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Sample records for prevent protein aggregation

  1. The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides.

    Science.gov (United States)

    Gillis, Judith; Schipper-Krom, Sabine; Juenemann, Katrin; Gruber, Anna; Coolen, Silvia; van den Nieuwendijk, Rian; van Veen, Henk; Overkleeft, Hermen; Goedhart, Joachim; Kampinga, Harm H; Reits, Eric A

    2013-06-14

    Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein aggregation, preventing polyQ peptide aggregation by chaperones should greatly improve polyQ clearance and prevent aggregate formation. Here we expressed polyQ peptides in cells and show that their intracellular aggregation is prevented by DNAJB6 and DNAJB8, members of the DNAJ (Hsp40) chaperone family. In contrast, HSPA/Hsp70 and DNAJB1, also members of the DNAJ chaperone family, did not prevent peptide-initiated aggregation. Intriguingly, DNAJB6 and DNAJB8 also affected the soluble levels of polyQ peptides, indicating that DNAJB6 and DNAJB8 inhibit polyQ peptide aggregation directly. Together with recent data showing that purified DNAJB6 can suppress fibrillation of polyQ peptides far more efficiently than polyQ expanded protein fragments in vitro, we conclude that the mechanism of DNAJB6 and DNAJB8 is suppression of polyQ protein aggregation by directly binding the polyQ tract.

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

    Directory of Open Access Journals (Sweden)

    Bani Kumar Pathak

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

  3. Protein aggregation caused by aminoglycoside action is prevented by a hydrogen peroxide scavenger.

    Science.gov (United States)

    Ling, Jiqiang; Cho, Chris; Guo, Li-Tao; Aerni, Hans R; Rinehart, Jesse; Söll, Dieter

    2012-12-14

    Protein mistranslation causes growth arrest in bacteria, mitochondrial dysfunction in yeast, and neurodegeneration in mammals. It remains poorly understood how mistranslated proteins cause such cellular defects. Here we demonstrate that streptomycin, a bactericidal aminoglycoside that increases ribosomal mistranslation, induces transient protein aggregation in wild-type Escherichia coli. We further determined the aggregated proteome using label-free quantitative mass spectrometry. To identify genes that reduce cellular mistranslation toxicity, we selected from an overexpression library protein products that increased resistance against streptomycin and kanamycin. The selected proteins were significantly enriched in members of the oxidation-reduction pathway. Overexpressing one of these proteins, alkyl hydroperoxide reductase subunit F (a protein defending bacteria against hydrogen peroxide), but not its inactive mutant suppressed aggregated protein formation upon streptomycin treatment and increased aminoglycoside resistance. This work provides in-depth analyses of an aggregated proteome caused by streptomycin and suggests that cellular defense against hydrogen peroxide lowers the toxicity of mistranslation. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides

    NARCIS (Netherlands)

    Gillis, J.; Schipper-Krom, S.; Juenemann, K.; Gruber, A.; Coolen, S.; van den Nieuwendijk, R.; van Veen, H.; Overkleeft, H.; Goedhart, J.; Kampinga, H.H.; Reits, E.A.

    2013-01-01

    Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein

  5. The DNAJB6 and DNAJB8 Protein Chaperones Prevent Intracellular Aggregation of Polyglutamine Peptides

    NARCIS (Netherlands)

    Gillis, Judith; Schipper-Krom, Sabine; Juenemann, Katrin; Gruber, Anna; Coolen, Silvia; van den Nieuwendijk, Rian; van Veen, Henk; Overkleeft, Hermen; Goedhart, Joachim; Kampinga, Harm H.; Reits, Eric A.

    2013-01-01

    Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein

  6. Protein Colloidal Aggregation Project

    Science.gov (United States)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

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

  7. 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.

  8. Proteins aggregation and human diseases

    International Nuclear Information System (INIS)

    Hu, Chin-Kun

    2015-01-01

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

  9. 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.

  10. Production of prone-to-aggregate proteins.

    Science.gov (United States)

    Lebendiker, Mario; Danieli, Tsafi

    2014-01-21

    Expression of recombinant proteins in Escherichia coli (E. coli) remains the most popular and cost-effective method for producing proteins in basic research and for pharmaceutical applications. Despite accumulating experience and methodologies developed over the years, production of recombinant proteins prone to aggregate in E. coli-based systems poses a major challenge in most research applications. The challenge of manufacturing these proteins for pharmaceutical applications is even greater. This review will discuss effective methods to reduce and even prevent the formation of aggregates in the course of recombinant protein production. We will focus on important steps along the production path, which include cloning, expression, purification, concentration, and storage. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. Disaggregases, molecular chaperones that resolubilize protein aggregates

    Directory of Open Access Journals (Sweden)

    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.

  12. Detergent-mediated protein aggregation.

    Science.gov (United States)

    Neale, Chris; Ghanei, Hamed; Holyoake, John; Bishop, Russell E; Privé, Gilbert G; Pomès, Régis

    2013-04-01

    Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Targeting Protein Aggregation for the Treatment of Degenerative Diseases

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

    2015-01-01

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

  14. Protein Aggregation Inhibitors for ALS Therapy

    Science.gov (United States)

    2013-07-01

    the underlying cause of protein misfolding and aggregation (oxidative stress damage ) and combating the aggregation caused by ROS that survive to...mechanisms of neuronal degeneration remain unknown in ALS, it has been postulated that protein misfolding and aggregation may be an early event that...of misfolded proteins . Of particular interest in this group is the proteasome ATPase, PSMC1. PSMC1 (also called Rpt2) has been reported to regulate

  15. Protein aggregates stimulate macropinocytosis facilitating their propagation.

    Science.gov (United States)

    Yerbury, Justin J

    2016-03-03

    Temporal and spatial patterns of pathological changes such as loss of neurons and presence of pathological protein aggregates are characteristic of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis, Frontotemporal Dementia, Alzheimer's disease and Parkinson's disease. These patterns are consistent with the propagation of protein misfolding and aggregation reminiscent of the prion diseases. There is a surge of evidence that suggests that large protein aggregates of a range of proteins are able to enter cells via macropinocytosis. Our recent work suggests that this process is activated by the binding of aggregates to the neuron cell surface. The current review considers the potential role of cell surface receptors in the triggering of macropinocytosis by protein aggregates and the possibility of utilizing macropinocytosis pathways as a therapeutic target.

  16. Griseofulvin-induced aggregation of microtubule protein.

    Science.gov (United States)

    Roobol, A; Gull, K; Pogson, C I

    1977-01-01

    Griseofulvin (7-chloro-2',4,6-trimethoxy-6'-methylspiro[benzofuran-2(3H),1'-[2]cyclohexene]-3,4'-dione) induces aggregation of microtubule protein at 0 degrees C. This aggregate contains approx. 90% of the microtubule-associated proteins originally present in the microtubule protein. The supernatant obtained after removal of the griseofulvin-induced aggregate does not form microtubules on warming at 37 degrees C. Addition of the griseofulvin-aggregated protein to this supernatant and warming to 37 degrees C gives rise to a limited amount of microtubule assembly. The possible involvement of griseofulvin-induced aggregation of microtubule protein at 0 degrees C in the inhibition by griseofulvin of microtubule assembly in vitro is discussed. Images PLATE 1 PLATE 2 PMID:588267

  17. Immunogenicity of therapeutic proteins: influence of aggregation.

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    Ratanji, Kirsty D; Derrick, Jeremy P; Dearman, Rebecca J; Kimber, Ian

    2014-01-01

    The elicitation of anti-drug antibodies (ADA) against biotherapeutics can have detrimental effects on drug safety, efficacy, and pharmacokinetics. The immunogenicity of biotherapeutics is, therefore, an important issue. There is evidence that protein aggregation can result in enhanced immunogenicity; however, the precise immunological and biochemical mechanisms responsible are poorly defined. In the context of biotherapeutic drug development and safety assessment, understanding the mechanisms underlying aggregate immunogenicity is of considerable interest. This review provides an overview of the phenomenon of protein aggregation, the production of unwanted aggregates during bioprocessing, and how the immune response to aggregated protein differs from that provoked by non-aggregated protein. Of particular interest is the nature of the interaction of aggregates with the immune system and how subsequent ADA responses are induced. Pathways considered here include 'classical' activation of the immune system involving antigen presenting cells and, alternatively, the breakdown of B-cell tolerance. Additionally, methods available to screen for aggregation and immunogenicity will be described. With an increased understanding of aggregation-enhanced immune responses, it may be possible to develop improved manufacturing and screening processes to avoid, or at least reduce, the problems associated with ADA.

  18. SUMO modulation of protein aggregation and degradation

    Directory of Open Access Journals (Sweden)

    Marco Feligioni

    2015-09-01

    Full Text Available Small ubiquitin-like modifier (SUMO conjugation and binding to target proteins regulate a wide variety of cellular pathways. The functional aspects of SUMOylation include changes in protein-protein interactions, intracellular trafficking as well as protein aggregation and degradation. SUMO has also been linked to specialized cellular pathways such as neuronal development and synaptic transmission. In addition, SUMOylation is associated with neurological diseases associated with abnormal protein accumulations. SUMOylation of the amyloid and tau proteins involved in Alzheimer's disease and other tauopathies may contribute to changes in protein solubility and proteolytic processing. Similar events have been reported for α-synuclein aggregates found in Parkinson's disease, polyglutamine disorders such as Huntington's disease as well as protein aggregates found in amyotrophic lateral sclerosis (ALS. This review provides a detailed overview of the impact SUMOylation has on the etiology and pathology of these related neurological diseases.

  19. Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation.

    Science.gov (United States)

    Lee, Jin-Haeng; Jeong, Jaeho; Jeong, Eui Man; Cho, Sung-Yup; Kang, Jeong Wook; Lim, Jisun; Heo, Jinbeom; Kang, Hyunsook; Kim, In-Gyu; Shin, Dong-Myung

    2014-04-01

    Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases.

  20. 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...

  1. Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A.

    Science.gov (United States)

    Fujita, Issei; Nobunaga, Mizuki; Seki, Takahiro; Kurauchi, Yuki; Hisatsune, Akinori; Katsuki, Hiroshi

    2017-07-22

    Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy. Copyright © 2017. Published by Elsevier Inc.

  2. 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

  3. Polysorbates, peroxides, protein aggregation, and immunogenicity – a growing concern

    Directory of Open Access Journals (Sweden)

    Edward T. Maggio

    2012-06-01

    Full Text Available Aggregation can have a number of deleterious effects on biotherapeutics including the loss of efficacy, the induction of unwanted immunogenicity, altered pharmacokinetics, and reduced shelf life. Aggregation is ameliorated by the inclusion of surfactants in biotherapeutics formulations, typically non-ionic polymeric ether surfactants. The most commonly used examples are Tween® 20 (Polysorbate 20 and Tween® 80 (Polysorbate 80. Others include Triton™ X-100, Pluronic® F-68, Pluronic® F-88, Pluronic® F-127 (poloxamers, and Brij 35 (polyoxyethylene alkyl ether. The usefulness of polysorbates, in particular in preventing protein aggregation in biotherapeutic formulations, is well accepted. However, polysorbates contain ether linkages and unsaturated alkyl chains that have been shown to auto-oxidize in aqueous solution to protein-damaging peroxides and reactive aldehydes including formaldehyde and acetaldehyde. The peroxides principally affect methionine and tryptophan moieties. The aldehydes react with primary amino groups on proteins and are known to induce immunogenicity of proteins in the absence of aggregation or adjuvants. Detection of protein aggregation and prevention of aggregation using polysorbates is relatively straightforward using light scattering or size exclusion chromatography methods. Detection of oxidative damage to amino acyl moieties or increased immunogenicity resulting from the reaction of biotherapeutics with the degradation products of polysorbates is considerably more difficult and has generally been ignored in the scientific literature. As an increasing number of biotherapeutic agents come into use in common clinical practice, including both as innovator and as biosimilar products, these latter issues will come under increased scrutiny. Substitution of non-ionic, non-ether-based surfactants, could offer significant improvements in stability, reduced immunogenicity, and shelf life, and represents a significant unmet

  4. Global analysis of protein aggregation in yeast during physiological conditions and arsenite stress

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    Sebastian Ibstedt

    2014-09-01

    Full Text Available Protein aggregation is a widespread phenomenon in cells and associated with pathological conditions. Yet, little is known about the rules that govern protein aggregation in living cells. In this study, we biochemically isolated aggregation-prone proteins and used computational analyses to identify characteristics that are linked to physiological and arsenite-induced aggregation in living yeast cells. High protein abundance, extensive physical interactions, and certain structural properties are positively correlated with an increased aggregation propensity. The aggregated proteins have high translation rates and are substrates of ribosome-associated Hsp70 chaperones, indicating that they are susceptible for aggregation primarily during translation/folding. The aggregation-prone proteins are enriched for multiple chaperone interactions, thus high protein abundance is probably counterbalanced by molecular chaperones to allow soluble expression in vivo. Our data support the notion that arsenite interferes with chaperone activity and indicate that arsenite-aggregated proteins might engage in extensive aberrant protein–protein interactions. Expression of aggregation-prone proteins is down-regulated during arsenite stress, possibly to prevent their toxic accumulation. Several aggregation-prone yeast proteins have human homologues that are implicated in misfolding diseases, suggesting that similar mechanisms may apply in disease- and non-disease settings.

  5. Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways

    OpenAIRE

    Douglas, Peter M; Summers, Daniel W; Cyr, Douglas M

    2009-01-01

    The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease protei...

  6. 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.

  7. Protein G, Protein A and Protein-A-Derived Peptides Inhibit the Agitation Induced Aggregation of IgG

    Science.gov (United States)

    Zhang, Jun; Topp, Elizabeth M.

    2012-01-01

    Controlling and preventing aggregation is critical to the development of safe and effective antibody drug products. The studies presented here test the hypothesis that Protein A and Protein G inhibit the agitation-induced aggregation of IgG. The hypothesis is motivated by the enhanced conformational stability of proteins upon ligand binding and the specific binding affinity of Protein A and Protein G to the Fc region of IgG. The aggregation of mixed human IgG from pooled human plasma was induced by agitation alone or in the presence of: (i) Protein A, (ii) Protein G or (iii) a library of 24 peptides derived from the IgG-binding domain of Protein A. Aggregation was assessed by UV spectroscopy, SDS-PAGE, high performance size-exclusion chromatography (HP-SEC), dynamic light scattering (DLS) and fluorescence spectroscopy. Additional information on IgG-ligand interactions was obtained using differential scanning fluorimetry (DSF) and competitive binding studies. The results demonstrate that Protein A provides near-complete inhibition of agitation-induced aggregation, while Protein G and two peptides from the peptide library show partial inhibition. The findings indicate that the IgG Protein A binding site is involved in the agitation-induced aggregation of IgG, and suggest a dominant role of colloidal interactions. PMID:22304418

  8. Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways.

