalization performance in contrast with single SVM, K-nearest neighbor, decision tree, Bagging, AdaBoost, and the state-of-the-art methods. Experiments also explored the effect of subspace size on prediction performance. Conclusions. The proposed RS_SVM method yielded superior performance in analyzing gene expression profiles, which demonstrates that RS_SVM provides a good channel for such biological data.

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

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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

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

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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

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

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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

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.

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.

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.

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

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

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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

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

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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.

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.

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.

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.

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

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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.

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

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

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

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

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)

PROFIL PROTEIN SUSU DAN PRODUK OLAHANNYA

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R. Susanti

2017-03-01

Full Text Available Penelitian ini bertujuan untuk menganalisis kadar protein dan profil protein pada beberapa susu (susu kedelai, susu kambing dan olahannya (yogurt, tofu. Kadar protein diukur dengan metode Lowry, sedangkan profil protein dianalisis menggunakan SDS PAGE. Data yang diperoleh dianalisis secara deskriptif. Kadar protein tertinggi pada sampel yang dianalisis terdapat pada produk yogurt A (579,5 mg/ml, disusul susu kedelai (289,99 mg/ml dan susu kambing (133,1 mg/ml. Analisis profil protein terlihat pita protein dengan mobilitas terendah sampai tertinggi terletak pada berat molekul 14-150 KDa. Pita protein khas yang hanya dimiliki susu kambing adalah pita 150kDa. Sementara pita protein khas yang hanya dimiliki susu kedelai adalah pita 44 kDa dan 55kDa. Pita protein yang khas hanya dimiliki yogurt A (dengan bakteri Lactobacillus bulgaricus dan Streptococcus thermophillus adalah pita 65Da. Semua jenis susu dan olahannya memiliki pita 70kDa, kecuali susu kedelai. Profil protein susu kedelai dan tofu menunjukkan profil protein yang sangat berbeda, namun keduanya memiliki pita 18kDa.This study aimed to observe protein level and profiles on some milks (soy milk, goat's milk and dairy (yogurt, tofu product. Protein content was observed by Lowry method, whereas the protein profiles were analyzed by polyacrylamide gel electrophoresis. Data were analyzed descriptively. The highest protein content of the observed sample was in yogurt A products (579,5 mg/ml, followed by soy milk (289,99 mg/ml and goat's milk (133,1 mg/ml. Analysis of protein profiles showed protein bands with lowest to highest mobility lies in the molecular weight of 14-150 KDa. Typical protein band of goat's milk was a 150kDa band. While the typical protein bands of soy milk were 44 kDa and 55kDa band. The typical protein band of yogurt A (with Lactobacillus bulgaricus and Streptococcus thermophillus bacterium was 65Da. All types of milks and dairy had 70kDa band, except for soy milk. Protein

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

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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.

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...

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

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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.

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

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

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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.

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

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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

Natural Biomolecules and Protein Aggregation: Emerging Strategies against Amyloidogenesis

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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.

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,

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.

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

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.

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

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

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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.

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.

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.

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.

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

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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.

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....

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.

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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.

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.

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

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)

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.

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.

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®

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

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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.

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

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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.

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

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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.

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

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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.

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...

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

Identifying seasonal mobility profiles from anonymized and aggregated mobile phone data. Application in food security.

Science.gov (United States)

Zufiria, Pedro J; Pastor-Escuredo, David; Úbeda-Medina, Luis; Hernandez-Medina, Miguel A; Barriales-Valbuena, Iker; Morales, Alfredo J; Jacques, Damien C; Nkwambi, Wilfred; Diop, M Bamba; Quinn, John; Hidalgo-Sanchís, Paula; Luengo-Oroz, Miguel

2018-01-01

We propose a framework for the systematic analysis of mobile phone data to identify relevant mobility profiles in a population. The proposed framework allows finding distinct human mobility profiles based on the digital trace of mobile phone users characterized by a Matrix of Individual Trajectories (IT-Matrix). This matrix gathers a consistent and regularized description of individual trajectories that enables multi-scale representations along time and space, which can be used to extract aggregated indicators such as a dynamic multi-scale population count. Unsupervised clustering of individual trajectories generates mobility profiles (clusters of similar individual trajectories) which characterize relevant group behaviors preserving optimal aggregation levels for detailed and privacy-secured mobility characterization. The application of the proposed framework is illustrated by analyzing fully anonymized data on human mobility from mobile phones in Senegal at the arrondissement level over a calendar year. The analysis of monthly mobility patterns at the livelihood zone resolution resulted in the discovery and characterization of seasonal mobility profiles related with economic activities, agricultural calendars and rainfalls. The use of these mobility profiles could support the timely identification of mobility changes in vulnerable populations in response to external shocks (such as natural disasters, civil conflicts or sudden increases of food prices) to monitor food security.

Identifying seasonal mobility profiles from anonymized and aggregated mobile phone data. Application in food security.

Directory of Open Access Journals (Sweden)

Pedro J Zufiria

Full Text Available We propose a framework for the systematic analysis of mobile phone data to identify relevant mobility profiles in a population. The proposed framework allows finding distinct human mobility profiles based on the digital trace of mobile phone users characterized by a Matrix of Individual Trajectories (IT-Matrix. This matrix gathers a consistent and regularized description of individual trajectories that enables multi-scale representations along time and space, which can be used to extract aggregated indicators such as a dynamic multi-scale population count. Unsupervised clustering of individual trajectories generates mobility profiles (clusters of similar individual trajectories which characterize relevant group behaviors preserving optimal aggregation levels for detailed and privacy-secured mobility characterization. The application of the proposed framework is illustrated by analyzing fully anonymized data on human mobility from mobile phones in Senegal at the arrondissement level over a calendar year. The analysis of monthly mobility patterns at the livelihood zone resolution resulted in the discovery and characterization of seasonal mobility profiles related with economic activities, agricultural calendars and rainfalls. The use of these mobility profiles could support the timely identification of mobility changes in vulnerable populations in response to external shocks (such as natural disasters, civil conflicts or sudden increases of food prices to monitor food security.

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

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.

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

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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.

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

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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.

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

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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.

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.

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

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.

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

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.

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

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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.

Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

Science.gov (United States)

Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

2013-01-01

Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

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®

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.

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.

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.

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

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)

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.

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.

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.

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.

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

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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

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.

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).

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).

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

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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.

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

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.

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.

Detection of ubiquitinated huntingtin species in intracellular aggregates

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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.

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...

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.

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

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

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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.

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.

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.

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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.

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

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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.

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

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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

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

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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.

Metagenome and Metatranscriptome Analyses Using Protein Family Profiles.

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Cuncong Zhong

2016-07-01

Full Text Available Analyses of metagenome data (MG and metatranscriptome data (MT are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE genes. Finally, HMM

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

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

Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

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Rom William N

2005-08-01

Full Text Available Abstract Background Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Methods Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD. Two-color rolling-circle amplification was used to measure protein abundance. Results Seven of the 84 antibodies gave a significant difference (p Conclusion Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

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.

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

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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

Analysis of protein profiles using fuzzy clustering methods

DEFF Research Database (Denmark)

Karemore, Gopal Raghunath; Ukendt, Sujatha; Rai, Lavanya

The tissue protein profiles of healthy volunteers and volunteers with cervical cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence  technique  (HPLC-LIF)  developed  in  our  lab.      We analyzed      the protein profile data using different...

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.

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

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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.

Light-induced aggregation of microbial exopolymeric substances.

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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.

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.

Changes in the protein profile of Habanero pepper ( Capsicum ...

African Journals Online (AJOL)

Protein profile was studied during the development of Capsicum chinense somatic embryos. The total protein content and profile of polypeptides (by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of somatic embryos at different developmental stages (globular, heart-shaped, torpedo and cotyledonary stages) ...

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.