    Science.gov (United States)

    Douglas, Peter M; Summers, Daniel W; Cyr, Douglas M

    2009-01-01

    The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates.

  9. Cellular Handling of Protein Aggregates by Disaggregation Machines.

    Science.gov (United States)

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

    2018-01-18

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

  10. Neurodegenerative diseases and widespread aggregation are associated with supersaturated proteins

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    Ciryam, Prajwal; Tartaglia, Gian Gaetano; Morimoto, Richard I.; Dobson, Christopher M.; Vendruscolo, Michele

    2013-01-01

    Summary The maintenance of protein solubility is a fundamental aspect of protein homeostasis, as aggregation is associated with cytotoxicity and a variety of human diseases. Numerous proteins unrelated in sequence and structure, however, can misfold and aggregate, and widespread aggregation can occur in living systems under stress or ageing. A crucial question in this context is why only certain proteins aggregate in vivo while others do not. We identify here the proteins most vulnerable to aggregation as those whose cellular concentrations are high relative to their solubilities. These supersaturated proteins represent a metastable sub-proteome involved in pathological aggregation during stress and ageing, and are overrepresented in biochemical processes associated with neurodegenerative disorders. Consequently, such cellular processes become dysfunctional when the ability to keep intrinsically supersaturated proteins soluble is compromised. Thus, the simultaneous analysis of abundance and solubility can rationalize the diverse cellular pathologies linked to neurodegenerative diseases and aging. PMID:24183671

  11. Protein aggregation kinetics during Protein A chromatography. Case study for an Fc fusion protein.

    Science.gov (United States)

    Shukla, Abhinav A; Gupta, Priyanka; Han, Xuejun

    2007-11-09

    Protein A chromatography has come to be widely adopted for large-scale purification of monoclonal antibodies and Fc fusion proteins. The low pH conditions required for Protein A elution can often lead to aggregation issues for these products. A concerted study of the kinetics of aggregate formation and their relation to chromatography on Protein A media has been lacking. This paper provides a framework to describe aggregation kinetics for an Fc fusion protein that was highly susceptible to aggregate formation under low pH conditions. In contrast to what is usually expected to be a higher order reaction, first order aggregation kinetics were observed for this protein over a wide range of conditions. A comparison of the rate constants of aggregation forms an effective means of comparing various stabilizing additives to the elution buffer with one another. Inclusion of urea in the elution buffer at moderate concentrations (Protein A column were both found to be effective solutions to the aggregation issue. Elution from the Protein A resin was found to increase the aggregation rate constants over and above what would be expected from exposure to low pH conditions in solution alone. This demonstrates that Protein A-Fc interactions can destabilize product structure and increase the tendency to aggregate. The results presented here are anticipated to assist the development of Protein A process conditions for products that are prone to form high molecular weight aggregates during column elution.

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

    Science.gov (United States)

    Dasgupta, Anushka

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

  13. Protein aggregates in Huntington’s disease

    Science.gov (United States)

    Arrasate, Montserrat; Finkbeiner, Steven

    2014-01-01

    Huntington’s disease (HD) is an incurable neurodegenerative disease characterized by abnormal motor movements, personality changes, and early death. HD is caused by a mutation in the IT-15 gene that expands abnormally the number of CAG nucleotide repeats. As a result, the translated protein huntingtin contains disease-causing expansions of glutamines (polyQ) that make it prone to misfold and aggregate. While the gene and mutations that cause HD are known, the mechanisms underlying HD pathogenesis are not. Here we will review the state of knowledge of HD, focusing especially on a hallmark pathological feature—intracellular aggregates of mutant Htt called inclusion bodies (IBs). We will describe the role of IBs in the disease. We speculate that IB formation could be just one component of a broader coping response triggered by misfolded Htt whose efficacy may depend on the extent to which it clears toxic forms of mutant Htt. We will describe how IB formation might be regulated and which factors could determine different coping responses in different subsets of neurons. A differential regulation of IB formation as a function of the cellular context could, eventually, explain part of the neuronal vulnerability observed in HD. PMID:22200539

  14. Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

    Science.gov (United States)

    Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

    2016-01-01

    Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.

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

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2009-01-01

    and that the outcome of IgG adsorption is much more sensitive to surface characteristics than the outcome of albumin adsorption. Using high concentrations of protein solution and hydrophobic polymer surfaces during adsorption can induce IgG aggregation, which is observed as extremely high IgG adsorptions. Besides......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...

  16. Removing protein aggregates: the role of proteolysis in neurodegeneration

    NARCIS (Netherlands)

    Nijholt, D.A.T.; de Kimpe, L.; Elfrink, H.L.; Hoozemans, J.J.M.; Scheper, W.

    2011-01-01

    A common characteristic of neurodegenerative diseases like Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD) is the accumulation of protein aggregates. This reflects a severe disturbance of protein homeostasis, the proteostasis. Here, we review the involvement of the

  17. Detection and Quantification of Protein Aggregates in Plants.

    Science.gov (United States)

    Planas-Marquès, Marc; Lema A, Saul; Coll, Núria S

    2016-01-01

    Plants are constantly exposed to a complex and changing environment that challenges their cellular homeostasis. Stress responses triggered as a consequence of unfavorable conditions result in increased protein aggregate formation at the cellular level. When the formation of misfolded proteins surpasses the capacity of the cell to remove them, insoluble protein aggregates accumulate. In the animal field, an enormous effort is being placed to uncover the mechanisms regulating aggregate formation because of its implications in many important human diseases. Because of its importance for cellular functionality and fitness, it is equally important to expand plant research in this field. Here, we describe a cell fractionation-based method to obtain very pure insoluble protein aggregate fractions that can be subsequently semiquantified using image analysis. This method can be used as a first step to evaluate whether a particular condition results in an alteration of protein aggregate formation levels.

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

    Science.gov (United States)

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

    2016-01-01

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

  19. Cadmium Causes Misfolding and Aggregation of Cytosolic Proteins in Yeast.

    Science.gov (United States)

    Jacobson, Therese; Priya, Smriti; Sharma, Sandeep K; Andersson, Stefanie; Jakobsson, Sofia; Tanghe, Robbe; Ashouri, Arghavan; Rauch, Sebastien; Goloubinoff, Pierre; Christen, Philipp; Tamás, Markus J

    2017-09-01

    Cadmium is a highly poisonous metal and is classified as a human carcinogen. While its toxicity is undisputed, the underlying in vivo molecular mechanisms are not fully understood. Here, we demonstrate that cadmium induces aggregation of cytosolic proteins in living Saccharomyces cerevisiae cells. Cadmium primarily targets proteins in the process of synthesis or folding, probably by interacting with exposed thiol groups in not-yet-folded proteins. On the basis of in vitro and in vivo data, we show that cadmium-aggregated proteins form seeds that increase the misfolding of other proteins. Cells that cannot efficiently protect the proteome from cadmium-induced aggregation or clear the cytosol of protein aggregates are sensitized to cadmium. Thus, protein aggregation may contribute to cadmium toxicity. This is the first report on how cadmium causes misfolding and aggregation of cytosolic proteins in vivo The proposed mechanism might explain not only the molecular basis of the toxic effects of cadmium but also the suggested role of this poisonous metal in the pathogenesis of certain protein-folding disorders. Copyright © 2017 American Society for Microbiology.

  20. How do Proteins Misfold and Aggregate?

    Indian Academy of Sciences (India)

    samrat

    Brain. Alzheimer's disease. Intrinsically disordered. Tau protein. Brain. Transmissible spongiform encephalopathy α-helical. Prion protein. Protein deposit. Disease. Type of structure. Amyloido- genic proteins. Skin & muscle. Injection-localized amyloidosis. Largely α - helical. Insulin. Brain. Parkinson's disease. Intrinsically.

  1. Sketching protein aggregation with a physics-based toy model

    Science.gov (United States)

    Enciso, Marta; Rey, Antonio

    2013-09-01

    We explore the applicability of a single-bead coarse-grained molecular model to describe the competition between protein folding and aggregation. We have designed very simple and regular sequences, based on our previous studies on peptide aggregation, that successfully fold into the three main protein structural families (all-α, all-β, and α + β). Thanks to equilibrium computer simulations, we evaluate how temperature and concentration promote aggregation. Aggregates have been obtained for all the amino acid sequences considered, showing that this process is common to all proteins, as previously stated. However, each structural family presents particular characteristics that can be related to its specific balance between hydrogen bond and hydrophobic interactions. The model is very simple and has limitations, yet it is able to reproduce both the cooperative folding of isolated polypeptide chains with regular sequences and the formation of different types of aggregates at high concentrations.

  2. Understanding curcumin-induced modulation of protein aggregation.

    Science.gov (United States)

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

    2017-07-01

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

  3. Niclosamide prevents the formation of large ubiquitin-containing aggregates caused by proteasome inhibition.

    Directory of Open Access Journals (Sweden)

    Esther Gies

    2010-12-01

    Full Text Available Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63 of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence

  4. 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

  5. p53 protein aggregation promotes platinum resistance in ovarian cancer.

    Science.gov (United States)

    Yang-Hartwich, Y; Soteras, M G; Lin, Z P; Holmberg, J; Sumi, N; Craveiro, V; Liang, M; Romanoff, E; Bingham, J; Garofalo, F; Alvero, A; Mor, G

    2015-07-01

    High-grade serous ovarian carcinoma (HGSOC), the most lethal gynecological cancer, often leads to chemoresistant diseases. The p53 protein is a key transcriptional factor regulating cellular homeostasis. A majority of HGSOCs have inactive p53 because of genetic mutations. However, genetic mutation is not the only cause of p53 inactivation. The aggregation of p53 protein has been discovered in different types of cancers and may be responsible for impairing the normal transcriptional activation and pro-apoptotic functions of p53. We demonstrated that in a unique population of HGSOC cancer cells with cancer stem cell properties, p53 protein aggregation is associated with p53 inactivation and platinum resistance. When these cancer stem cells differentiated into their chemosensitive progeny, they lost tumor-initiating capacity and p53 aggregates. In addition to the association of p53 aggregation and chemoresistance in HGSOC cells, we further demonstrated that the overexpression of a p53-positive regulator, p14ARF, inhibited MDM2-mediated p53 degradation and led to the imbalance of p53 turnover that promoted the formation of p53 aggregates. With in vitro and in vivo models, we demonstrated that the inhibition of p14ARF could suppress p53 aggregation and sensitize cancer cells to platinum treatment. Moreover, by two-dimensional gel electrophoresis and mass spectrometry we discovered that the aggregated p53 may function uniquely by interacting with proteins that are critical for cancer cell survival and tumor progression. Our findings help us understand the poor chemoresponse of a subset of HGSOC patients and suggest p53 aggregation as a new marker for chemoresistance. Our findings also suggest that inhibiting p53 aggregation can reactivate p53 pro-apoptotic function. Therefore, p53 aggregation is a potential therapeutic target for reversing chemoresistance. This is paramount for improving ovarian cancer patients' responses to chemotherapy, and thus increasing their

  6. 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

  7. Effect of αB-Crystallin on Protein Aggregation in Drosophila

    Directory of Open Access Journals (Sweden)

    Nguyen Trong Tue

    2012-01-01

    Full Text Available Disorganisation and aggregation of proteins containing expanded polyglutamine (polyQ repeats, or ectopic expression of α-synuclein, underlie neurodegenerative diseases including Alzheimer’s, Parkinson, Huntington, Creutzfeldt diseases. Small heat-shock proteins, such as αB-crystallin, act as chaperones to prevent protein aggregation and play a key role in the prevention of such protein disorganisation diseases. In this study, we have explored the potential for chaperone activity of αB-crystallin to suppress the formation of protein aggregates. We tested the ability of αB-crystallin to suppress the aggregation of a polyQ protein and α-synuclein in Drosophila. We found that αB-crystallin suppresses both the compound eye degeneration induced by polyQ and the α-synuclein-induced rough eye phenotype. Furthermore, by using histochemical staining we have determined that αB-crystallin inhibits the aggregation of polyQ in vivo. These data provide a clue for the development of therapeutics for neurodegenerative diseases.

  8. Molecular dynamics studies of protein folding and aggregation

    Science.gov (United States)

    Ding, Feng

    This thesis applies molecular dynamics simulations and statistical mechanics to study: (i) protein folding; and (ii) protein aggregation. Most small proteins fold into their native states via a first-order-like phase transition with a major free energy barrier between the folded and unfolded states. A set of protein conformations corresponding to the free energy barrier, Delta G >> kBT, are the folding transition state ensemble (TSE). Due to their evasive nature, TSE conformations are hard to capture (probability ∝ exp(-DeltaG/k BT)) and characterize. A coarse-grained discrete molecular dynamics model with realistic steric constraints is constructed to reproduce the experimentally observed two-state folding thermodynamics. A kinetic approach is proposed to identify the folding TSE. A specific set of contacts, common to the TSE conformations, is identified as the folding nuclei which are necessary to be formed in order for the protein to fold. Interestingly, the amino acids at the site of the identified folding nuclei are highly conserved for homologous proteins sharing the same structures. Such conservation suggests that amino acids that are important for folding kinetics are under selective pressure to be preserved during the course of molecular evolution. In addition, studies of the conformations close to the transition states uncover the importance of topology in the construction of order parameter for protein folding transition. Misfolded proteins often form insoluble aggregates, amyloid fibrils, that deposit in the extracellular space and lead to a type of disease known as amyloidosis. Due to its insoluble and non-crystalline nature, the aggregation structure and, thus the aggregation mechanism, has yet to be uncovered. Discrete molecular dynamics studies reveal an aggregate structure with the same structural signatures as in experimental observations and show a nucleation aggregation scenario. The simulations also suggest a generic aggregation mechanism

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

    Science.gov (United States)

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

    2016-02-15

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

  10. Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway.

    Science.gov (United States)

    Chuang, Wen-Ying; Kung, Po-Hsiung; Kuo, Chih-Yun; Wu, Chin-Chung

    2013-06-01

    Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

  11. Hierarchical organization in aggregates of protein molecules

    DEFF Research Database (Denmark)

    Bohr, Henrik; Kyhle, Anders; Sørensen, Alexis Hammer

    1997-01-01

    of the solution and the density of protein are varied shows the existence of specific growth processes resulting in different branch-like structures. The resulting structures are strongly influenced by the shape of each protein molecule. Lysozyme and ribonuclease are found to form spherical structures...

  12. How do Proteins Misfold and Aggregate?

    Indian Academy of Sciences (India)

    samrat

    The Central Dogma of Molecular Biology. DNA. RNA. PROTEIN. FUNCTION. Data storage Readout. Execution. Flow of genetic information ... Wheat germ agglutinin. Proteins are elegantly and intricately folded bio-macromolecules .... Rapid conformational averaging: Fluctuations are much faster than the transit time of the ...

  13. Impact of aggregate formation on the viscosity of protein solutions.

    Science.gov (United States)

    Nicoud, Lucrèce; Lattuada, Marco; Yates, Andrew; Morbidelli, Massimo

    2015-07-21

    Gaining knowledge on the stability and viscosity of concentrated therapeutic protein solutions is of great relevance to the pharmaceutical industry. In this work, we borrow key concepts from colloid science to rationalize the impact of aggregate formation on the changes in viscosity of a concentrated monoclonal antibody solution. In particular, we monitor the kinetics of aggregate growth under thermal stress by static and dynamic light scattering, and we follow the rise in solution viscosity by measuring the diffusion coefficient of tracer nanoparticles with dynamic light scattering. Moreover, we characterize aggregate morphology in the frame of the fractal geometry. We show that the curves of the increase in viscosity with time monitored at three different protein concentrations collapse on one single master curve when the reaction profiles are normalized based on an effective volume fraction occupied by the aggregates, which depends on the aggregate size, concentration and morphology. Importantly, we find that the viscosity of an aggregate sample is lower than the viscosity of a monomeric sample of a similar occupied volume fraction due to the polydispersity of the aggregate distribution.

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

    Science.gov (United States)

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

    2018-03-01

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

  15. The effects of excipients on protein aggregation during agitation: an interfacial shear rheology study.

    Science.gov (United States)

    Liu, Lu; Qi, Wei; Schwartz, Daniel K; Randolph, Theodore W; Carpenter, John F

    2013-08-01

    We investigated the effects of excipients in solutions of keratinocyte growth factor 2 (KGF-2) on protein aggregation during agitation as well as on interfacial shear rheology at the air-water interface. Samples were incubated with or without agitation, and in the presence or absence of the excipients heparin, sucrose, or polysorbate 80 (PS80). The effect of excipients on the extent of protein aggregation was determined by UV-visible spectroscopy and micro-flow imaging. Interfacial shear rheology was used to detect the gelation time and strength of protein gels at the air-water interface. During incubation, protein particles of size ≥1 μm and insoluble aggregates formed faster for KGF-2 solutions subjected to agitation. Addition of either heparin or sucrose promoted protein aggregation during agitation. In contrast, PS80 substantially inhibited agitation-induced KGF-2 aggregation but facilitated protein particulate formation in quiescent solutions. The combination of PS80 and heparin or sucrose completely prevented protein aggregation during both nonagitated and agitated incubations. Interfacial rheological measurements showed that KGF-2 in buffer alone formed an interfacial gel within a few minutes. In the presence of heparin, KGF-2 interfacial gels formed too quickly for gelation time to be determined. KGF-2 formed gels in about 10 min in the presence of sucrose. The presence of PS80 in the formulation inhibited gelation of KGF-2. Furthermore, the interfacial gels formed by the protein in the absence of PS80 were reversible when PS80 was added to the samples after gelation. Therefore, there is a correspondence between formulations that exhibited interfacial gelation and formulations that exhibited agitation-induced aggregation. Copyright © 2013 Wiley Periodicals, Inc.

  16. Protein/Peptide Aggregation and Amyloidosis on Biointerfaces

    Directory of Open Access Journals (Sweden)

    Qi Lu

    2016-08-01

    Full Text Available Recently, studies of protein/peptide aggregation, particularly the amyloidosis, have attracted considerable attention in discussions of the pathological mechanisms of most neurodegenerative diseases. The protein/peptide aggregation processes often occur at the membrane–cytochylema interface in vivo and behave differently from those occurring in bulk solution, which raises great interest to investigate how the interfacial properties of artificial biomaterials impact on protein aggregation. From the perspective of bionics, current progress in this field has been obtained mainly from four aspects: (1 hydrophobic–hydrophilic interfaces; (2 charged surface; (3 chiral surface; and (4 biomolecule-related interfaces. The specific physical and chemical environment provided by these interfaces is reported to strongly affect the adsorption of proteins, transition of protein conformation, and diffusion of proteins on the biointerface, all of which are ultimately related to protein assembly. Meanwhile, these compelling results of in vitro experiments can greatly promote the development of early diagnostics and therapeutics for the relevant neurodegenerative diseases. This paper presents a brief review of these appealing studies, and particular interests are placed on weak interactions (i.e., hydrogen bonding and stereoselective interactions that are also non-negligible in driving amyloid aggregation at the interfaces. Moreover, this paper also proposes the future perspectives, including the great opportunities and challenges in this field as well.

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

    Science.gov (United States)

    Chong, Song-Ho; Ham, Sihyun

    2015-04-21

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

  18. Intrinsic disorder modulates protein self-assembly and aggregation.

    Science.gov (United States)

    De Simone, Alfonso; Kitchen, Craig; Kwan, Ann H; Sunde, Margaret; Dobson, Christopher M; Frenkel, Daan

    2012-05-01

    Protein molecules have evolved to adopt distinctive and well-defined functional and soluble states under physiological conditions. In some circumstances, however, proteins can self-assemble into fibrillar aggregates designated as amyloid fibrils. In vivo these processes are normally associated with severe pathological conditions but can sometimes have functional relevance. One such example is the hydrophobins, whose aggregation at air-water interfaces serves to create robust protein coats that help fungal spores to resist wetting and thus facilitate their dispersal in the air. We have performed multiscale simulations to address the molecular determinants governing the formation of functional amyloids by the class I fungal hydrophobin EAS. Extensive samplings of full-atom replica-exchange molecular dynamics and coarse-grained simulations have allowed us to identify factors that distinguish aggregation-prone from highly soluble states of EAS. As a result of unfavourable entropic terms, highly dynamical regions are shown to exert a crucial influence on the propensity of the protein to aggregate under different conditions. More generally, our findings suggest a key role that specific flexible structural elements can play to ensure the existence of soluble and functional states of proteins under physiological conditions.

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

    Science.gov (United States)

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

    2011-11-04

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

  20. 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...

  1. Polymorphism of Protein Aggregation: From Amyloid Fibrils to Crystallization

    Science.gov (United States)

    Safari, Mohammad S.; Conrad, Jacinta C.; Vekilov, Peter G.

    Protein aggregation is commonly observed in neurological diseases and in different types of cancer. Despite the established mechanism of amyloid formation, the polymorphism of aggregation is not very well understood; improved knowledge of mechanisms for aggregation that operate in vivo or under physiological conditions is likely to inform therapeutic design. Here we show that reduction of disulfide bonds in lysozyme can lead to formation of gel-like oligomers that are precursors for protein crystal nucleation events. The growth in size of oligomers follows slow first-order kinetics, suggesting that monomers with free thiol contribute to formation of clusters. Free thiol concentration measurements showed that the thiol concentration was relatively stable over 12 hr, confirming the slow kinetics was due to gelation inside the clusters. We probed the hydrophobicity of the clusters using ANS and ThT assays, and showed that the protein conformation in these clusters differs from that of thermally denatured aggregates. Although partial unfolding aids the formation of precursors to both amyloids and crystals, our results suggest that these pathways exhibit distinct signatures even at the earliest stages. NASA.

  2. Protein-encapsulated gold cluster aggregates: the case of lysozyme

    Science.gov (United States)

    Baksi, Ananya; Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; Goswami, N.; Pal, S. K.; Pradeep, T.

    2013-02-01

    We report the evolution and confinement of atomically precise and luminescent gold clusters in a small protein, lysozyme (Lyz) using detailed mass spectrometric (MS) and other spectroscopic investigations. A maximum of 12 Au0 species could be bound to a single Lyz molecule irrespective of the molar ratio of Lyz : Au3+ used for cluster growth. The cluster-encapsulated protein also forms aggregates similar to the parent protein. Time dependent studies reveal the emergence of free protein and the redistribution of detached Au atoms, at specific Lyz to Au3+ molar ratios, as a function of incubation time, proposing inter-protein metal ion transfer. The results are in agreement with the studies of inter-protein metal transfer during cluster growth in similar systems. We believe that this study provides new insights into the growth of clusters in smaller proteins.We report the evolution and confinement of atomically precise and luminescent gold clusters in a small protein, lysozyme (Lyz) using detailed mass spectrometric (MS) and other spectroscopic investigations. A maximum of 12 Au0 species could be bound to a single Lyz molecule irrespective of the molar ratio of Lyz : Au3+ used for cluster growth. The cluster-encapsulated protein also forms aggregates similar to the parent protein. Time dependent studies reveal the emergence of free protein and the redistribution of detached Au atoms, at specific Lyz to Au3+ molar ratios, as a function of incubation time, proposing inter-protein metal ion transfer. The results are in agreement with the studies of inter-protein metal transfer during cluster growth in similar systems. We believe that this study provides new insights into the growth of clusters in smaller proteins. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33180b

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Maňásková, Pavla; Tichá, M.; Jonáková, Věra

    2003-01-01

    Roč. 32, - (2003), s. 99-107 ISSN 0141-8130 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 : proteinase inhibitors, aggregated forms, boar seminal plasma proteins Subject RIV: CE - Biochemistry Impact factor: 1.427, year: 2003

  5. 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.

  6. Inhibitory effect of corcin on aggregation of 1N/4R human tau protein in vitro

    Directory of Open Access Journals (Sweden)

    Ali Mohammadi Karakani

    2015-05-01

    Full Text Available Objective(s:Alzheimer's disease (AD is the most common age-related neurodegenerative disorder. One of the hallmarks of AD is an abnormal accumulation of fibril forms of tau protein which is known as a microtubule associated protein. In this regard, inhibition of tau aggregation has been documented to be a potent therapeutic approach in AD and tauopathies. Unfortunately, the available synthetic drugs have modest beneficial efficacy with several side effects. Therefore, pipeline drugs from natural sources with anti-aggregation properties can be useful in the prevention and treatment of AD. Among medicinal plants, saffron (Crocus sativus, L., as a traditional herbal medicine has different pharmacological properties and can be used as treatment for several nervous system impairment including depression and dementia. Crocin as a major constituent of saffron is the glycosylated form of crocetin. Materials and Methods:  In this study, we investigated the inhibitory effect of crocin on aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods and cell culture. Results:  Results revealed that tau protein under the fibrillation condition and in the presence of crocin had enough stability with low tendency for aggregation. Crocin inhibited tau aggregation with IC50 of 100 µg/ml.  Furthermore, transmission electron microscopy images confirmed that crocin could suppress the formation of tau protein filaments. Conclusion: Inhibitory effect of crocin could be related to its interference with nucleation phase that led to increases in monomer species of tau protein. Based on our results, crocin is recommended as a proper candidate to be used in AD treatment.

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

    Directory of Open Access Journals (Sweden)

    Antonella Sgarbossa

    2012-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Della C David

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

  9. Protein Aggregates May Differ in Water Entrapment but Are Comparable in Water Confinement

    NARCIS (Netherlands)

    Urbonaite, V.; Jongh, De H.H.J.; Linden, Van Der E.; Pouvreau, L.

    2015-01-01

    Aggregate size and density are related to gel morphology. In the context of the water distribution in complex food systems, in this study, it was aimed to investigate whether protein aggregates varying in size and density differ in entrapped and confined water. Heat-set soy protein aggregates

  10. Organelle-based aggregation and retention of damaged proteins in asymmetrically dividing cells.

    Science.gov (United States)

    Zhou, Chuankai; Slaughter, Brian D; Unruh, Jay R; Guo, Fengli; Yu, Zulin; Mickey, Kristen; Narkar, Akshay; Ross, Rhonda Trimble; McClain, Melainia; Li, Rong

    2014-10-23

    Aggregation of damaged or misfolded proteins is a protective mechanism against proteotoxic stress, abnormalities of which underlie many aging-related diseases. Here, we show that in asymmetrically dividing yeast cells, aggregation of cytosolic misfolded proteins does not occur spontaneously but requires new polypeptide synthesis and is restricted to the surface of ER, which harbors the majority of active translation sites. Protein aggregates formed on ER are frequently also associated with or are later captured by mitochondria, greatly constraining aggregate mobility. During mitosis, aggregates are tethered to well-anchored maternal mitochondria, whereas mitochondria acquired by the bud are largely free of aggregates. Disruption of aggregate-mitochondria association resulted in increased mobility and leakage of mother-accumulated aggregates into the bud. Cells with advanced replicative age exhibit gradual decline of aggregates-mitochondria association, likely contributing to their diminished ability to rejuvenate through asymmetric cell division.

  11. Stabilizing two IgG1 monoclonal antibodies by surfactants: Balance between aggregation prevention and structure perturbation.

    Science.gov (United States)

    Wang, Shujing; Wu, Guoliang; Zhang, Xinyi; Tian, Zhou; Zhang, Ning; Hu, Tao; Dai, Weiguo; Qian, Feng

    2017-05-01

    Surfactants are widely used as stabilizers in the biopharmaceutical formulations to minimize protein aggregation. Under a fixed stress condition, the protecting and destabilizing effects of surfactants are hypothesized to be highly dependent on the species and concentrations of surfactants and mAb. Therefore, we here studied the aggregation-prevention and structure-perturbation effects of eight commonly used surfactants (Tw20, Tw80, Brij35, Chaps, TrX-100, SDS, Pluronic F68 and F127) on two IgG1 solution formulations under agitation, using analytical methodologies including visual inspection, OD 350 measurement, HPLC-SEC, circular dicroism, fluorescence spectroscopy and differential scanning calorimetry. We found that: (1) With concentrations range from 0.02 to 2mg/mL, nonionic surfactants were found to offer efficient aggregation-prevention effect, which is superior than the ionic surfactants; and higher surfactant concentration prevented mAb aggregation better especially under prolonged stability test under stress conditions. (2) The surfactant induced structure-perturbation emerged when even higher surfactant concentration (≥2mg/mL) was used, and such effect was surfactant-property dependent; and (3) the two IgG1 demonstrated different aggregation mechanisms and surfactant dependency, especially at high mAb concentrations. In conclusion, surfactants usage in mAb formulations, including the types and concentrations, should strike an optimal balance between the desirable aggregation-prevention and the detrimental structure-perturbation effects, while the consideration of mAb aggregation mechanism and concentration is also required for surfactant assessment. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  13. Modeling Protein Aggregation and the Heat Shock Response in ALS iPSC-Derived Motor Neurons.