Finite size effects in simulations of protein aggregation.

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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.

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

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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.

Curcumin inhibits aggregation of alpha-synuclein.

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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.

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

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.

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.

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

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

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.

Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

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Xiaolin Wu

Full Text Available Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs, mainly mannose-binding lectin (agglutinin, exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE. Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.

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

Science.gov (United States)

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.

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.

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...

Collective Rationality in Graph Aggregation

NARCIS (Netherlands)

Endriss, U.; Grandi, U.; Schaub, T.; Friedrich, G.; O'Sullivan, B.

2014-01-01

Suppose a number of agents each provide us with a directed graph over a common set of vertices. Graph aggregation is the problem of computing a single “collective” graph that best represents the information inherent in this profile of individual graphs. We consider this aggregation problem from the

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

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

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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.

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.

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

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.

Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

International Nuclear Information System (INIS)

Gao, Wei-Min; Haab, Brian B; Hanash, Samir M; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S

2005-01-01

Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer

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

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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.

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.

Differences of protein profile before and after orthodontic treatment

Science.gov (United States)

Nasri, Farah Amirah Mohd; Wahab, Rohaya Megat Abdul; Karsani, Saiful Anuar; Ariffin, Shahrul Hisham Zainal

2016-11-01

Mechanical forces in orthodontic treatment used to treat malocclusion can cause inflamed gingival tissue and the process of tooth movement may resorb dental root. Root resorption is an iatrogenic effect of orthodontic treatment but it can be monitored using protein biomarker. This study aims to investigate the differences of protein profile before and after orthodontic treatment using different staining methods. Human gingival crevicular fluid and saliva were collected from orthodontic patients before and after treatment. Protein profile were observed using SDS-PAGE. Our study shows down regulation of proteins after 3 months of treatment. Hence, there are potential values from this study to aid in investigation for specific biomarkers for root resorption.

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)

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

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

O-GlcNAc profiling: from proteins to proteomes

Science.gov (United States)

2014-01-01

O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases. PMID:24593906

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

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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.

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.

Analysis of Proximate and Protein Profile of Kefir from Fermented Goat and Cow Milk

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Erwin Hidayat

2015-09-01

Full Text Available This research aims to analyze the characteristics of proximate and protein profile in kefir from fermented goat milk and cow milk with different concentration of kefir grains. The research design was true experimental with Completely Randomized Design (CRD of 3 repetitions. The research procedures consisted of kefir production, proximate analysis and protein profile characterization. Proximate assay result was analyzed by using LSD, whereas the protein profile was analyzed by descriptive qualitative method. Based on the analysis of kefir proximate levels, the kefir grain (5% showed the highest proximate level of both kefirs from goat milk and cow milk. The analysis of protein profile of cow milk kefir showed 75 kDa of protein ribbon, while the goat milk kefir showed 48 kDa, 60 kDa and 75 kDa. Therefore it can be concluded that the proximate level of goat and cow milk kefir with different concentration of kefir grains showed significant differences in the nutrition content as well as its protein profiles.Tujuan dari penelitian ini adalah menganalisis karakteristik proksimat dan profil protein pada kefir hasil fermentasi susu kambing dan susu sapi dengan konsentrasi biji kefir yang berbeda-beda. Penelitian ini adalah eksperimen murni, dengan Rancangan Acak Lengkap (RAL 3 kali ulangan. Prosedur penelitian meliputi pembuatan kefir, analisis proksimat dan profil protein. Data hasil proksimat dianalisi uji BNT, sedangkan profil protein dianalisis deskriptif kualitatif. Berdasarkan analisis kadar proksimat kefir, kefir grains 5% menunjukan kadar proksimat paling tinggi baik pada kefir susu kambing dan susu sapi. Sedangkan analisis profil protein kefir susu sapi menunjukan pita protein 75 kDa, pada kefir susu kambing yaitu 48 kDa, 60 kDa dan 75 kDa. Simpulan dari penelitian ini bahwa kadar proksimat kefir susu kambing dan susu sapi dengan konsentrasi kefir grains yang berbeda menunjukan perbedaan kandungan yang berbeda secara signifikan dengan

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

Automatic selection of reference taxa for protein-protein interaction prediction with phylogenetic profiling

DEFF Research Database (Denmark)

Simonsen, Martin; Maetschke, S.R.; Ragan, M.A.

2012-01-01

Motivation: Phylogenetic profiling methods can achieve good accuracy in predicting protein–protein interactions, especially in prokaryotes. Recent studies have shown that the choice of reference taxa (RT) is critical for accurate prediction, but with more than 2500 fully sequenced taxa publicly......: We present three novel methods for automating the selection of RT, using machine learning based on known protein–protein interaction networks. One of these methods in particular, Tree-Based Search, yields greatly improved prediction accuracies. We further show that different methods for constituting...... phylogenetic profiles often require very different RT sets to support high prediction accuracy....

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

Science.gov (United States)

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

Proteomic characterization of the human centrosome by protein correlation profiling

DEFF Research Database (Denmark)

Andersen, Jens S; Wilkinson, Christopher J; Mayor, Thibault

2003-01-01

chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human...... centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel...... components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity....

Hierarchical partitioning of metazoan protein conservation profiles provides new functional insights.

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Jonathan Witztum

Full Text Available The availability of many complete, annotated proteomes enables the systematic study of the relationships between protein conservation and functionality. We explore this question based solely on the presence or absence of protein homologues (a.k.a. conservation profiles. We study 18 metazoans, from two distinct points of view: the human's and the fly's. Using the GOrilla gene ontology (GO analysis tool, we explore functional enrichment of the "universal proteins", those with homologues in all 17 other species, and of the "non-universal proteins". A large number of GO terms are strongly enriched in both human and fly universal proteins. Most of these functions are known to be essential. A smaller number of GO terms, exhibiting markedly different properties, are enriched in both human and fly non-universal proteins. We further explore the non-universal proteins, whose conservation profiles are consistent with the "tree of life" (TOL consistent, as well as the TOL inconsistent proteins. Finally, we applied Quantum Clustering to the conservation profiles of the TOL consistent proteins. Each cluster is strongly associated with one or a small number of specific monophyletic clades in the tree of life. The proteins in many of these clusters exhibit strong functional enrichment associated with the "life style" of the related clades. Most previous approaches for studying function and conservation are "bottom up", studying protein families one by one, and separately assessing the conservation of each. By way of contrast, our approach is "top down". We globally partition the set of all proteins hierarchically, as described above, and then identify protein families enriched within different subdivisions. While supporting previous findings, our approach also provides a tool for discovering novel relations between protein conservation profiles, functionality, and evolutionary history as represented by the tree of life.

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...

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.

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

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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

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

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Mo Min

2008-05-01

Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

Multiplex single-molecule interaction profiling of DNA barcoded proteins

Science.gov (United States)

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage

Science.gov (United States)

Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

2016-03-01

The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1 ×, DE3 ×, ME3 ×, NEL3 ×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P = 0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P = 0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P = 0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P = 0.09), RD of PB2 (29 vs. 62 g/kg CP, P = 0.02), RD of CB2 (251 vs. 198 g/kg DM, P = 0.06), RD of CB3 (236 vs. 261 g/kg CHO, P = 0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P = 0.06), and metabolic bioenergy (P = 0.095). As to protein molecular structure, there were differences in protein structure 1st

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.

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.

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

Science.gov (United States)

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2016-09-01

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Soil aggregation under different management systems

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Cibele Mascioli Rebello Portella

2012-12-01

Full Text Available Considering that the soil aggregation reflects the interaction of chemical, physical and biological soil factors, the aim of this study was evaluate alterations in aggregation, in an Oxisol under no-tillage (NT and conventional tillage (CT, since over 20 years, using as reference a native forest soil in natural state. After analysis of the soil profile (cultural profile in areas under forest management, samples were collected from the layers 0-5, 5-10, 10-20 and 20-40 cm, with six repetitions. These samples were analyzed for the aggregate stability index (ASI, mean weighted diameter (MWD, mean geometric diameter (MGD in the classes > 8, 8-4, 4-2, 2-1, 1-0.5, 0.5-0.25, and < 0.25 mm, and for physical properties (soil texture, water dispersible clay (WDC, flocculation index (FI and bulk density (Bd and chemical properties (total organic carbon - COT, total nitrogen - N, exchangeable calcium - Ca2+, and pH. The results indicated that more intense soil preparation (M < NT < PC resulted in a decrease in soil stability, confirmed by all stability indicators analyzed: MWD, MGD, ASI, aggregate class distribution, WDC and FI, indicating the validity of these indicators in aggregation analyses of the studied soil.

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.

HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features.

Science.gov (United States)

Zaman, Rianon; Chowdhury, Shahana Yasmin; Rashid, Mahmood A; Sharma, Alok; Dehzangi, Abdollah; Shatabda, Swakkhar

2017-01-01

DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM) as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.

HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features

Directory of Open Access Journals (Sweden)

Rianon Zaman

2017-01-01

Full Text Available DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.

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

Correlations between RNA and protein expression profiles in 23 human cell lines

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Pontén Fredrik

2009-08-01

Full Text Available Abstract Background The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays and protein profiles (determined immunohistochemically in tissue microarrays of 1066 gene products in 23 human cell lines. Results A high mean correlation coefficient (0.52 was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Conclusion Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.

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

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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.

The first decade of MALDI protein profiling

DEFF Research Database (Denmark)

Albrethsen, Jakob

2011-01-01

MALDI protein profiling has identified several important challenges in omics-based biomarker research. First, research into the analytical performance of a novel omics-platform of potential diagnostic impact must be carried out in a critical manner and according to common guidelines. Evaluation s...

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

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

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

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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.

What favors convective aggregation and why?

Science.gov (United States)

Muller, Caroline; Bony, Sandrine

2015-07-01

The organization of convection is ubiquitous, but its physical understanding remains limited. One particular type of organization is the spatial self-aggregation of convection, taking the form of cloud clusters, or tropical cyclones in the presence of rotation. We show that several physical processes can give rise to self-aggregation and highlight the key features responsible for it, using idealized simulations. Longwave radiative feedbacks yield a "radiative aggregation." In that case, sufficient spatial variability of radiative cooling rates yields a low-level circulation, which induces the upgradient energy transport and radiative-convective instability. Not only do vertically integrated radiative budgets matter but the vertical profile of cooling is also crucial. Convective aggregation is facilitated when downdrafts below clouds are weak ("moisture-memory aggregation"), and this is sufficient to trigger aggregation in the absence of longwave radiative feedbacks. These results shed some light on the sensitivity of self-aggregation to various parameters, including resolution or domain size.

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

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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

SOD1 aggregation in ALS mice shows simplistic test tube behavior.

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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.

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.

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

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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.

Aggregates in monoclonal antibody manufacturing processes.

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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.

Biomarkers for ragwort poisoning in horses: identification of protein targets

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Beynon Robert J

2008-08-01

Full Text Available Abstract Background Ingestion of the poisonous weed ragwort (Senecio jacobea by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods. Results One dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. Using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin. Conclusion These findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. The high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. This model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock.

Reproducibility of mass spectrometry based protein profiles for diagnosis of breast cancer across clinical studies

DEFF Research Database (Denmark)

Callesen, Anne Kjærgaard; Vach, Werner; Jørgensen, Per E

2008-01-01

Serum protein profiling by mass spectrometry has achieved attention as a promising technology in oncoproteomics. We performed a systematic review of published reports on protein profiling as a diagnostic tool for breast cancer. The MEDLINE, EMBASE, and COCHRANE databases were searched for original...... studies reporting discriminatory protein peaks for breast cancer as either protein identity or as m/ z values in the period from January 1995 to October 2006. To address the important aspect of reproducibility of mass spectrometry data across different clinical studies, we compared the published lists...... of potential discriminatory peaks with those peaks detected in an original MALDI MS protein profiling study performed by our own research group. A total of 20 protein/peptide profiling studies were eligible for inclusion in the systematic review. Only 3 reports included information on protein identity...

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.

Two-Step Amyloid Aggregation: Sequential Lag Phase Intermediates

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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.

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.

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.

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.

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.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

Science.gov (United States)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

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

Pharmacology profiling of chemicals and proteins

DEFF Research Database (Denmark)

Kringelum, Jens Vindahl

between pharmaceuticals and proteins in vivo potential leads to unwanted adverse effects, toxicity and reduced half-life, but can also lead to novel therapeutic effects of already approved pharmaceuticals. Hence identification of in vivo targets is of importance in discovery, development and repurposing....... This limitation complicates adverse effect assessment in the early drug-development phase, thus contributing to drugattrition. Prediction models offer the possibility to close these gaps and provide more complete pharmacology profiles, however improvements in performances are required for these tools to serve...... to its nonself origin, which potentially alters the pharmacology profile of the substance. The neutralization of biopharmaceuticals by antidrug antibodies (ADAs) is an important element in the immune response cascade, however studies of ADA binding site on biopharmaceuticals, referred to as B...

Injury Profile SIMulator, a qualitative aggregative modelling framework to predict crop injury profile as a function of cropping practices, and the abiotic and biotic environment. I. Conceptual bases.

Directory of Open Access Journals (Sweden)

Jean-Noël Aubertot

Full Text Available The limitation of damage caused by pests (plant pathogens, weeds, and animal pests in any agricultural crop requires integrated management strategies. Although significant efforts have been made to i develop, and to a lesser extent ii combine genetic, biological, cultural, physical and chemical control methods in Integrated Pest Management (IPM strategies (vertical integration, there is a need for tools to help manage Injury Profiles (horizontal integration. Farmers design cropping systems according to their goals, knowledge, cognition and perception of socio-economic and technological drivers as well as their physical, biological, and chemical environment. In return, a given cropping system, in a given production situation will exhibit a unique injury profile, defined as a dynamic vector of the main injuries affecting the crop. This simple description of agroecosystems has been used to develop IPSIM (Injury Profile SIMulator, a modelling framework to predict injury profiles as a function of cropping practices, abiotic and biotic environment. Due to the tremendous complexity of agroecosystems, a simple holistic aggregative approach was chosen instead of attempting to couple detailed models. This paper describes the conceptual bases of IPSIM, an aggregative hierarchical framework and a method to help specify IPSIM for a given crop. A companion paper presents a proof of concept of the proposed approach for a single disease of a major crop (eyespot on wheat. In the future, IPSIM could be used as a tool to help design ex-ante IPM strategies at the field scale if coupled with a damage sub-model, and a multicriteria sub-model that assesses the social, environmental, and economic performances of simulated agroecosystems. In addition, IPSIM could also be used to help make diagnoses on commercial fields. It is important to point out that the presented concepts are not crop- or pest-specific and that IPSIM can be used on any crop.

Injury Profile SIMulator, a qualitative aggregative modelling framework to predict crop injury profile as a function of cropping practices, and the abiotic and biotic environment. I. Conceptual bases.