    Science.gov (United States)

    Seminary, Emily R; Sison, Samantha L; Ebert, Allison D

    2018-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by the selective loss of the upper and lower motor neurons. Only 10% of all cases are caused by a mutation in one of the two dozen different identified genes, while the remaining 90% are likely caused by a combination of as yet unidentified genetic and environmental factors. Mutations in C9orf72, SOD1 , or TDP-43 are the most common causes of familial ALS, together responsible for at least 60% of these cases. Remarkably, despite the large degree of heterogeneity, all cases of ALS have protein aggregates in the brain and spinal cord that are immunopositive for SOD1, TDP-43, OPTN, and/or p62. These inclusions are normally prevented and cleared by heat shock proteins (Hsps), suggesting that ALS motor neurons have an impaired ability to induce the heat shock response (HSR). Accordingly, there is evidence of decreased induction of Hsps in ALS mouse models and in human post-mortem samples compared to unaffected controls. However, the role of Hsps in protein accumulation in human motor neurons has not been fully elucidated. Here, we generated motor neuron cultures from human induced pluripotent stem cell (iPSC) lines carrying mutations in SOD1, TDP-43 , or C9orf72 . In this study, we provide evidence that despite a lack of overt motor neuron loss, there is an accumulation of insoluble, aggregation-prone proteins in iPSC-derived motor neuron cultures but that content and levels vary with genetic background. Additionally, although iPSC-derived motor neurons are generally capable of inducing the HSR when exposed to a heat stress, protein aggregation itself is not sufficient to induce the HSR or stress granule formation. We therefore conclude that ALS iPSC-derived motor neurons recapitulate key early pathological features of the disease and fail to endogenously upregulate the HSR in response to increased protein burden.

  14. MBAR-enhanced lattice Monte Carlo simulation of the effect of helices on membrane protein aggregation

    Science.gov (United States)

    Xu, Yuanwei; Rodger, P. Mark

    2017-03-01

    We study the effect of helical structure on the aggregation of proteins using a simplified lattice protein model with an implicit membrane environment. A recently proposed Monte Carlo approach, which exploits the proven statistical optimality of the MBAR estimator in order to improve simulation efficiency, was used. The results show that with both two and four proteins present, the tendency to aggregate is strongly expedited by the presence of amphipathic helix (APH), whereas a transmembrane helix (TMH) slightly disfavours aggregation. When four protein molecules are present, partially aggregated states (dimers and trimers) were more common when the APH was present, compared with the cases where no helices or only the TMH is present.

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

    Directory of Open Access Journals (Sweden)

    Maggie P Wear

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

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

    Science.gov (United States)

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

    2016-02-01

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

  17. Finite size effects in simulations of protein aggregation.

    Directory of Open Access Journals (Sweden)

    Amol Pawar

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

  18. Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding.

    Science.gov (United States)

    Żwirowski, Szymon; Kłosowska, Agnieszka; Obuchowski, Igor; Nillegoda, Nadinath B; Piróg, Artur; Ziętkiewicz, Szymon; Bukau, Bernd; Mogk, Axel; Liberek, Krzysztof

    2017-03-15

    Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp-substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp-substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions. © 2017 The Authors.

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

    Science.gov (United States)

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

    2013-09-01

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

  20. Specific-ion effects on the aggregation mechanisms and protein-protein interactions for anti-streptavidin immunoglobulin gamma-1.

    Science.gov (United States)

    Barnett, Gregory V; Razinkov, Vladimir I; Kerwin, Bruce A; Laue, Thomas M; Woodka, Andrea H; Butler, Paul D; Perevozchikova, Tatiana; Roberts, Christopher J

    2015-05-07

    Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the relationship(s) among protein-protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate. Aggregate size and shape were determined using a combination of laser light scattering and small-angle neutron or X-ray scattering. Protein-protein interactions were quantified in terms of the protein-protein Kirkwood-Buff integral (G22) determined from static light scattering and in terms of the protein effective charge (Zeff) measured using electrophoretic light scattering. Changing from citrate to acetate resulted in significantly different protein-protein interactions as a function of pH for low NaCl concentrations when the protein displayed positive Zeff. Overall, the results suggest that electrostatic repulsions between proteins were lessened because of preferential accumulation of citrate anions, compared to acetate anions, at the protein surface. The predominant aggregation mechanisms correlated well with G22, indicating that ion-specific effects beyond traditional mean-field descriptions of electrostatic protein-protein interactions are important for predicting qualitative shifts in protein aggregation state diagrams. Interestingly, while solution conditions dictated which mechanisms predominated, aggregate average molecular weight and size displayed a common scaling behavior across both citrate- and acetate-based systems.

  1. AGGRESCAN3D (A3D): server for prediction of aggregation properties of protein structures.

    Science.gov (United States)

    Zambrano, Rafael; Jamroz, Michal; Szczasiuk, Agata; Pujols, Jordi; Kmiecik, Sebastian; Ventura, Salvador

    2015-07-01

    Protein aggregation underlies an increasing number of disorders and constitutes a major bottleneck in the development of therapeutic proteins. Our present understanding on the molecular determinants of protein aggregation has crystalized in a series of predictive algorithms to identify aggregation-prone sites. A majority of these methods rely only on sequence. Therefore, they find difficulties to predict the aggregation properties of folded globular proteins, where aggregation-prone sites are often not contiguous in sequence or buried inside the native structure. The AGGRESCAN3D (A3D) server overcomes these limitations by taking into account the protein structure and the experimental aggregation propensity scale from the well-established AGGRESCAN method. Using the A3D server, the identified aggregation-prone residues can be virtually mutated to design variants with increased solubility, or to test the impact of pathogenic mutations. Additionally, A3D server enables to take into account the dynamic fluctuations of protein structure in solution, which may influence aggregation propensity. This is possible in A3D Dynamic Mode that exploits the CABS-flex approach for the fast simulations of flexibility of globular proteins. The A3D server can be accessed at http://biocomp.chem.uw.edu.pl/A3D/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

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

    2017-12-10

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

  3. Specific Ion Effects on Protein Thermal Aggregation from Dilute Solutions to Crowded Environments.

    Science.gov (United States)

    Li, Shuling; Ye, Shuji; Liu, Guangming

    2018-03-29

    We have investigated specific ion effects on protein thermal aggregation from dilute solutions to crowded environments. Ovalbumin and poly(ethylene glycol) have been employed as the model protein and crowding agent, respectively. Our studies demonstrate that the rate-limiting step of ovalbumin thermal aggregation is changed from the aggregation of unfolded protein molecules to the unfolding of the protein molecules, when the solution conditions are varied from a dilute solution to a crowded environment. The specific ion effects acting on the thermal aggregation of ovalbumin generated by kosmotropic and chaotropic ions are different. The thermal aggregation of ovalbumin molecules is promoted by kosmotropic anions in dilute solutions via an increase in protein hydrophobic interactions. In contrast, ovalbumin thermal aggregation is facilitated by chaotropic ions in crowded environments through accelerated unfolding of protein molecules. Therefore, there are distinct mechanisms causing the ion specificities of protein thermal aggregation between dilute solutions and crowded environments. The ion specificities are dominated by ion-specific hydrophobic interactions between protein molecules and ion-specific unfolding of protein molecules in dilute solutions and crowded environments, respectively.

  4. A novel Escherichia coli solubility enhancer protein for fusion expression of aggregation-prone heterologous proteins.

    Science.gov (United States)

    Song, Jong-Am; Lee, Dae-Sung; Park, Jin-Seung; Han, Kyung-Yeon; Lee, Jeewon

    2011-07-10

    Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. 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)

  6. Fluctuations in Protein Aggregation: Design of Preclinical Screening for Early Diagnosis of Neurodegenerative Disease

    Science.gov (United States)

    Costantini, Giulio; Budrikis, Zoe; Taloni, Alessandro; Buell, Alexander K.; Zapperi, Stefano; La Porta, Caterina A. M.

    2016-09-01

    Autocatalytic fibril nucleation has recently been proposed to be a determining factor for the spread of neurodegenerative diseases, but the same process could also be exploited to amplify minute quantities of protein aggregates in a diagnostic context. Recent advances in microfluidic technology allow the analysis of protein aggregation in micron-scale samples, potentially enabling such diagnostic approaches, but the theoretical foundations for the analysis and interpretation of such data are, so far, lacking. Here, we study computationally the onset of protein aggregation in small volumes and show that the process is ruled by intrinsic fluctuations whose volume-dependent distribution we also estimate theoretically. Based on these results, we develop a strategy to quantify in silico the statistical errors associated with the detection of aggregate-containing samples. Our work explores a different perspective on the forecasting of protein aggregation in asymptomatic subjects.

  7. [Inhibition of aggregation of dissociated embryonic Amphibian cells by inhibitors of protein and RNA synthesis].

    Science.gov (United States)

    Grunz, Horst

    1969-06-01

    1. Aggregation of embryonic Amphibian cells dissociated by Trypsin or EDTA is supressed reversibly by actidion (cycloheximid 0.5-2 Μg/ml), an inhibitor of protein synthesis. Protein synthesis is inhibited more than 90% at these concentrations. The inhibition by actidion was still reversible after 48 hours, when the cells were transferred to normal medium. Actidion added to cells, cultured in Flickinger solution for 3 h, was no longer able to stop further aggregation. 2. Puromycin reversibly suppresses aggregation at a concentration of 20 (Μg/ml, when protein synthesis is inhibited to 66 %. 3. After EDTA-dissociation the formation of initial small clusters of 5-10 cells (primary aggregation) occurred even in the presence of inhibitors of protein synthesis. The primary aggregation is however suppressed if actidion or puromycin are already added during the dissociation procedure. 4. In the presence of actinomycin-D (1-2 Μg/ml) the cells continue to aggregate like the controls. After 30 h further aggregation is stopped and the aggregates loose single cells. 5. Differences in the aggregation of cells after EDTA and Trypsin treatment are discussed.

  8. Spatially organized aggregation of misfolded proteins as cellular stress defense strategy.

    Science.gov (United States)

    Miller, Stephanie B M; Mogk, Axel; Bukau, Bernd

    2015-04-10

    An evolutionary conserved response of cells to proteotoxic stress is the organized sequestration of misfolded proteins into subcellular deposition sites. In Saccharomyces cerevisiae, three major sequestration sites for misfolded proteins exist, IPOD (insoluble protein deposit), INQ (intranuclear quality control compartment) [former JUNQ (juxtanuclear quality control compartment)] and CytoQ. IPOD is perivacuolar and predominantly sequesters amyloidogenic proteins. INQ and CytoQs are stress-induced deposits for misfolded proteins residing in the nucleus and the cytosol, respectively, and requiring cell-compartment-specific aggregases, nuclear Btn2 and cytosolic Hsp42 for formation. The organized aggregation of misfolded proteins is proposed to serve several purposes collectively increasing cellular fitness and survival under proteotoxic stress. These include (i) shielding of cellular processes from interference by toxic protein conformers, (ii) reducing the substrate burden for protein quality control systems upon immediate stress, (iii) orchestrating chaperone and protease functions for efficient repair or degradation of damaged proteins [this involves initial extraction of aggregated molecules via the Hsp70/Hsp104 bi-chaperone system followed by either refolding or proteasomal degradation or removal of entire aggregates by selective autophagy (aggrephagy) involving the adaptor protein Cue5] and (iv) enabling asymmetric retention of protein aggregates during cell division, thereby allowing for damage clearance in daughter cells. Regulated protein aggregation thus serves cytoprotective functions vital for the maintenance of cell integrity and survival even under adverse stress conditions and during aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Jing Kang

    2010-01-01

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

  10. 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

    OpenAIRE

    Haina Qin; Liangzhong Lim; Yuanyuan Wei; Garvita Gupta; Jianxing Song

    2013-01-01

    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 environm...

  11. Molecular perspective of antibody aggregates and their adsorption on Protein A resin.

    Science.gov (United States)

    Yu, Deqiang; Song, Yuanli; Huang, Richard Y-C; Swanson, Ryan K; Tan, Zhijun; Schutsky, Elizabeth; Lewandowski, Angela; Chen, Guodong; Li, Zheng Jian

    2016-07-29

    Antibody aggregate is a common issue in therapeutic antibodies, which may compromise product efficacy and cause adverse effects. Antibody aggregate level is normally controlled in bioprocessing by polishing steps after Protein A capture. This paper studied the Higher Order Structures (HOS) of antibody aggregates (dimer H1 and H2) and their adsorption on Protein A resin and thus elucidated the mechanism using Protein A capture for enhanced aggregate removal. The HOS of antibody aggregates and their complex with Protein A were characterized using HDX-MS combined with SEC-MALS, Protein Conformational Array (PCA), and molecular modeling. The aggregate size and Protein A binding ratio suggested that H2 has much more compact structure than H1. HDX-MS and PCA further revealed that H1 was formed by single Fab-Fab interaction while H2 formed by Fab-Fab and likely Fc-Fc interaction. On Protein A resin, both the molar binding ratio and the correlation between protein size and ligand distance support that each monomer can only bind one Protein A ligand, while each dimer can bind two ligands, thus resulting in stronger resin binding. Furthermore, dimer H2 binds stronger than dimer H1 due to its compact structure. By integrating biophysical analysis and molecular modeling with process development, this study revealed the antibody aggregate structures and the mechanism of aggregate removal using Protein A chromatography. It also provided a general strategy for in-depth product and process understanding in antibody and other biologics development. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. 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

  13. 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

  14. 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

  15. Aldosterone and angiotensin II induce protein aggregation in renal proximal tubules.

    Science.gov (United States)

    Cheema, Muhammad U; Poulsen, Ebbe T; Enghild, Jan J; Hoorn, Ewout J; Hoorn, Ewout; Fenton, Robert A; Praetorius, Jeppe

    2013-09-01

    Renal tubules are highly active transporting epithelia and are at risk of protein aggregation due to high protein turnover and/or oxidative stress. We hypothesized that the risk of aggregation was increased upon hormone stimulation and assessed the state of the intracellular protein degradation systems in the kidney from control rats and rats receiving aldosterone or angiotensin II treatment for 7 days. Control rats formed both aggresomes and autophagosomes specifically in the proximal tubules, indicating a need for these structures even under baseline conditions. Fluorescence sorted aggresomes contained various rat keratins known to be expressed in renal tubules as assessed by protein mass spectrometry. Aldosterone administration increased the abundance of the proximal tubular aggresomal protein keratin 5, the ribosomal protein RPL27, ataxin-3, and the chaperone heat shock protein 70-4 with no apparent change in the aggresome-autophagosome markers. Angiotensin II induced aggregation of RPL27 specifically in proximal tubules, again without apparent change in antiaggregating proteins or the aggresome-autophagosome markers. Albumin endocytosis was unaffected by the hormone administration. Taken together, we find that the renal proximal tubules display aggresome formation and autophagy. Despite an increase in aggregation-prone protein load in these tubules during hormone treatment, renal proximal tubules seem to have sufficient capacity for removing protein aggregates from the cells.

  16. Formation of J-Aggregates of an Anionic Oxacarbocyanine Dye Upon Interaction with Proteins and Polyelectrolytes

    Science.gov (United States)

    Pronkin, P. G.; Tatikolov, A. S.

    2017-05-01

    J-aggregation of the anionic oxacarbocyanine dye 3,3'-di-(γ-sulfopropyl)-5,5'-diphenyl-9-ethyloxacarbocyanine betaine was studied in aqueous solutions in the presence of proteins (collagens, immunoglobulin G, serum albumins) and polyelectrolytes (polyethyleneimine, polyvinylpyrrolidone). It was found that denaturation of human serum albumin by urea stimulated J-aggregation of the dye. The dye formed two types of J-aggregates in the presence of denatured albumin and polyethyleneimine. J-aggregates formed in the presence of polyethyleneimine rearranged over time.

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

    Science.gov (United States)

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

    2017-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Martin J. Egan

    2015-04-01

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

  19. 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

  20. Phosphorylation of the 19S regulatory particle ATPase subunit, Rpt6, modifies susceptibility to proteotoxic stress and protein aggregation.

    Directory of Open Access Journals (Sweden)

    Esther Magdalena Marquez-Lona

    Full Text Available The ubiquitin proteasome system (UPS is a highly conserved and tightly regulated biochemical pathway that degrades the majority of proteins in eukaryotic cells. Importantly, the UPS is responsible for counteracting altered protein homeostasis induced by a variety of proteotoxic stresses. We previously reported that Rpt6, the ATPase subunit of the 19S regulatory particle (RP of the 26S proteasome, is phosphorylated in mammalian neurons at serine 120 in response to neuronal activity. Furthermore, we found that Rpt6 S120 phosphorylation, which regulates the activity and distribution of proteasomes in neurons, is relevant for proteasome-dependent synaptic remodeling and function. To better understand the role of proteasome phosphorylation, we have constructed models of altered Rpt6 phosphorylation in S. cerevisiae by introducing chromosomal point mutations that prevent or mimic phosphorylation at the conserved serine (S119. We find that mutants which prevent Rpt6 phosphorylation at this site (rpt6-S119A, had increased susceptibility to proteotoxic stress, displayed abnormal morphology and had reduced proteasome activity. Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington's disease (HD related huntingtin (Htt protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead (rpt6-S119A and phospho-mimetic (rpt6-S119D mutants. We showed Htt103Q aggregate size to be significantly larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D strains. Furthermore, we observed that phosphorylation of endogenous Rpt6 at S119 is increased in response to various stress conditions. Together, these data suggest that Rpt6 phosphorylation at S119 may play an important function in proteasome-dependent relief of proteotoxic stress that can be critical in protein aggregation pathologies.

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

    Directory of Open Access Journals (Sweden)

    Karl eScheu

    2014-08-01

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

  2. 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

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

    Science.gov (United States)

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

    2016-08-01

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

  4. Aggregation of whey protein hydrolysate using Alcalase 2.4 L.

    Directory of Open Access Journals (Sweden)

    Chunhong Liu

    Full Text Available Here, we describe peptide aggregation, which is also known as enzymatic protein resynthesis. Whey protein hydrolysate (WPH is the starting material for assembling peptides. Analyses of the involved amino acids, intrinsic fluorescence, fluorescence phase diagram, secondary structure, turbidity, and surface hydrophobicity were performed to investigate the reaction process. The aggregation mechanism consists of two parts: 1 formation and 2 aggregation of the building blocks that form the ordered secondary β-sheet structure. Constructing the building blocks requires at least one intermediate state, which is formed after 0.5 hours. Non-synergistic changes in the secondary and tertiary structures then allow the intermediate state to emerge.

  5. Aggregation of whey protein hydrolysate using Alcalase 2.4 L.

    Science.gov (United States)

    Liu, Chunhong; Liu, Wen; Feng, Zhibiao; Li, Dongmei

    2014-01-01

    Here, we describe peptide aggregation, which is also known as enzymatic protein resynthesis. Whey protein hydrolysate (WPH) is the starting material for assembling peptides. Analyses of the involved amino acids, intrinsic fluorescence, fluorescence phase diagram, secondary structure, turbidity, and surface hydrophobicity were performed to investigate the reaction process. The aggregation mechanism consists of two parts: 1) formation and 2) aggregation of the building blocks that form the ordered secondary β-sheet structure. Constructing the building blocks requires at least one intermediate state, which is formed after 0.5 hours. Non-synergistic changes in the secondary and tertiary structures then allow the intermediate state to emerge.

  6. 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.

  7. Ubiquitin ligase ITCH recruitment suppresses the aggregation and cellular toxicity of cytoplasmic misfolded proteins.

    Science.gov (United States)

    Chhangani, Deepak; Upadhyay, Arun; Amanullah, Ayeman; Joshi, Vibhuti; Mishra, Amit

    2014-05-28

    The protein quality control (QC) system protects cells against cellular toxicity induced by misfolded proteins and maintains overall cellular fitness. Inefficient clearance of or failure to degrade damaged proteins causes several diseases, especially age-linked neurodegenerative disorders. Attenuation of misfolded protein degradation under severe stress conditions leads to the rapid over-accumulation of toxic proteinaceous aggregates in the cytoplasmic compartment. However, the precise cytoplasmic quality control degradation mechanism is unknown. In the present study, we demonstrate that the Nedd4-like E3 ubiquitin ligase ITCH specifically interacts with mutant bona fide misfolded proteins and colocalizes with their perinuclear aggregates. In a cell culture model, we demonstrate ITCH recruitment by cytoplasmic inclusions containing polyglutamine-expanded huntingtin or ataxin-3 proteins. Transient overexpression of ITCH dramatically induced the degradation of thermally denatured misfolded luciferase protein. Partial depletion of ITCH increased the rate of aggregate formation and cell death generated by expanded polyglutamine proteins. Finally, we demonstrate that overexpression of ITCH alleviates the cytotoxic potential of expanded polyglutamine proteins and reduces aggregation. These observations indicate that ITCH is involved in the cytosolic quality control pathway and may help to explain how abnormal proteins are targeted by QC ubiquitin-protein ligases.

  8. Epigallocatechin-3-gallate and related phenol compounds redirect the amyloidogenic aggregation pathway of ataxin-3 towards non-toxic aggregates and prevent toxicity in neural cells and Caenorhabditis elegans animal model.

    Science.gov (United States)

    Visentin, Cristina; Pellistri, Francesca; Natalello, Antonino; Vertemara, Jacopo; Bonanomi, Marcella; Gatta, Elena; Penco, Amanda; Relini, Annalisa; De Gioia, Luca; Airoldi, Cristina; Regonesi, Maria E; Tortora, Paolo

    2017-09-01

    The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. Glycation of Lysozyme by Glycolaldehyde Provides New Mechanistic Insights in Diabetes-Related Protein Aggregation.

    Science.gov (United States)

    Mariño, Laura; Maya-Aguirre, Carlos Andrés; Pauwels, Kris; Vilanova, Bartolomé; Ortega-Castro, Joaquin; Frau, Juan; Donoso, Josefa; Adrover, Miquel

    2017-04-21

    Glycation occurs in vivo as a result of the nonenzymatic reaction of carbohydrates (and/or their autoxidation products) with proteins, DNA, or lipids. Protein glycation causes loss-of-function and, consequently, the development of diabetic-related diseases. Glycation also boosts protein aggregation, which can be directly related with the higher prevalence of aggregating diseases in diabetic people. However, the molecular mechanism connecting glycation with aggregation still remains unclear. Previously we described mechanistically how glycation of hen egg-white lysozyme (HEWL) with ribose induced its aggregation. Here we address the question of whether the ribose-induced aggregation is a general process or it depends on the chemical nature of the glycating agent. Glycation of HEWL with glycolaldehyde occurs through two different scenarios depending on the HEWL concentration regime (both within the micromolar range). At low HEWL concentration, non-cross-linking fluorescent advanced glycation end-products (AGEs) are formed on Lys side chains, which do not change the protein structure but inhibit its enzymatic activity. These AGEs have little impact on HEWL surface hydrophobicity and, therefore, a negligible effect on its aggregation propensity. Upon increasing HEWL concentration, the glycation mechanism shifts toward the formation of intermolecular cross-links, which triggers a polymerization cascade involving the formation of insoluble spherical-like aggregates. These results notably differ with the aggregation-modulation mechanism of ribosylated HEWL directed by hydrophobic interactions. Additionally, their comparison constitutes the first experimental evidence showing that the mechanism underlying the aggregation of a glycated protein depends on the chemical nature of the glycating agent.

  10. Influence of the N-terminal domain on the aggregation properties of the prion protein

    OpenAIRE

    Frankenfield, Kristen N.; Powers, Evan T.; Kelly, Jeffery W.

    2005-01-01

    Prion diseases appear to be caused by the aggregation of the cellular prion protein (PrPC) into an infectious form denoted PrPSc. The in vitro aggregation of the prion protein has been extensively investigated, yet many of these studies utilize truncated polypeptides. Because the C-terminal portion of PrPSc is protease-resistant and retains infectivity, it is assumed that studies on this fragment are most relevant. The full-length protein can be distinguished from the truncated protein becaus...

  11. The role of macropinocytosis in the propagation of protein aggregation associated with neurodegenerative diseases.

    Science.gov (United States)

    Zeineddine, Rafaa; Yerbury, Justin J

    2015-01-01

    With the onset of the rapidly aging population, the impact of age related neurodegenerative diseases is becoming a predominant health and economic concern. Neurodegenerative diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease (CJD), Parkinson's disease, Huntington's disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) result from the loss of a specific subsets of neurons, which is closely associated with accumulation and deposition of specific protein aggregates. Protein aggregation, or fibril formation, is a well-studied phenomenon that occurs in a nucleation-dependent growth reaction. Recently, there has been a swell of literature implicating protein aggregation and its ability to propagate cell-to-cell in the rapid progression of these diseases. In order for protein aggregation to be kindled in recipient cells it is a requisite that aggregates must be able to be released from one cell and then taken up by others. In this article we will explore the relationship between protein aggregates, their propagation and the role of macropinocytosis in their uptake. We highlight the ability of neurons to undergo stimulated macropinocytosis and identify potential therapeutic targets.

  12. The Role of Macropinocytosis in the Propagation of Protein Aggregation Associated with Neurodegenerative Diseases.

    Directory of Open Access Journals (Sweden)

    Rafaa eZeineddine

    2015-10-01

    Full Text Available With the onset of the rapidly ageing population, the impact of age related neurodegenerative diseases is becoming a predominant health and economic concern. Neurodegenerative diseases such as Alzheimer’s disease, Creutzfeldt-Jakob disease, Parkinson’s disease, Huntington’s disease, frontotemporal dementia and amyotrophic lateral sclerosis result from the loss of a specific subsets of neurons, which is closely associated with accumulation and deposition of specific protein aggregates. Protein aggregation, or fibril formation, is a well-studied phenomenon that occurs in a nucleation-dependent growth reaction. Recently, there has been a swell of literature implicating protein aggregation and its ability to propagate cell-to-cell in the rapid progression of these diseases. In order for protein aggregation to be kindled in recipient cells it is a requisite that aggregates must be able to be released from one cell and then taken up by others. In this article we will explore the relationship between protein aggregates, their propagation and the role of macropinocytosis in their uptake. We highlight the ability of neurons to undergo stimulated macropinocytosis and identify potential therapeutic targets.

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

    Directory of Open Access Journals (Sweden)

    Michael Brunsteiner

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

  14. Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates.

    Science.gov (United States)

    Hübner, S; Eam, J E; Hübner, A; Jans, D A

    2006-01-15

    Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.

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

    Science.gov (United States)

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

    2018-09-01

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

  16. Proteins isolated from regenerating sciatic nerves of rats form aggregates following posttranslational amino acid modification

    International Nuclear Information System (INIS)

    Ingoglia, N.A.; Chakroborty, G.; Yu, M.; Luo, D.; Sturman, J.A.

    1991-01-01

    Soluble proteins of regenerating sciatic nerves of rats can be posttranslationally, covalently modified by a variety of radioactive amino acids. The present study shows that once modified by a mixture of 15 amino acids, many of those proteins form aggregates that are unable to pass through a 0.45-micron filter and pellet following 20,000g centrifugation (suggesting a size of greater than 2 x 10(6) Da). Aggregation of proteins also occurs following modification by Arg or Lys alone, but does not occur following protein modification in nonregenerating nerves or in brain. Aggregates are not disrupted by treatment with 100 mM beta mercaptoethanol or by exposure to 1.0 M NaCl, but aggregates are solubilized by treatment with urea and by boiling in 1.5% SDS. Amino acid analysis of proteins modified by a mixture of [3H]amino acids shows a similar proportion of posttranslationally incorporated Ser, Pro, Val, Ala, Leu, Phe, Lys, and Arg in the soluble and pelletable fractions. Two-dimensional PAGE profiles of soluble and pelletable modified proteins show that the modified proteins in both fractions are in similar pI and molecular weight ranges, except that the soluble modified proteins include a high-molecular-weight component that is absent in the pelleted modified proteins. Kinetic studies show that while half-maximal levels of protein modification occur within 30 seconds of incubation, the appearance of the pelletable modified protein fraction is delayed significantly. These results indicate that amino acid modification of soluble proteins in regenerating sciatic nerves of rats results in physical changes in those proteins so that they form high-molecular-weight aggregates

  17. Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Huang, Richard Y.-C.; Iacob, Roxana E.; Krystek, Stanley R.; Jin, Mi; Wei, Hui; Tao, Li; Das, Tapan K.; Tymiak, Adrienne A.; Engen, John R.; Chen, Guodong

    2017-05-01

    Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.