Science.gov (United States)

Aubertot, Jean-Noël; Robin, Marie-Hélène

2013-01-01

The limitation of damage caused by pests (plant pathogens, weeds, and animal pests) in any agricultural crop requires integrated management strategies. Although significant efforts have been made to i) develop, and to a lesser extent ii) combine genetic, biological, cultural, physical and chemical control methods in Integrated Pest Management (IPM) strategies (vertical integration), there is a need for tools to help manage Injury Profiles (horizontal integration). Farmers design cropping systems according to their goals, knowledge, cognition and perception of socio-economic and technological drivers as well as their physical, biological, and chemical environment. In return, a given cropping system, in a given production situation will exhibit a unique injury profile, defined as a dynamic vector of the main injuries affecting the crop. This simple description of agroecosystems has been used to develop IPSIM (Injury Profile SIMulator), a modelling framework to predict injury profiles as a function of cropping practices, abiotic and biotic environment. Due to the tremendous complexity of agroecosystems, a simple holistic aggregative approach was chosen instead of attempting to couple detailed models. This paper describes the conceptual bases of IPSIM, an aggregative hierarchical framework and a method to help specify IPSIM for a given crop. A companion paper presents a proof of concept of the proposed approach for a single disease of a major crop (eyespot on wheat). In the future, IPSIM could be used as a tool to help design ex-ante IPM strategies at the field scale if coupled with a damage sub-model, and a multicriteria sub-model that assesses the social, environmental, and economic performances of simulated agroecosystems. In addition, IPSIM could also be used to help make diagnoses on commercial fields. It is important to point out that the presented concepts are not crop- or pest-specific and that IPSIM can be used on any crop.

Self-aggregation of magnetic semiconductor EuS nanocrystals

International Nuclear Information System (INIS)

Tanaka, Atsushi; Hasegawa, Yasuchika; Kamikubo, Hironari; Kataoka, Mikio; Kawai, Tsuyoshi

2009-01-01

Controlled formation of aggregates having organized structure of cube-shaped EuS nanocrystals is reported. The EuS aggregates in liquid media (methanol) were obtained by means of van der Waals interaction between EuS nanocrystals. The packing structure of the EuS aggregates is characterized with transmission electron microscopy (TEM) and small angle X-ray scattering measurements (SAXS). TEM image indicates the EuS nanocrystals form self-aggregated 2D orthogonal lattice structure. The diffraction peak of (111) of SAXS profile shows that the cube-shaped EuS form 3D cubic superlattice. We successfully demonstrated that the aggregates of cube-shaped EuS nanocrystals formed cubic stacking structure.

Profil Protein Trypanosoma evansi dari Daerah Geografis Berbeda di Indonesia Tahun 2012-2014 dengan Sodium Dodecil Sulphate Polyacrylamide Gel Electrophoresis (TRYPANOSOMA EVANSI PROTEIN PROFILE OF DIFFERENT GEOGRAPHICAL AREAS ORIGIN IN INDONESIA

Directory of Open Access Journals (Sweden)

Fitrine Ekawasti

2018-01-01

Full Text Available Surra outbreak in 2012 has led to more than 1,700 animals have died in the province of East Nusa Tenggara (NTT Indonesia. Surra case sporadically continues throughout the year in various areas, especially Kalimantan, Banten as well as other areas. Some reports reveal differences in protein profiles among multiple isolates of T. evansi. Therefore the purpose of this research were to find out the protein profile of each isolate T. evansi in Indonesia and the possible biological differences among them. Eleven isolates originating from the province of East Nusa Tenggara, South Kalimantan and Central Kalimantan, Banten, Lampung and Bengkulu has been isolated and purified Using DEAE. Trypanosoma isolate were frezeethawing repeatedly to obtain soluble protein. Furthermore, soluble protein is treated with heating or without heating and then each was run on SDS PAGE with Coomassie Blue staining. The protein profiles of all isolates were compared each other. The results showed that eleven isolates of T. evansi in Indonesia has a very diverse protein profile. Then for the purposes of development of diagnostic kit can be used whole lysate cell (WCL as stock antigen in serological test process.

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)

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.

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.

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.

Pretreatment of flaxseed protein isolate by high hydrostatic pressure: Impacts on protein structure, enzymatic hydrolysis and final hydrolysate antioxidant capacities.

Science.gov (United States)

Perreault, Véronique; Hénaux, Loïc; Bazinet, Laurent; Doyen, Alain

2017-04-15

The effect of high hydrostatic pressure (HHP) on flaxseed protein structure and peptide profiles, obtained after protein hydrolysis, was investigated. Isolated flaxseed protein (1%, m/v) was subjected to HHP (600MPa, 5min or 20min at 20°C) prior to hydrolysis with trypsin only and trypsin-pronase. The results demonstrated that HHP treatment induced dissociation of flaxseed proteins and generated higher molecular weight aggregates as a function of processing duration. Fluorescence spectroscopy showed that HHP treatment, as well as processing duration, had an impact on flaxseed protein structure since exposition of hydrophobic amino acid tyrosine was modified. Except for some specific peptides, the concentrations of which were modified, similar peptide profiles were obtained after hydrolysis of pressure-treated proteins using trypsin. Finally, hydrolysates obtained using trypsin-pronase had a greater antioxidant capacity (ORAC) than control samples; these results confirmed that HHP enhanced the generation of antioxidant peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.

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...

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

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

Science.gov (United States)

Lim, Sanghyun; Chisholm, Kenneth; Coffin, Robert H; Peters, Rick D; Al-Mughrabi, Khalil I; Wang-Pruski, Gefu; Pinto, Devanand M

2012-04-06

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

Effects of Holder pasteurization on the protein profile of human milk.

Science.gov (United States)

Peila, Chiara; Coscia, Alessandra; Bertino, Enrico; Cavaletto, Maria; Spertino, Stefano; Icardi, Sara; Tortone, Claudia; Visser, Gerard H A; Gazzolo, Diego

2016-04-07

The most widespread method for the treatment of donor milk is the Holder pasteurization (HoP). The available literature data show that HoP may cause degradation of some bioactive components. The aim of this study was to determine the effect of HoP on the protein profile of human milk (HM) using a GeLC-MS method, a proteomic approach and a promising technique able to offer a qualitative HM protein profile. HM samples were collected by standardized methods from 20 mothers carrying both preterm and term newborns. A aliquot of each sample was immediately frozen at -80 °C, whilst another one was Holder pasteurized and then frozen. All samples were then analyzed by GeLC-MS. The protein bands of interest were excised from the gel, digested with trypsin and identified by nano-HPLC-MS/MS analysis. The protein profile before and after HoP showed qualitative differences only in 6 samples out of 20, while in the remaining 14 no detectable differences were found. The differences interested only colostrums and transitional milk samples and regarded the decrease of the electrophoretic bands corresponding to alpha and beta-casein, tenascin, lactoferrin and immunoglobulin. In the majority of samples, HoP did not cause any modification, thereby preserving the biological activity of HM proteins.

Effects of Pineal Proteins on Biochemical, Enzyme Profile and Non ...

African Journals Online (AJOL)

Effects of Pineal Proteins on Biochemical, Enzyme Profile and Non-Specific Immune Response of Indian Goats under Thermal Stress. ... Total precipitated pineal proteins successfully and significantly relieved the animals from adverse effects of heat stress and metyrapone treatment. There is evidence that most of the ...

PROFIL PROTEIN HIPOFISA SAPI PERAH PERANAKAN FRIES HOLLAND (PFH BETINA FASE FOLIKULER DAN LUTEA

Directory of Open Access Journals (Sweden)

Nurul Isnaini

2012-04-01

Full Text Available ABSTRAK   Penelitian dilakukan dengan tujuan untuk mengetahui profil protein hipofisa sapi perah PFH betina fase folikuler dan fase luteal dengan menggunakan metode SDS-PAGE. Hasil penelitian ini diharapkan dapat digunakan sebagai acuan untuk melaksanakan pengujian jenis protein tertentu yang terdapat dalam hipofisa sapi perah PFH.Materi yang digunakan dalam penelitian ini adalah hipofisa sapi perah PFH betina Fase Folikuler dan Fase Luteal. Sampel hipofisa didapatkan dari Rumah Potong Hewan (RPH Singosari Malang. Metode penelitian yang digunakan dalam penelitian ini adalah metode observasi. Dimana sampel hipofisa sapi perah PFH fase folikuler dan fase luteal dilakukan isolasi protein, dan ditentukan Berat Molekul (BM proteinnya. Data yang diperoleh dianalisis dengan menggunakan analisis diskriptif. Hasil penelitian menunjukkan bahwa profil protein hipofisa fase folikuler dan fase luteal sapi perah PFH memiliki perbedaan. Pada hipofisa fase folikuler memiliki 12 pita protein, yaitu 163; 112,3; 101,3; 85,8; 80,6; 71,2; 53,3; 45,2; 39,9; 32,4; 29,8; dan 24,8 kDa. Pada hipofisa fase luteal memiliki 12 pita protein, yaitu 163; 112,3; 101,3; 85,8; 71,2; 60,3; 53,3; 45,2; 39,9; 32,4; 29,8; dan 24,8 kDa. Pita protein yang membedakan adalah pita dengan berat molekul 80,6 kDa hanya terdapat pada hipofisa fase folikuler, dan pita dengan berat molekul 60,3 kDa hanya terdapat pada hipofisa fase luteal. Kesimpulan yang diperoleh dari penelitian ini adalah terdapat perbedaan profil protein yang dihasilkan pada hipofisa fase folikuler dengan hipofisa fase luteal. Saran yang diberikan adalah perlu dilakukan penelitian lanjutan yaitu uji immunoblothing atau westernblothing (WB untuk memastikan keberadaan  protein-protein tertentu.   Kata kunci: Hipofisa, folikuler, luteal, berat molekul protein. HIPOPHISIS PROTEIN PROFILE OF CROSSBREED FRIESH HOLLAND DAIRY CATTLE IN FOLLICULAR AND LUTEAL PHASE   ABSTRACT   The aim of this research was to identification of

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.

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...

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.

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.

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...

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.

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.

Protein Profile study of clinical samples using Laser Induced Fluorescence as the detection method

DEFF Research Database (Denmark)

Karemore, Gopal Raghunath; Raja, Sujatha N.; Rai, Lavanya

2009-01-01

  Protein profiles of tissue homogenates were recorded using HPLC separation and LIF detection method. The samples were collected from volunteers with clinically normal or cervical cancer conditions. It is shown that the protein profile can be classified as belonging to malignant or normal state ...

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.

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.

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.

Effect of soy protein on serum lipid profile and some lipid ...

African Journals Online (AJOL)

The effect of soy protein on serum lipid profile and some lipid metabolizing enzymes in rats fed with cholesterol diets was examined in this study. Rats were subjected to feeding trial over a period of six weeks on formulated diets containing: 20% soy protein with 0% cholesterol (group A), 20% soy protein with 5% cholesterol ...

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.

Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

Science.gov (United States)

Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

2016-04-01

We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

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.

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.

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

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.

Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

Institute of Scientific and Technical Information of China (English)

Yi Feng; Zhong-Min Tian; Ming-Xi Wan; Zhao-Bin Zheng

2007-01-01

AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.METHODS: Total proteins from human hepatocarcinomacell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite.Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)and database searching.RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin,endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein Ⅰ,peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

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.

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

Commercial Building Tenant Energy Usage Aggregation and Privacy

Energy Technology Data Exchange (ETDEWEB)

Livingston, Olga V.; Pulsipher, Trenton C.; Anderson, David M.; Wang, Na

2014-10-31

A growing number of building owners are benchmarking their building energy use. This requires the building owner to acquire monthly whole-building energy usage information, which can be challenging for buildings in which individual tenants have their own utility meters and accounts with the utility. Some utilities and utility regulators have turned to aggregation of customer energy use data (CEUD) as a way to give building owners whole-building energy usage data while protecting customer privacy. Meter profile aggregation adds a layer of protection that decreases the risk of revealing CEUD as the number of meters aggregated increases. The report statistically characterizes the similarity between individual energy usage patterns and whole-building totals at various levels of meter aggregation.

Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?

DEFF Research Database (Denmark)

Callesen, Anne K; Madsen, Jonna S; Vach, Werner

2009-01-01

Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and ...

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.

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.

Molecular simulations of beta-amyloid protein near hydrated lipids (PECASE).

Energy Technology Data Exchange (ETDEWEB)

Thompson, Aidan Patrick; Han, Kunwoo (Texas A& M University, College Station, TX); Ford, David M. (Texas A& M University, College Station, TX)

2005-12-01

We performed molecular dynamics simulations of beta-amyloid (A{beta}) protein and A{beta} fragment(31-42) in bulk water and near hydrated lipids to study the mechanism of neurotoxicity associated with the aggregation of the protein. We constructed full atomistic models using Cerius2 and ran simulations using LAMMPS. MD simulations with different conformations and positions of the protein fragment were performed. Thermodynamic properties were compared with previous literature and the results were analyzed. Longer simulations and data analyses based on the free energy profiles along the distance between the protein and the interface are ongoing.

Serum Protein Profile Study of Clinical Samples Using High Performance Liquid Chromatography-Laser Induced Fluorescence

DEFF Research Database (Denmark)

Karemore, Gopal Raghunath; Ukendt, Sujatha; Rai, Lavanya

2009-01-01

The serum protein profiles of normal subjects, patients diagnosed with cervical cancer, and oral cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence detection (HPLC-LIF). Serum protein profiles of the above three classes were tested for estab...

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.

Ecological and morphological profile of floating spherical Cladophora socialis aggregations in central Thailand.

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Isao Tsutsui

Full Text Available The unique beauty of spherical aggregation forming algae has attracted much attention from both the scientific and lay communities. Several aegagropilous seaweeds have been identified to date, including the plants of genus Cladophora and Chaetomorpha. However, this phenomenon remains poorly understood. In July 2013, a mass occurrence of spherical Cladophora aggregations was observed in a salt field reservoir in Central Thailand. The aims of the present study were to describe the habitat of the spherical aggregations and confirm the species. We performed a field survey, internal and external morphological observations, pyrenoid ultrastructure observations, and molecular sequence analysis. Floating spherical Cladophora aggregations (1-8 cm in diameter were observed in an area ~560 m2, on the downwind side of the reservoir where there was water movement. Individual filaments in the aggregations were entangled in each other; consequently, branches growing in different directions were observed within a clump. We suggest that water movement and morphological characteristics promote the formation of spherical aggregations in this species. The molecular sequencing results revealed that the study species was highly homologous to both C. socialis and C. coelothrix. However, the diameter of the apical cells in the study species was less than that of C. coelothrix. The pyrenoid ultrastructure was more consistent with that of C. socialis. We conclude that the study species is C. socialis. This first record of spherical aggregations in this species advances our understanding of these formations. However, further detailed physical measurements are required to fully elucidate the mechanism behind these spherical formations.

Ecological and morphological profile of floating spherical Cladophora socialis aggregations in central Thailand.

Science.gov (United States)

Tsutsui, Isao; Miyoshi, Tatsuo; Sukchai, Halethichanok; Pinphoo, Piyarat; Aue-Umneoy, Dusit; Meeanan, Chonlada; Songphatkaew, Jaruwan; Klomkling, Sirimas; Yamaguchi, Iori; Ganmanee, Monthon; Sudo, Hiroyuki; Hamano, Kaoru

2015-01-01

The unique beauty of spherical aggregation forming algae has attracted much attention from both the scientific and lay communities. Several aegagropilous seaweeds have been identified to date, including the plants of genus Cladophora and Chaetomorpha. However, this phenomenon remains poorly understood. In July 2013, a mass occurrence of spherical Cladophora aggregations was observed in a salt field reservoir in Central Thailand. The aims of the present study were to describe the habitat of the spherical aggregations and confirm the species. We performed a field survey, internal and external morphological observations, pyrenoid ultrastructure observations, and molecular sequence analysis. Floating spherical Cladophora aggregations (1-8 cm in diameter) were observed in an area ~560 m2, on the downwind side of the reservoir where there was water movement. Individual filaments in the aggregations were entangled in each other; consequently, branches growing in different directions were observed within a clump. We suggest that water movement and morphological characteristics promote the formation of spherical aggregations in this species. The molecular sequencing results revealed that the study species was highly homologous to both C. socialis and C. coelothrix. However, the diameter of the apical cells in the study species was less than that of C. coelothrix. The pyrenoid ultrastructure was more consistent with that of C. socialis. We conclude that the study species is C. socialis. This first record of spherical aggregations in this species advances our understanding of these formations. However, further detailed physical measurements are required to fully elucidate the mechanism behind these spherical formations.