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

    Science.gov (United States)

    Castellanos, Maria Monica

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

  19. Ubiquitin ligase ITCH recruitment suppresses the aggregation and cellular toxicity of cytoplasmic misfolded proteins

    OpenAIRE

    Chhangani, Deepak; Upadhyay, Arun; Amanullah, Ayeman; Joshi, Vibhuti; Mishra, Amit

    2014-01-01

    The protein quality control (QC) system protects cells against cellular toxicity induced by misfolded proteins and maintains overall cellular fitness. Inefficient clearance of or failure to degrade damaged proteins causes several diseases, especially age-linked neurodegenerative disorders. Attenuation of misfolded protein degradation under severe stress conditions leads to the rapid over-accumulation of toxic proteinaceous aggregates in the cytoplasmic compartment. However, the precise cytopl...

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

    Directory of Open Access Journals (Sweden)

    Nicholas J Bradshaw

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

  1. 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....

  2. Confinement to organelle-associated inclusion structures mediates asymmetric inheritance of aggregated protein in budding yeast.

    Science.gov (United States)

    Spokoini, Rachel; Moldavski, Ofer; Nahmias, Yaakov; England, Jeremy L; Schuldiner, Maya; Kaganovich, Daniel

    2012-10-25

    The division of the S. cerevisiae budding yeast, which produces one mother cell and one daughter cell, is asymmetric with respect to aging. Remarkably, the asymmetry of yeast aging coincides with asymmetric inheritance of damaged and aggregated proteins by the mother cell. Here, we show that misfolded proteins are retained in the mother cell by being sequestered in juxtanuclear quality control compartment (JUNQ) and insoluble protein deposit (IPOD) inclusions, which are attached to organelles. Upon exposure to stress, misfolded proteins accumulate in stress foci that must be disaggregated by Hsp104 in order to be degraded or processed to JUNQ and IPOD. Cells that fail to deliver aggregates to an inclusion pass on aggregates to subsequent generations. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Confinement to Organelle-Associated Inclusion Structures Mediates Asymmetric Inheritance of Aggregated Protein in Budding Yeast

    Directory of Open Access Journals (Sweden)

    Rachel Spokoini

    2012-10-01

    Full Text Available The division of the S. cerevisiae budding yeast, which produces one mother cell and one daughter cell, is asymmetric with respect to aging. Remarkably, the asymmetry of yeast aging coincides with asymmetric inheritance of damaged and aggregated proteins by the mother cell. Here, we show that misfolded proteins are retained in the mother cell by being sequestered in juxtanuclear quality control compartment (JUNQ and insoluble protein deposit (IPOD inclusions, which are attached to organelles. Upon exposure to stress, misfolded proteins accumulate in stress foci that must be disaggregated by Hsp104 in order to be degraded or processed to JUNQ and IPOD. Cells that fail to deliver aggregates to an inclusion pass on aggregates to subsequent generations.

  4. 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...

  5. Aldosterone and angiotensin II induced protein aggregation in renal proximal tubules

    DEFF Research Database (Denmark)

    Cheema, Muhammad Umar; Poulsen, Ebbe Toftgaard; Enghild, Jan J

    2013-01-01

    systems in the kidney from control rats and rats receiving aldosterone or angiotensin II treatment for 7 days. Control rats formed both aggresomes and autophagosomes specifically in the proximal tubules, indicating a need for these structures even under baseline conditions. Fluorescence sorted aggresomes......Renal tubules are highly active transporting epithelia and are at risk of protein aggregation due to high protein turnover and/or oxidative stress. We hypothesized that the risk of aggregation was increased upon hormone stimulation and assessed the state of the intracellular protein degradation...... contained various rat keratins known to be expressed in renal tubules as assessed by protein mass spectrometry. Aldosterone administration increased the abundance of the proximal tubular aggresomal protein keratin 5, the ribosomal protein RPL27, ataxin-3, and the chaperone heat shock protein 70...

  6. Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Hassan Ramshini

    Full Text Available The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i low pH with salts, (ii pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.

  7. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions

    OpenAIRE

    Cieplak, Andrzej Stanis?aw

    2017-01-01

    Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of foldin...

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

    Directory of Open Access Journals (Sweden)

    Erik Kvam

    2009-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Ashley A. Zurawel

    2016-09-01

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

  10. Inhibition of Mutant αB Crystallin-Induced Protein Aggregation by a Molecular Tweezer.

    Science.gov (United States)

    Xu, Na; Bitan, Gal; Schrader, Thomas; Klärner, Frank-Gerrit; Osinska, Hanna; Robbins, Jeffrey

    2017-08-08

    Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. The development of therapeutics directed at proteotoxicity-based pathology in heart disease is just beginning. The molecular tweezer CLR01 is a broad-spectrum inhibitor of abnormal self-assembly of amyloidogenic proteins, including amyloid β-protein, tau, and α-synuclein. This small molecule interferes with aggregation by binding selectively to lysine side chains, changing the charge distribution of aggregation-prone proteins and thereby disrupting aggregate formation. However, the effects of CLR01 in cardiomyocytes undergoing proteotoxic stress have not been explored. Here we assess whether CLR01 can decrease cardiac protein aggregation catalyzed by cardiomyocyte-specific expression of mutated αB-crystallin (CryAB R 120G ). A proteotoxic model of desmin-related cardiomyopathy caused by cardiomyocyte-specific expression of CryAB R 120G was used to test the efficacy of CLR01 therapy in the heart. Neonatal rat cardiomyocytes were infected with adenovirus expressing either wild-type CryAB or CryAB R 120G . Subsequently, the cells were treated with different doses of CLR01 or a closely related but inactive derivative, CLR03. CLR01 decreased aggregate accumulation and attenuated cytotoxicity caused by CryAB R 120G expression in a dose-dependent manner, whereas CLR03 had no effect. Ubiquitin-proteasome system function was analyzed using a ubiquitin-proteasome system reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein. CLR01 improved proteasomal function in CryAB R 120G cardiomyocytes but did not alter autophagic flux. In vivo, CLR01 administration also resulted in reduced protein aggregates in CryAB R 120G transgenic mice. CLR01 can inhibit CryAB R 120G aggregate formation and decrease cytotoxicity in cardiomyocytes undergoing proteotoxic stress

  11. Autophagy Dysregulation in ALS: When Protein Aggregates Get Out of Hand

    Directory of Open Access Journals (Sweden)

    Nandini Ramesh

    2017-08-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a neurodegenerative disorder that results from the loss of upper and lower motor neurons. One of the key pathological hallmarks in diseased neurons is the mislocalization of disease-associated proteins and the formation of cytoplasmic aggregates of these proteins and their interactors due to defective protein quality control. This apparent imbalance in the cellular protein homeostasis could be a crucial factor in causing motor neuron death in the later stages of the disease in patients. Autophagy is a major protein degradation pathway that is involved in the clearance of protein aggregates and damaged organelles. Abnormalities in autophagy have been observed in numerous neurodegenerative disorders, including ALS. In this review, we discuss the contribution of autophagy dysfunction in various in vitro and in vivo models of ALS. Furthermore, we examine the crosstalk between autophagy and other cellular stresses implicated in ALS pathogenesis and the therapeutic implications of regulating autophagy in ALS.

  12. Relationship between functional properties and aggregation changes of whey protein induced by high pressure microfluidization.

    Science.gov (United States)

    Liu, Cheng-Mei; Zhong, Jun-Zhen; Liu, Wei; Tu, Zong-Cai; Wan, Jie; Cai, Xiao-Fei; Song, Xin-Yun

    2011-05-01

    Aggregation changes of whey protein induced by high-pressure microfluidization (HPM) treatment have been investigated in relation with their functional properties. Whey protein was treated with HPM under pressure from 40 to 160 MPa. Functional properties (solubility, foaming, and emulsifying properties) of whey protein concentrate (WPC) ultrafiltered from fluid whey were evaluated. The results showed significant modifications in the solubility (30% to 59%) and foaming properties (20% to 65%) of WPC with increasing pressure. However, emulsifying property of WPC treated at different pressures was significantly worse than untreated sample. To better understand the mechanism of the modification by HPM, the HPM-induced aggregation changes were examined using particle size distribution, scanning electron microscopy, and hydrophobicity. It was indicated that HPM induced 2 kinds of aggregation changes on WPC: deaggregation and reaggregation of WPC, which resulted in the changes of functional properties of WPC modified by HPM. © 2011 Institute of Food Technologists®

  13. 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

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

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2012-01-01

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

  15. 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.

  16. The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation

    Directory of Open Access Journals (Sweden)

    Marcel Stark

    2014-01-01

    Full Text Available The standardized Ginkgo biloba extract EGb 761 has well-described antioxidative activities and effects on different cytoprotective signaling pathways. Consequently, a potential use of EGb 761 in neurodegenerative diseases has been proposed. A common characteristic feature of a variety of such disorders is the pathologic formation of protein aggregates, suggesting a crucial role for protein homeostasis. In this study, we show that EGb 761 increased the catalytic activity of the proteasome and enhanced protein degradation in cultured cells. We further investigated this effect in a cellular model of Huntington’s disease (HD by employing cells expressing pathologic variants of a polyglutamine protein (polyQ protein. We show that EGb 761 affected these cells by (i increasing proteasome activity and (ii inducing a more efficient degradation of aggregation-prone proteins. These results demonstrate a novel activity of EGb 761 on protein aggregates by enhancing proteasomal protein degradation, suggesting a therapeutic use in neurodegenerative disorders with a disturbed protein homeostasis.

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

    Directory of Open Access Journals (Sweden)

    Haina Qin

    2013-10-01

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

  18. The implication of topology for protein structure and aggregation

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik

    1997-01-01

    For proteins, it is discussed which intrinsic dynamical modes can be present when all static forces are screened. Excitations involving a wringing of the polypeptide chain around its path exists. It is suggested that the existence of such wringons is the underlying cause for the uniqueness...

  19. Protein quaternary structure and aggregation in relation to allergenicity

    NARCIS (Netherlands)

    Boxtel, van E.L.

    2007-01-01

    In order to induce systemic food allergic reactions in humans, proteins after digestion in the human gastro-intestinal tract should still be able to bind IgE. The aim of the work presented in this thesis was to determine the effects of heating on the structure and digestibility of cupin and prolamin

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

    Science.gov (United States)

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

    2016-01-01

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

  1. 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.

  2. Heavy Metals and Metalloids As a Cause for Protein Misfolding and Aggregation

    Directory of Open Access Journals (Sweden)

    Markus J. Tamás

    2014-02-01

    Full Text Available 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.

  3. Real-time protein aggregation monitoring with a Bloch surface wave-based approach

    Science.gov (United States)

    Santi, Sara; Barakat, Elsie; Descrovi, Emiliano; Neier, Reinhard; Herzig, Hans Peter

    2014-05-01

    The misfolding and aggregation of amyloid proteins has been associated with incurable diseases such as Alzheimer's or Parkinson's disease. In the specific case of Alzheimer's disease, recent studies have shown that cell toxicity is caused by soluble oligomeric forms of aggregates appearing in the early stages of aggregation, rather than by insoluble fibrils. Research on new strategies of diagnosis is imperative to detect the disease prior to the onset of clinical symptoms. Here, we propose the use of an optical method for protein aggregation dynamic studies using a Bloch surface wave based approach. A one dimension photonic crystal made of a periodic stack of silicon oxide and silicon nitride layers is used to excite a Bloch surface wave, which is sensitive to variation of the refractive index of an aqueous solution. The aim is to detect the early dynamic events of protein aggregation and fibrillogenesis of the amyloid-beta peptide Aβ42, which plays a central role in the onset of the Alzheimer's disease. The detection principle relies on the refractive index changes caused by the depletion of the Aβ42 monomer concentration during oligomerization and fibrillization. We demonstrate the efficacy of the Bloch surface wave approach by monitoring in real-time the first crucial steps of Aβ42 oligomerization.

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

    International Nuclear Information System (INIS)

    Michaels, Thomas C. T.; Lazell, Hamish W.; Arosio, Paolo; Knowles, Tuomas P. J.

    2015-01-01

    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

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

    Science.gov (United States)

    Michaels, Thomas C. T.; Lazell, Hamish W.; Arosio, Paolo; Knowles, Tuomas P. J.

    2015-08-01

    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.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Paola C Rosas

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

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

    Directory of Open Access Journals (Sweden)

    Baoru Yin

    2015-03-01

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

  9. 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

  10. Molecular mechanisms of protein aggregation from global fitting of kinetic models.

    Science.gov (United States)

    Meisl, Georg; Kirkegaard, Julius B; Arosio, Paolo; Michaels, Thomas C T; Vendruscolo, Michele; Dobson, Christopher M; Linse, Sara; Knowles, Tuomas P J

    2016-02-01

    The elucidation of the molecular mechanisms by which soluble proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity associated with aggregation reaction networks, the analysis of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quantitative kinetic assays and global fitting, to determine and to verify a molecular mechanism for aggregation reactions that is compatible with experimental kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic experiments that measure the concentration of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic experiment measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-separated text file. We also outline general experimental strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the analysis, we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global analysis of kinetic data without the need for extensive programming or detailed mathematical knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic analysis: determination of constraints to be placed on the aggregation mechanism based on the concentration dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and

  11. 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)

  12. 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

  13. The p62 antibody reveals various cytoplasmic protein aggregates in spinocerebellar ataxia Type 6

    NARCIS (Netherlands)

    Seidel, K.; Brunt, E. R. P.; de Vos, R. A. I.; Dijk, F.; van der Want, H. J. L.; Kampinga, H. H.; Rueb, U.; den Dunnen, W. F. A.

    2009-01-01

    Neuronal protein aggregates are considered as pathological hallmarks of various human neurodegenerative diseases, including the so-called CAG-repeat disorders, such as spinocerebellar ataxia Type 6 (SCA6). Since the immunocytochemical findings of an initial post-mortem study using a specific

  14. 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

  15. Effect of Protein Charge on the Generation of Aggregation-Prone Conformers

    NARCIS (Netherlands)

    Broersen, K.; Weijers, M.; Groot, de J.; Hamer, R.J.; Jongh, de H.H.J.

    2007-01-01

    This study describes how charge modification affects aggregation of ovalbumin, thereby distinguishing the role of conformational and electrostatic stability in the process. Ovalbumin variants were engineered using chemical methylation or succinylation to obtain a range of protein net charge from -1

  16. 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

  17. 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.

  18. Effects of succinylation on the structure and thermal aggregation of soy protein isolate.

    Science.gov (United States)

    Wan, Yangling; Liu, Jingyuan; Guo, Shuntang

    2018-04-15

    The structures of soy protein isolate, beta-conglycinin, and glycinin at increasing succinylation levels (0-94.88%) were determined to control the formation of soy protein thermal aggregates. In addition, the thermal aggregation was investigated under various temperatures (70-100 °C) and ionic strengths (0-1.0 mol/L NaCl) at pH 7.0. Results showed that soy protein isolate, beta-conglycinin, and glycinin underwent obvious structural changes when their succinylation degrees reached around 60%, 30%, and 65%, respectively. After which, the acylation rates markedly declined. During succinylation, soy proteins, particularly glycinin, endured gradual damages in its secondary and tertiary structures. Consequently, the thermal stability of glycinin was reduced, whereas that of beta-conglycinin was hardly affected. However, as the colloid stability of succinylated soy protein isolate was enhanced significantly, its thermal aggregation was markedly suppressed. Thus, succinylation could be used to improve the stability of soy proteins after heating. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-05-28

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

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

    Science.gov (United States)

    Myant, Connor William; Cann, Philippa

    2014-06-01

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

  1. Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates.

    Science.gov (United States)

    Tharad, Sudarat; Toca-Herrera, José L; Promdonkoy, Boonhiang; Krittanai, Chartchai

    2016-10-01

    Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane. © 2016 The Author(s).

  2. Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile re

    NARCIS (Netherlands)

    Sutter, M.; Oliveira, S.; Sanders, N.N.; Lucas, B.; Hoek, van A.; Hink, M.A.; Visser, A.J.W.G.; Smedt, de S.C.; Hennink, W.E.; Jiskoot, W.

    2007-01-01

    The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein ß-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of ß-galactosidase below and above the protein¿s unfolding temperature of

  3. Prion protein insertional mutations increase aggregation propensity but not fiber stability

    Directory of Open Access Journals (Sweden)

    True Heather L

    2008-03-01

    Full Text Available Abstract Background Mutations in the PRNP gene account for ~15% of all prion disease cases. Little is understood about the mechanism of how some of these mutations in PRNP cause the protein to aggregate into amyloid fibers or cause disease. We have taken advantage of a chimeric protein system to study the oligopeptide repeat domain (ORD expansions of the prion protein, PrP, and their effect on protein aggregation and amyloid fiber formation. We replaced the ORD of the yeast prion protein Sup35p with that from wild type and expanded ORDs of PrP and compared their biochemical properties in vitro. We previously determined that these chimeric proteins maintain the [PSI+] yeast prion phenotype in vivo. Interestingly, we noted that the repeat expanded chimeric prions seemed to be able to maintain a stronger strain of [PSI+] and convert from [psi-] to [PSI+] with a much higher frequency. In this study we have attempted to understand the biochemical properties of these chimeric proteins and to establish a system to study the properties of the ORD of PrP both in vivo and in vitro. Results Investigation of the chimeric proteins in vitro reveals that repeat-expansions increase aggregation propensity and that the kinetics of fiber formation depends on the number of repeats. The fiber formation reactions are promiscuous in that the chimeric protein containing 14 repeats can readily cross-seed fiber formation of proteins that have the wild type number of repeats. Morphologically, the amyloid fibers formed by repeat-expanded proteins associate with each other to form large clumps that were not as prevalent in fibers formed by proteins containing the wild type number of repeats. Despite the increased aggregation propensity and lateral association of the repeat expanded proteins, there was no corresponding increase in the stability of the fibers formed. Therefore, we predict that the differences in fibers formed with different repeat lengths may not be due to

  4. Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models

    Directory of Open Access Journals (Sweden)

    Natalia V. Povarova

    2017-07-01

    Full Text Available Despite great advances in practical applications of fluorescent proteins (FPs, their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed “trio-FPs” consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.

  5. Combined dynamic light scattering and Raman spectroscopy approach for characterizing the aggregation of therapeutic proteins.

    Science.gov (United States)

    Lewis, E Neil; Qi, Wei; Kidder, Linda H; Amin, Samiul; Kenyon, Stacy M; Blake, Steven

    2014-12-12

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

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

    Directory of Open Access Journals (Sweden)

    E. Neil Lewis

    2014-12-01

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

  7. [Role of protein kinases of human red cell membrane in deformability and aggregation changes].

    Science.gov (United States)

    Murav'ev, A V; Maĭmistova, A A; Tikhomirova, I A; Bulaeva, S V; Mikhaĭlov, P V; Murav'ev, A A

    2012-01-01

    The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p RBCA) was significantly decreased under these conditions (p RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

  8. The physical chemistry of the amyloid phenomenon: thermodynamics and kinetics of filamentous protein aggregation.

    Science.gov (United States)

    Buell, Alexander K; Dobson, Christopher M; Knowles, Tuomas P J

    2014-01-01

    In this chapter, we present an overview of the kinetics and thermodynamics of protein aggregation into amyloid fibrils. The perspective we adopt is largely experimental, but we also discuss recent developments in data analysis and we show that only a combination of well-designed experiments with appropriate theoretical modelling is able to provide detailed mechanistic insight into the complex pathways of amyloid formation. In the first part of the chapter, we describe measurements of the thermodynamic stability of the amyloid state with respect to the soluble state of proteins, as well as the magnitude and origin of this stability. In the second part, we discuss in detail the kinetics of the individual molecular steps in the overall mechanism of the conversion of soluble protein into amyloid fibrils. Finally, we highlight the effects of external factors, such as salt type and concentration, chemical denaturants and molecular chaperones on the kinetics of aggregation.

  9. 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.

  10. Choosing the right protein A affinity chromatography media can remove aggregates efficiently.

    Science.gov (United States)

    Yada, Tomokazu; Nonaka, Koichi; Yabuta, Masayuki; Yoshimoto, Noriko; Yamamoto, Shuichi

    2017-01-01

    Protein A chromatography (PAC) is commonly used as an efficient capture step in monoclonal antibody (mAb) separation processes. Usually dynamic binding capacity is used for choosing the right PAC. However, if aggregates can be efficiently removed during elution, it can make the following polishing steps easier. In this study a method for choosing the right PAC media in terms of mAb aggregate removal is proposed. Linear pH gradient elution experiments of two different mAbs on various PAC columns are carried out, where the elution behavior of aggregates as well as the monomer is measured. Aggregates of one mAb are more strongly retained compared with the mAb monomer. Another mAb showed different elution behavior, where the aggregates are eluted as both the weakly and strongly retained peaks. In order to remove the two types of aggregates by stepwise elution two protocols are tested. The first protocol A consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. No detectable peaks are observed during the wash stage of protocol A whereas significant peaks are monitored during the arginine wash of protocol B. One of the PAC columns showed a smaller peak during the arginine wash. In addition, both aggregate removal and monomer yield are higher with protocol B compared with the other PAC columns. This method is found to be useful for choosing the right PAC column. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. 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.

  12. Tau Protein Hyperphosphorylation and Aggregation in Alzheimer's Disease and Other Tauopathies, and Possible Neuroprotective Strategies.

    Science.gov (United States)

    Šimić, Goran; Babić Leko, Mirjana; Wray, Selina; Harrington, Charles; Delalle, Ivana; Jovanov-Milošević, Nataša; Bažadona, Danira; Buée, Luc; de Silva, Rohan; Di Giovanni, Giuseppe; Wischik, Claude; Hof, Patrick R

    2016-01-06

    Abnormal deposition of misprocessed and aggregated proteins is a common final pathway of most neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized by the extraneuronal deposition of the amyloid β (Aβ) protein in the form of plaques and the intraneuronal aggregation of the microtubule-associated protein tau in the form of filaments. Based on the biochemically diverse range of pathological tau proteins, a number of approaches have been proposed to develop new potential therapeutics. Here we discuss some of the most promising ones: inhibition of tau phosphorylation, proteolysis and aggregation, promotion of intra- and extracellular tau clearance, and stabilization of microtubules. We also emphasize the need to achieve a full understanding of the biological roles and post-translational modifications of normal tau, as well as the molecular events responsible for selective neuronal vulnerability to tau pathology and its propagation. It is concluded that answering key questions on the relationship between Aβ and tau pathology should lead to a better understanding of the nature of secondary tauopathies, especially AD, and open new therapeutic targets and strategies.

  13. Thermal aggregation behaviour of soy protein: characteristics of different polypeptides and sub-units.

    Science.gov (United States)

    He, Xiu-Ting; Yuan, De-Bao; Wang, Jin-Mei; Yang, Xiao-Quan

    2016-03-15

    Due to the differences in structure and composition of glycinin and β-conglycinin, they exhibit different characteristics during heat treatment. In present study, the thermal aggregation behaviour of glycinin, β-conglycinin and their isolated sub-units was investigated at pH 7.0. Acidic polypeptides, basic polypeptides, αα' and β sub-units of soy protein were denatured during the isolation process. The degree of aggregation of protein fractions after heat treatment was in the order: denatured basic polypeptides > native glycinin > denatured β sub-unit > native β-conglycinin > denatured acidic polypeptides > denatured αα' sub-units. Glycinin, β-conglycinin, acidic polypeptides and αα'/β sub-units exhibited different changing trends of surface hydrophobicity with increasing temperature. The αα' sub-units showed higher ability to suppress thermal aggregation of basic polypeptides than β sub-units during heat treatment. The β sub-units were shown to form soluble aggregates with glycinin after heating. The interaction mechanism of αα' and β sub-units heated with basic polypeptides was proposed. For the β sub-units-basic polypeptides mixed system, more hydrophobic chains were binding together and buried inside during heat treatment, which resulted in lower surface hydrophobicity. The αα' sub-units-basic polypeptides mixed system was considered to be a stable system with higher surface hydrophobicity after being heated. © 2015 Society of Chemical Industry.

  14. Administration of DNA Plasmid Coding Protein Aggregating Domain Induces Inflammatory Bone Loss.

    Science.gov (United States)

    Agas, Dimitrios; Concetti, Fabio; Capitani, Melania; Lacava, Giovanna; Concetti, Antonio; Marchetti, Luigi; Laus, Fulvio; Marchegiani, Andrea; Azevedo, Vasco; Sabbieti, Maria Giovanna; Venanzi, Franco Maria

    2016-01-01

    Plasmids coding protein aggregation polypeptides from different sources have been proposed as genetic adjuvants for DNA vaccines. We reported that a plasmid (pATRex), encompassing the DNA sequence for the von Willebrand A (vWA/A) domain of the Anthrax Toxin Receptor-1 (ANTXR-1, alias TEM8, Tumor Endothelial Marker 8), acts as strong immune adjuvant by inducing formation of insoluble intracellular aggregates and subsequent cell death. In the present study we addressed the question of whether there is any substantial immunotoxicity associated with the use of self-aggregating proteins as genetic adjuvants. Here we report, by mean of histology, X-ray and molecular examinations of bone specimens, the unexpected finding that intramuscular injection of pATRex in mice triggers, per se, severe bone loss (osteoporosis) independently from the sex and genotype of the treated animals. Even though the study suggests that proteinaceous "sticky " adjuvants are unlikely to find their way into practical vaccination, the information gained is of value as ATRex injections could provide an additional, simplified, mouse model of osteoporosis. Moreover, our results provide experimental support to the hypothesis that proteotoxic aggregates chronically activate the innate immune system in amyloid and aggregosome associated disorders.

  15. Sugar-Terminated Nanoparticle Chaperones Are 102-105Times Better Than Molecular Sugars in Inhibiting Protein Aggregation and Reducing Amyloidogenic Cytotoxicity.

    Science.gov (United States)

    Pradhan, Nibedita; Shekhar, Shashi; Jana, Nihar R; Jana, Nikhil R

    2017-03-29

    Sugar-based osmolyte molecules are known to stabilize proteins under stress, but usually they have poor chaperone performance in inhibiting protein aggregation. Here, we show that the nanoparticle form of sugars molecule can enhance their chaperone performance typically by 10 2 -10 5 times, compared to molecular sugar. Sugar-based plate-like nanoparticles of 20-40 nm hydrodynamic size have been synthesized by simple heating of acidic aqueous solution of glucose/sucrose/maltose/trehalose. These nanoparticles have excitation-dependent green/yellow/orange emission and surface chemistry identical to the respective sugar molecule. Fibrillation of lysozyme/insulin/amyloid beta in extracellular space, aggregation of mutant huntingtin protein inside model neuronal cell, and cytotoxic effect of fibrils are investigated in the presence of these sugar nanoparticles. We found that sugar nanoparticles are 10 2 -10 5 times efficient than respective sugar molecules in inhibiting protein fibrillation and preventing cytotoxicity arising of fibrils. We propose that better performance of the nanoparticle form is linked to its stronger binding with fibril structure and enhanced cell uptake. This result suggests that nanoparticle form of osmolyte can be an attractive option in prevention and curing of protein aggregation-derived diseases.

  16. Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

    Science.gov (United States)

    Courtois, Fabienne; Agrawal, Neeraj J; Lauer, Timothy M; Trout, Bernhardt L

    2016-01-01

    The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions. PMID:26514585

  17. PARKINSON’S DISEASE MODELS OF ABNORMAL PROTEIN AGGREGATION IN THE GOTTINGEN MINIPIG CNS

    DEFF Research Database (Denmark)

    Glud, Andreas Nørgaard; Landau, Anne M.; Lillethorup, Thea Pinholt

    2015-01-01

    Background: Parkinson's disease (PD) animal models are important translational steps toward clinical applications. The Göttingen minipig(GM) has a large gyrencephalic brain (6x5x4cm) that can be examined using conventional scanning modalities. Preclinical neuromodulatory devices can be evaluated...... in GM models. aSYN is a known pathological protein in PD. Aggregation or overexpression occurs in human PD and has been used to induce PD in other animal models. Aim: We aim to develop and validate novel models of PD in the GM based on abnormal protein aggregation. Methods: Using human intended MRI......-guided stereotaxic neurosurgery, we are inducing a PD-syndrome in GM via overexpression of mutated human aSYN using viral vectors or intracerebroventricular administration of an ubiquitin proteasome system inhibitor. The resulting pathological changes are quantified by neurological tests, PET-imaging of monaminergic...