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.

ANALISIS PROFIL PROTEIN DARAH ANAK KAMBING PERANAKAN ETAWAH DENGAN PEMBERIAN PAKAN SUBSTITUSI SUSU SAPI

Directory of Open Access Journals (Sweden)

Teguh Wicaksono

2017-08-01

Full Text Available The objective of this study is to determine the protein profile of pre-weaning kids fed with cow's milk as a substitute for dam’s milk. The materials used were 18 Etawah Descendant (PE kids born the twin at the age of 5-13 days from 3-4-year-old dams. This experimental design was a completely randomized design with three treatments with six replications per treatment, namely the control (T0 fed 100% goat’s milk, treatment 1 (T1 fed 50% goat’s milk and 50% cow’s milk, treatment 2 (T2 fed 100% cow’s milk. The protein profile serum was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE method, 12,5% of the resolving gel and 3% of the stacking gel were used. The protein profile of the 5-14 days old PE kids were 19 protein bands with the molecular weight ranging from 15-160 kDa. The kids fed with 100% goat milk (T0 and those substituted by 50% cow's milk (T1, it was produced 19 protein bands with molecular weights ranging from 15 kDa to 155 kDa, while those fed with 100 % cow's milk (T2, it was produced 17 protein bands with molecular weights ranging from 13 kDa to 160 kDa. It can be concluded that the dam's milk substitute using cow's milk at the 50% level does not affect the blood protein profile of goat kids, while the 100% substitute produces the different number and types of protein

Protein profiles of hatchery egg shell membrane.

Science.gov (United States)

Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

2016-01-01

Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

Inflammation Induces TDP-43 Mislocalization and Aggregation.

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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.

Genotypic variability and mutant identification in cicer arietinum L. by seed storage protein profiling

International Nuclear Information System (INIS)

Hameed, A.; Iqbal, N.; Shah, T.M.

2012-01-01

A collection of thirty-four chickpea genotypes, including five kabuli and twenty-nine desi, were analyzed by SDS-PAGE for seed storage protein profiling. Total soluble seed proteins were resolved on 12% gels. A low level of variability was observed in desi as compared to kabuli genotypes. Dendrogram based on electrophoretic data clustered the thirty-four genotypes in four major groups. As large number of desi genotypes illustrated identical profiles, therefore could not be differentiated on the basis of seed storage protein profiles. One kabuli genotype ILC-195 found to be the most divergent showing 86% similarity with all other genotypes. ILC-195 can be distinguished from its mutant i.e., CM-2000 and other kabuli genotypes on the basis of three peptides i.e. SSP-66, SSP-43 and SSP-39. Some proteins peptides were found to be genotype specific like SSP-26 for ICCV-92311. Uniprot and NCBI protein databases were searched for already reported and characterized seed storage proteins in chickpea. Among 33 observed peptides, only six seed storages proteins from chickpea source were available in databases. On the basis of molecular weight similarity, identified peptides were SSP-64 as Serine/Threonine dehydratase, SSP-56 as Alpha-amylase inhibitor, SSP-50 as Provicillin, SSP-39 as seed imbibition protein, SSP-35 as Isoflavane reductase and SSP-19 as lipid transport protein. Highest variability was observed in vicillin subunits and beta subunits of legumins and its polymorphic forms. In conclusion, seed storage profiling can be economically used to asses the genetic variation, phylogenetic relationship and as markers to differentiate mutants from their parents. (author)

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,

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

Directory of Open Access Journals (Sweden)

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.

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.

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

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.

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.

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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.

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

Serum protein profile of Malaria patients through SDS-PAGE method ...

African Journals Online (AJOL)

Serum protein profile of Malaria patients through SDS-PAGE method. ... reliable method in the diagnosis of antibodies produced against Plasmodium spps. ... of malaria patients may be undertaken for study to develop possible future vaccine.

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.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

Energy Technology Data Exchange (ETDEWEB)

Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris; Hyman, Michael R.; Löffler, F. E.

2016-01-29

Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH₃) to nitrite (NO₂^₋) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH₄⁺-dependent O₂uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and

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...

Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

Energy Technology Data Exchange (ETDEWEB)

Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

2017-12-06

Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

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.

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.

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.

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...

Analysis of protein profiles in diabetic rat blood plasma that induced by alloxan

Science.gov (United States)

Hidayati, Dewi; Abdulgani, Nurlita; Setiyawan, Hengki; Trisnawati, Indah; Ashuri, Nova Maulidina; Sa'adah, Noor Nailis

2017-06-01

Proteomics is the study to identify the proteins involved in physiological metabolic pathway. The protein profiles of blood plasma from alloxan-induced diabetic rats has investigated using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Data were analyzed descriptively based on variations of the type and intensity of the protein. There were identified the similarity of protein variant between diabetic and control rats included ankyrin (200kDa), IgG (150kDa), nephrin (136 kDa), IDE (112 kDA), albumin (66 kDa), prealbumin (55 kDA), CICP (43 kDa), ApoA-V (39 kDa), GAPDH (35 kDa), C-RP (27,1 kDa), leptin (16 kDa) and apelin (13 kDa). However, the apelin profile at diabetic rats shows the higher intensity than control.

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...

Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

Czech Academy of Sciences Publication Activity Database

Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

2015-01-01

Roč. 16, č. 11 (2015), s. 1616-1621 ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity-based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.850, year: 2015

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

Long-term forecasting of hourly electricity load: Identification of consumption profiles and segmentation of customers

DEFF Research Database (Denmark)

Møller Andersen, Frits; Larsen, Helge V.; Boomsma, Trine Krogh

2013-01-01

, to model and forecast long-term changes in the aggregated electricity load profile, we identify profiles for different categories of customers and link these to projections of the aggregated annual consumption by categories of customers. Long-term projection of the aggregated load is important for future......Data for aggregated hourly electricity demand shows systematic variations over the day, week, and seasons, and forecasting of aggregated hourly electricity load has been the subject of many studies. With hourly metering of individual customers, data for individual consumption profiles is available....... Using this data and analysing the case of Denmark, we show that consumption profiles for categories of customers are equally systematic but very different for distinct categories, that is, distinct categories of customers contribute differently to the aggregated electricity load profile. Therefore...

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization.

Science.gov (United States)

Raju, Murugesan; Mooney, Brian P; Thakkar, Kavi M; Giblin, Frank J; Schey, Kevin L; Sharma, K Krishna

2015-03-01

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Changes in the serum protein profile during radiotherapy to the upper respiratory and gastro-intestinal tracts

International Nuclear Information System (INIS)

David, M.; Lobera, A.; Legrand, E.

1984-01-01

Patients with a cancer of the upper airways of upper gastro-intestinal tract present a state of malnutrition as a result of the disease itself and, more importantly, as a result of its localisation. Loco-regional radiotherapy often leads to an aggravation, of this state. The protein profile, consisting of nine serum proteins, was determined each week in 54 patients with cancer of the upper respirato-gastro-intestinal tract receiving radiotherapy. During the course of radiotherapy, the already altered nutritional state of these patients deteriorated further, as shown by a regular and significant downturn in the weight curve. The weekly monitoring of the protein profile showed a gradual and significant decrease in the levels of nutritional proteins (prealbumin, retinol binding protein, transferrin) and immunoglobulins (IgM, IgA) and a small variation in the levels of inflammatory proteins (haptoglobin, orosomucoid, C3 complement fraction, alpha 1 -antitrypsin). The protein profile, established on the basis of carefully selected proteins, can provide useful information in the monitoring of a patient's nutritional state [fr

Changes in the serum protein profile during radiotherapy to the upper respiratory and gastro-intestinal tracts

Energy Technology Data Exchange (ETDEWEB)

David, M; Lobera, A; Legrand, E [Fondation Bergorie, Bordeaux (France)

1984-01-01

Patients with a cancer of the upper airways on upper gastro-intestinal tract present a state of malnutrition as a result of the disease itself and, more importantly, as a result of its localisation. Loco-regional radiotherapy often leads to an aggravation, of this state. The protein profile, consisting of nine serum proteins, was determined each week in 54 patients with cancer of the upper respirato-gastro-intestinal tract receiving radiotherapy. During the course of radiotherapy, the already altered nutritional state of these patients deteriorated further, as shown by a regular and significant downturn in the weight curve. The weekly monitoring of the protein profile showed a gradual and significant decrease in the levels of nutritional proteins (prealbumin, retinol binding protein, transferrin) and immunoglobulins (IgM, IgA) and a small variation in the levels of inflammatory proteins (haptoglobin, orosomucoid, C3 complement fraction, alpha/sub 1/-antitrypsin). The protein profile, established on the basis of carefully selected proteins, can provide useful information in the monitoring of a patient's nutritional state.

Modulation of invasive phenotype by interstitial pressure-driven convection in aggregates of human breast cancer cells.

Directory of Open Access Journals (Sweden)

Joe Tien

Full Text Available This paper reports the effect of elevated pressure on the invasive phenotype of patterned three-dimensional (3D aggregates of MDA-MB-231 human breast cancer cells. We found that the directionality of the interstitial pressure profile altered the frequency of invasion by cells located at the surface of an aggregate. In particular, application of pressure at one end of an aggregate suppressed invasion at the opposite end. Experimental alteration of the configuration of cell aggregates and computational modeling of the resulting flow and solute concentration profiles revealed that elevated pressure inhibited invasion by altering the chemical composition of the interstitial fluid near the surface of the aggregate. Our data reveal a link between hydrostatic pressure, interstitial convection, and invasion.

Maternal serum protein profile and immune response protein subunits as markers for non-invasive prenatal diagnosis of trisomy 21, 18, and 13

KAUST Repository

Narasimhan, Kothandaraman

2013-02-01

Objectives: To use proteomics to identify and characterize proteins in maternal serum from patients at high-risk for fetal trisomy 21, trisomy 18, and trisomy 13 on the basis of ultrasound and maternal serum triple tests. Methods: We performed a comprehensive proteomic analysis on 23 trisomy cases and 85 normal cases during the early second trimester of pregnancy. Protein profiling along with conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis/Tandem mass spectrometry analysis was carried out to characterize proteins associated with each trisomy condition and later validated using Western blot. Results: Protein profiling approach using surface enhanced laser desorption/ionization time-of-flight mass (SELDI-TOF/MS) spectrometry resulted in the identification of 37 unique hydrophobic proteomic features for three trisomy conditions. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Matrix Assisted Laser Desorption Ionization - Time of Flight/Time of Flight (MALDI-TOF/TOF) and western blot, glyco proteins such as alpha-1-antitrypsin, apolipoprotein E, apolipoprotein H, and serum carrier protein transthyretin were identified as potential maternal serum markers for fetal trisomy condition. The identified proteins showed differential expression at the subunit level. Conclusions: Maternal serum protein profiling using proteomics may allow non-invasive diagnostic testing for the most common trisomies and may complement ultrasound-based methods to more accurately determine pregnancies with fetal aneuploidies. © 2013 John Wiley & Sons, Ltd.

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.

Quantitative analysis of gender stereotypes and information aggregation in a national election.

Directory of Open Access Journals (Sweden)

Michele Tumminello

Full Text Available By analyzing a database of a questionnaire answered by a large majority of candidates and elected in a parliamentary election, we quantitatively verify that (i female candidates on average present political profiles which are more compassionate and more concerned with social welfare issues than male candidates and (ii the voting procedure acts as a process of information aggregation. Our results show that information aggregation proceeds with at least two distinct paths. In the first case candidates characterize themselves with a political profile aiming to describe the profile of the majority of voters. This is typically the case of candidates of political parties which are competing for the center of the various political dimensions. In the second case, candidates choose a political profile manifesting a clear difference from opposite political profiles endorsed by candidates of a political party positioned at the opposite extreme of some political dimension.

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.

Assessing Basal and Acute Autophagic Responses in the Adult Drosophila Nervous System: The Impact of Gender, Genetics and Diet on Endogenous Pathway Profiles.

Directory of Open Access Journals (Sweden)

Eric P Ratliff

Full Text Available The autophagy pathway is critical for the long-term homeostasis of cells and adult organisms and is often activated during periods of stress. Reduced pathway efficacy plays a central role in several progressive neurological disorders that are associated with the accumulation of cytotoxic peptides and protein aggregates. Previous studies have shown that genetic and transgenic alterations to the autophagy pathway impacts longevity and neural aggregate profiles of adult Drosophila. In this study, we have identified methods to measure the acute in vivo induction of the autophagy pathway in the adult fly CNS. Our findings indicate that the genotype, age, and gender of adult flies can influence pathway responses. Further, we demonstrate that middle-aged male flies exposed to intermittent fasting (IF had improved neuronal autophagic profiles. IF-treated flies also had lower neural aggregate profiles, maintained more youthful behaviors and longer lifespans, when compared to ad libitum controls. In summary, we present methodology to detect dynamic in vivo changes that occur to the autophagic profiles in the adult Drosophila CNS and that a novel IF-treatment protocol improves pathway response in the aging nervous system.

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.

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

Science.gov (United States)

Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

2012-01-01

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

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.

Distinct profile of vascular progenitor attachment to extracellular matrix proteins in cancer patients.

Science.gov (United States)

Labonté, Laura; Li, Yuhua; Addison, Christina L; Brand, Marjorie; Javidnia, Hedyeh; Corsten, Martin; Burns, Kevin; Allan, David S

2012-04-01

Vascular progenitor cells (VPCs) facilitate angiogenesis and initiate vascular repair by homing in on sites of damage and adhering to extracellular matrix (ECM) proteins. VPCs also contribute to tumor angiogenesis and induce angiogenic switching in sites of metastatic cancer. In this study, the binding of attaching cells in VPC clusters that form in vitro on specific ECM proteins was investigated. VPC cluster assays were performed in vitro on ECM proteins enriched in cancer cells and in remodelling tissue. Profiles of VPC clusters from patients with cancer were compared to healthy controls. The role of VEGF and integrin-specific binding of angiogenic attaching cells was addressed. VPC clusters from cancer patients were markedly increased on fibronectin relative to other ECM proteins tested, in contrast to VPC clusters from control subjects, which formed preferentially on laminin. Specific integrin-mediated binding of attaching cells in VPC clusters was matrix protein-dependent. Furthermore, cancer patients had elevated plasma VEGF levels compared to healthy controls and VEGF facilitated preferential VPC cluster formation on fibronectin. Incubating cells from healthy controls with VEGF induced a switch from the 'healthy' VPC binding profile to the profile observed in cancer patients with a marked increase in VPC cluster formation on fibronectin. The ECM proteins laminin and fibronectin support VPC cluster formation via specific integrins on attaching cells and can facilitate patterns of VPC cluster formation that are distinct in cancer patients. Larger studies, however, are needed to gain insight on how tumor angiogenesis may differ from normal repair processes.

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.

Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

Science.gov (United States)

Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

2016-02-18

Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease

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.

Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography.

Science.gov (United States)

Kim, Soung Min; Jeong, Dasul; Kim, Min Keun; Lee, Sang Shin; Lee, Suk Keun

2017-08-08

Oral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods. The present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC). Two types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin. The increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction

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...

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

Science.gov (United States)

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

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.

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.

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.

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...

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

ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles.

Science.gov (United States)

Ghouzam, Yassine; Postic, Guillaume; Guerin, Pierre-Edouard; de Brevern, Alexandre G; Gelly, Jean-Christophe

2016-06-20

Protein structure prediction based on comparative modeling is the most efficient way to produce structural models when it can be performed. ORION is a dedicated webserver based on a new strategy that performs this task. The identification by ORION of suitable templates is performed using an original profile-profile approach that combines sequence and structure evolution information. Structure evolution information is encoded into profiles using structural features, such as solvent accessibility and local conformation -with Protein Blocks-, which give an accurate description of the local protein structure. ORION has recently been improved, increasing by 5% the quality of its results. The ORION web server accepts a single protein sequence as input and searches homologous protein structures within minutes. Various databases such as PDB, SCOP and HOMSTRAD can be mined to find an appropriate structural template. For the modeling step, a protein 3D structure can be directly obtained from the selected template by MODELLER and displayed with global and local quality model estimation measures. The sequence and the predicted structure of 4 examples from the CAMEO server and a recent CASP11 target from the 'Hard' category (T0818-D1) are shown as pertinent examples. Our web server is accessible at http://www.dsimb.inserm.fr/ORION/.

Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry

Science.gov (United States)

Souda, Puneet; Ryan, Christopher M.; Cramer, William A.; Whitelegge, Julian

2011-01-01

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein’s native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electroncapture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. PMID:21982782

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.

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.

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)

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.

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.)

Multivariate analysis of protein profiles of metal hyperaccumulator Thlaspi caerulescens accessions.

NARCIS (Netherlands)

Tuomainen, M.H.; Nunan, N.; Lehesranta, S.J.; Tervahauta, A.I.; Hassinen, V.H.; Schat, H.; Koistinen, K.M.; Auriola, S.; McNicol, J.; Karenlampi, S.O.

2006-01-01

Thlaspi caerulescens is increasingly acknowledged as one of the best models for studying metal hyperaccumulation in plants. In order to study the mechanisms underlying metal hyper-accumulation, we used proteomic profiling to identify differences in protein intensities among three T caerulescens

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.

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

Science.gov (United States)

da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

2017-08-09

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

Protein profiles of serum, brain regions and hypophyses of pubertal ...

African Journals Online (AJOL)

The effects of dietary fumonisin B1 (FB1 ), a toxin produced mainly by Fusarium verticillioides and F. proliferatum that grow on maize worldwide, on protein profiles of serum, brain regions and hypophyses were studied in 24 male Large White weanling pigs randomly divided into four groups (n = 6). In a completely ...

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.

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.

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

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...

Bioorthogonal chemistry: applications in activity-based protein profiling.

Science.gov (United States)

Willems, Lianne I; van der Linden, Wouter A; Li, Nan; Li, Kah-Yee; Liu, Nora; Hoogendoorn, Sascha; van der Marel, Gijs A; Florea, Bogdan I; Overkleeft, Herman S

2011-09-20

of chemical biology research include contributions from many areas of the multifaceted discipline of chemistry, and particularly from organic chemistry. Researchers apply knowledge inherent to organic chemistry, such as reactivity and selectivity, to the manipulation of specific biomolecules in biological samples (cell extracts, living cells, and sometimes even animal models) to gain insight into the biological phenomena in which these molecules participate. In this Account, we highlight some of the recent developments in chemical biology research driven by organic chemistry, with a focus on bioorthogonal chemistry in relation to activity-based protein profiling. The rigorous demands of bioorthogonality have not yet been realized in a truly bioorthogonal reagent pair, but remarkable progress has afforded a range of tangible contributions to chemical biology research. Activity-based protein profiling, which aims to obtain information on the workings of a protein (or protein family) within the larger context of the full biological system, has in particular benefited from these advances. Both activity-based protein profiling and bioorthogonal chemistry have been around for approximately 15 years, and about 8 years ago the two fields very profitably intersected. We expect that each discipline, both separately and in concert, will continue to make important contributions to chemical biology research. © 2011 American Chemical Society

Tunneling electron induced molecular electroluminescence from individual porphyrin J-aggregates

Energy Technology Data Exchange (ETDEWEB)

Meng, Qiushi; Zhang, Chao; Zhang, Yang, E-mail: zhyangnano@ustc.edu.cn, E-mail: zcdong@ustc.edu.cn; Zhang, Yao; Liao, Yuan; Dong, Zhenchao, E-mail: zhyangnano@ustc.edu.cn, E-mail: zcdong@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at the Microscale and Synergetic Innovation Center of Quantum Information and Quantum Physics, University of Science and Technology of China, Hefei, Anhui 230026 (China)

2015-07-27

We investigate molecular electroluminescence from individual tubular porphyrin J-aggregates on Au(111) by tunneling electron excitations in an ultrahigh-vacuum scanning tunneling microscope (STM). High-resolution STM images suggest a spiral tubular structure for the porphyrin J-aggregate with highly ordered “brickwork”-like arrangements. Such aggregated nanotube is found to behave like a self-decoupled molecular architecture and shows red-shifted electroluminescence characteristics of J-aggregates originated from the delocalized excitons. The positions of the emission peaks are found to shift slightly depending on the excitation sites, which, together with the changes in the observed spectral profiles with vibronic progressions, suggest a limited exciton coherence number within several molecules. The J-aggregate electroluminescence is also found unipolar, occurring only at negative sample voltages, which is presumably related to the junction asymmetry in the context of molecular excitations via the carrier injection mechanism.

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...

Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

Science.gov (United States)

Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

2011-01-01

Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

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.

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.

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.

Design and application of natural product derived probes for activity based protein profiling

OpenAIRE

Battenberg, Oliver Alexander

2015-01-01

The identification of new antibacterial protein targets by activity based protein profiling (ABPP) is an important approach to face the increasing emergence of resistant bacteria. The scope of this work focuses on three new strategies for the labeling of antibacterial protein-targets with natural product derived ABPP-probes: A.) Evaluation of the intrinsic photo-reactivity of α-pyrones and pyrimidones for use as photo-crosslinkers. B.) Synthesis of a benzophenone-tag that combines photo-cross...

Spall Strength Measurements of Concrete for Varying Aggregate Sizes

International Nuclear Information System (INIS)

Chhabildas, Lalit C.; Kipp, Marlin E.; Reinhart, William D.; Wilson, Leonard T.

1999-01-01

Controlled impact experiments have been performed to determine the spall strength of four different concrete compositions. The four concrete compositions are identified as, 'SAC-5, CSPC', (''3/4'') large, and (''3/8'') small, Aggregate. They differ primarily in aggregate size but with average densities varying by less than five percent. Wave profiles from sixteen experiments, with shock amplitudes of 0.07 to 0.55 GPa, concentrate primarily within the elastic regime. Free-surface particle velocity measurements indicate consistent pullback signals in the release profiles, denoting average span strength of approximately 40 MPa. It is the purpose of this paper to present spall measurements under uniaxial strain loading. Notwithstanding considerable wave structure that is a unique characteristic to the heterogeneous nature of the scaled concrete, the spall amplitudes appear reproducible and consistent over the pressure range reported in this study