  18. 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.

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

    Science.gov (United States)

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

    2016-05-01

    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 p H, 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.

  20. The role of anionic peptide fragments in 1N4R human tau protein aggregation.

    Science.gov (United States)

    Khalili, Mohammad Ali Nasiri; Riazi, Gholamhossein; Ahmadian, Shahin; Khodarahmi, Reza; Khodadadi, Sirus; Afrasiabi, Ali; Karima, Oveis; Mokhtari, Farzad; Hoveizi, Elham

    2014-06-01

    Cellular protein degradation systems are necessary to avoid the accumulation of misfolded or damaged proteins. Deficiency in these systems might cause to partial degradation of misfolded proteins and generation of amyloidogenic fragments. Protein misfolding is believed to be the primary cause of neurodegenerative disorders such as Alzheimer's disease (AD). In this study, we investigate effect of two anionic peptide fragments including, an acidic fragment of human Aβ (Aβ1-11) and a phosphorylated fragment of β-Casein (Tetraphosphopeptide), on tau protein aggregation. According to our results, these peptide fragments, induced tau fibrillization in vitro. In sum, we suggest that structural and conformational characters of inducer are as important as charge distribution on anionic inducer molecules however more experiments would be need to exactly confirm this suggestion.

  1. 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®

  2. Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies.

    Science.gov (United States)

    Biasini, Emiliano; Seegulam, M Esa; Patti, Brianna N; Solforosi, Laura; Medrano, Andrea Z; Christensen, Heather M; Senatore, Assunta; Chiesa, Roberto; Williamson, R Anthony; Harris, David A

    2008-06-01

    The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP(C)) into an infectious, aggregated isoform (PrP(Sc)) that has a high content of beta-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP(Sc) but not PrP(C), as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19-33, 89-112, and 136-158) have been previously reported to react specifically with infectious PrP(Sc) but not PrP(C). In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.

  3. Proteostasis and the Regulation of Intra- and Extracellular Protein Aggregation by ATP-Independent Molecular Chaperones: Lens α-Crystallins and Milk Caseins.

    Science.gov (United States)

    Carver, John A; Ecroyd, Heath; Truscott, Roger J W; Thorn, David C; Holt, Carl

    2018-03-20

    Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in

  4. Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing.

    Science.gov (United States)

    Winkler, Juliane; Seybert, Anja; König, Lars; Pruggnaller, Sabine; Haselmann, Uta; Sourjik, Victor; Weiss, Matthias; Frangakis, Achilleas S; Mogk, Axel; Bukau, Bernd

    2010-03-03

    The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Insights in understanding aggregate formation and dissociation in cation exchange chromatography for a structurally unstable Fc-fusion protein.

    Science.gov (United States)

    Chen, Zhiqiang; Huang, Chao; Chennamsetty, Naresh; Xu, Xuankuo; Li, Zheng Jian

    2016-08-19

    Cation-exchange chromatography (CEX) of a structurally unstable Fc-fusion protein exhibited multi-peak elution profile upon a salt-step elution due to protein aggregation during intra-column buffer transition where low pH and high salt coexisted. The protein exhibited a single-peak elution behavior during a pH-step elution; nevertheless, the levels of soluble aggregates (i.e. high molecular weight species, HMW) in the CEX eluate were still found up to 12-fold higher than that for the load material. The amount of the aggregates formed upon the pH-step elution was dependent on column loading with maximum HMW achieved at intermediate loading levels, supporting the hypothesis that the aggregation was the result of both the conformational changes of the bound protein and the solution concentration of the aggregation-susceptible proteins during elution. Factors such as high load pH, short protein/resin contact time, hydrophilic resin surface, and weak ionizable ligand were effective, to some extent, to reduce aggregate formation by improving the structural integrity of the bound protein. An orthogonal technique, differential scanning fluorimetry (DSF) using Sypro Orange dye confirmed that the bound protein exposed more hydrophobic area than the native molecule in free solution, especially in the pH 4-5 range. The Sypro Orange dye study of resin surface property also demonstrated that the poly[styrene-divinylbenzene]-based Poros XS with polyhydroxyl surface coating is more hydrophobic compared to the agarose-based CM Sepharose FF and SP Sepharose FF. The hydrophobic property of Poros XS contributed to stronger interactions with the partially unfolded bound protein and consequently to the higher aggregate levels seen in Poros XS eluate. This work also investigates the aggregation reversibility in CEX eluate where up to 66% of the aggregates were observed to dissociate into native monomers over a period of 120h, and links the aggregate stability to such conditions as resin

  7. Aggregate structure and effect of phthalic anhydride modified soy protein on the mechanical properties of styrene-butadiene copolymer

    Science.gov (United States)

    The aggregate structure of phthalic anhydride (PA) modified soy protein isolate (SPI) was investigated by estimating its fractal dimension from the equilibrated dynamic strain sweep experiments. The estimated fractal dimensions of the filler aggregates were less than 2, indicating that these partic...

  8. Fluorescence lifetime dynamics of eGFP in protein aggregates with expanded polyQ

    Science.gov (United States)

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2009-02-01

    Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues (97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the process of polyQ protein aggregation per se.

  9. Effect of Dentin Bonding Agent on the Prevention of Tooth Discoloration Produced by Mineral Trioxide Aggregate

    Directory of Open Access Journals (Sweden)

    Majid Akbari

    2012-01-01

    Full Text Available Objective. Determination of the effect of dentin bonding agent (DBA on the prevention of tooth discoloration produced by mineral trioxide aggregate (MTA. Methods. 50 teeth were endodontically treated and after removal of 3 mm of obturating materials were divided into five groups. In white MTA (WMTA and grey MTA (GMTA groups, these materials were placed in root canal below the orifice. In DBA + WMTA and DBA + GMTA groups, DBAs were applied in the access cavity. Then, 3 mm of WMTA and GMTA was placed. The last 10 teeth served as control. All of teeth were restored and color measurement was recorded for each specimen at this time and 6 months later. Results. The mean tooth discoloration in WMTA and GMTA groups was significantly more than DBA + WMTA and DBA + GMTA groups, respectively. There was no significant difference between DBA + WMTA and DBA + GMTA groups and control group. Conclusion. Application of DBA before MTA may prevent tooth discoloration.

  10. 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......) exhibited a less severe temperature-sensitive folding defect. Based on the magnitude of in vitro defects, these SCAD proteins are characterized as folding-defective variants and mild folding variants, respectively. Pulse-chase experiments demonstrated that the variant SCAD proteins either triggered...... 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....

  11. 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

  12. Food protein aggregates as vitamin-matrix carriers: impact of processing conditions.

    Science.gov (United States)

    Relkin, Perla; Shukat, Rizwan

    2012-10-15

    We studied the ability of protein aggregates for loading and protection of α-tocopherol, a model of heat- and light-sensitive bioactive compounds. Aqueous dispersions of whey proteins (4.5 wt.%, pH 6.7) in the absence and presence of α-tocopherol (4 wt.%) were prepared using an ultradisperser (10,000 rpm for 10 min and 65 °C), and then submitted to further high-pressure homogenisation (HPH) at 300 or 1200 bar for 12 cycles. Relative to free-vitamin dispersions, increasing HPH conditions in the presence of vitamin led to higher protein denaturation, more tryptophan quenching and wavelength blue-shift (by 10nm), in parallel with increased zeta potential values (by -10 mV), particle sizes (by 50%), and newly formed protein dimers, trimers and high molecular weight aggregates. As a result, the degree of vitamin degradation under increasing HPH and long-term storage was shown to decrease from 66% (ultradisper) to 50%, or to 30% (subject to further treatments at 300 or 1200 bar, respectively). Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions.

    Science.gov (United States)

    Cieplak, Andrzej Stanisław

    2017-01-01

    Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer's amyloid β and tau, Parkinson's α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i) structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii) effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and chirality of solid

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  15. 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.

  16. 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

  17. Aggregation of model amyloid insulin protein in crowding environments and under ac-electric fields

    Science.gov (United States)

    Zheng, Zhongli; Jing, Benxin; Murray, Brian; Sorci, Mirco; Belfort, Georges; Zhu, Y.

    2013-03-01

    In vitro experiments have been widely used to characterize the misfolding/unfolding pathway characteristic of amylodogenic proteins. Conversion from natively folded amyloidogenic proteins to oligomers via nucleation is the accepted path to fibril formation upon heating over a certain lag time period. In this work, we investigate the effect of crowing environment and external electric fields on the pathway and kinetics of insulin, a well-established amyloid model protein by single fluorescence spectroscopy and imaging. With added co-solutes, such as glycerol and polyvinylpyrrolidone (PVP), to mimic the cellular crowding environments, we have observed that the lag time can be significantly prolonged. The lag time increases with increasing co-solute concentration, yet showing little dependence on solution viscosity. Conversely, applied ac-electric fields can considerably shorten the lag timewhen a critical ac-voltage is exceeded. The strong dependence of lag time on ac-frequency over a narrow range of 500 Hz-5 kHz indicates the effect of ac-electroosmosis on the diffusion controlled process of insulin nucleation. Yet, no conformational structure is detected with insulin under applied ac-fields, suggesting the equivalence of ac-polarization to the conventional thermal activation process for insulin aggregation. These finding suggest that at least the aggregation kinetics of insulin can be altered by local solution condition or external stimuli, which gives new insight to the treatment of amyloid related diseases.

  18. 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.

  19. 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

    2015-10-09

    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. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  20. 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

  1. Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS.

    Directory of Open Access Journals (Sweden)

    Zhihui Sun

    2011-04-01

    Full Text Available TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS and frontotemporal lobar degeneration (FTLD. Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.

  2. Propeptide of aminopeptidase 1 protein mediates aggregation and vesicle formation in cytoplasm-to-vacuole targeting pathway.

    Science.gov (United States)

    Morales Quinones, Mariana; Winston, Jared T; Stromhaug, Per E

    2012-03-23

    Misfolded protein aggregation causes disease and aging; autophagy counteracts this by eliminating damaged components, enabling cells to survive starvation. The cytoplasm-to-vacuole targeting pathway in yeast encompasses the aggregation of the premature form of aminopeptidase 1 (prApe1) in cytosol and its sequestration by autophagic proteins into a vesicle for vacuolar transport. We show that the propeptide of Ape1 is important for aggregation and vesicle formation and that it is sufficient for binding to prApe1 and Atg19. Defective aggregation disrupts vacuolar transport, suggesting that aggregate shape is important in vesicle formation, whereas Atg19 binding is not sufficient for vacuolar transport. Aggregation involves hydrophobicity, whereas Atg19 binding requires additional electrostatic interactions. Ape1 dodecamerization may cluster propeptides into trimeric structures, with sufficient affinity to form propeptide hexamers by binding to other dodecamers, causing aggregation. We show that Ape1 aggregates bind Atg19 and Atg8 in vitro; this could be used as a scaffold for an in vitro assay of autophagosome formation to elucidate the mechanisms of autophagy.

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

    Directory of Open Access Journals (Sweden)

    Yasusi eYamamoto

    2013-10-01

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

  4. Heat-induced aggregation of whey proteins: comparison of cheese WPC with acid WPC and relevance of mineral composition.

    Science.gov (United States)

    Havea, Palatasa; Singh, Harjinder; Creamer, Lawrence K

    2002-07-31

    Heat-induced aggregation of whey proteins in solutions made from two commercial whey protein concentrates (WPCs), one derived from mineral acid whey (acid WPC) and the other from cheese whey (cheese WPC), was studied using polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), and transmission electron microscopy (TEM). Heat treatment (75 degrees C) of acid WPC solutions (12.0%, w/w, pH 6.9) resulted in formation of relatively small "soluble" aggregates that were predominantly disulfide-linked. By contrast, heat treatment of the cheese WPC solutions (under the same conditions) caused formation of relatively large aggregates, containing high proportions of aggregates linked by noncovalent associations. The rate of aggregation of both beta-lactoglobulin and alpha-lactalbumin at 75 degrees C, measured as the loss of native proteins by PAGE, was higher in the cheese WPC solution than in the acid WPC solution. Cross dialysis of the two WPC solutions resulted in alteration of the mineral composition of each WPC solution and reversing their heat-induced aggregation behavior. The results demonstrated that the mineral composition is very important in controlling the aggregation behavior of WPC products.

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

    Directory of Open Access Journals (Sweden)

    Wüstner Daniel

    2012-11-01

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

  6. Identification of Structural Features of Condensed Tannins That Affect Protein Aggregation

    Science.gov (United States)

    Ropiak, Honorata M.; Lachmann, Peter; Ramsay, Aina; Green, Rebecca J.; Mueller-Harvey, Irene

    2017-01-01

    A diverse panel of condensed tannins was used to resolve the confounding effects of size and subunit composition seen previously in tannin-protein interactions. Turbidimetry revealed that size in terms of mean degree of polymerisation (mDP) or average molecular weight (amw) was the most important tannin parameter. The smallest tannin with the relatively largest effect on protein aggregation had an mDP of ~7. The average size was significantly correlated with aggregation of bovine serum albumin, BSA (mDP: r = -0.916; amw: r = -0.925; p<0.01; df = 27), and gelatin (mDP: r = -0.961; amw: r = -0.981; p<0.01; df = 12). The procyanidin/prodelphinidin and cis-/trans-flavan-3-ol ratios gave no significant correlations. Tryptophan fluorescence quenching indicated that procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface and in the hydrophobic binding pocket (r = 0.677; p<0.05; df = 9 and r = 0.887; p<0.01; df = 9, respectively). Circular dichroism revealed that higher proportions of prodelphinidins decreased the apparent α-helix content (r = -0.941; p<0.01; df = 5) and increased the apparent β-sheet content (r = 0.916; p<0.05; df = 5) of BSA. PMID:28125657

  7. Identification of Structural Features of Condensed Tannins That Affect Protein Aggregation.

    Directory of Open Access Journals (Sweden)

    Honorata M Ropiak

    Full Text Available A diverse panel of condensed tannins was used to resolve the confounding effects of size and subunit composition seen previously in tannin-protein interactions. Turbidimetry revealed that size in terms of mean degree of polymerisation (mDP or average molecular weight (amw was the most important tannin parameter. The smallest tannin with the relatively largest effect on protein aggregation had an mDP of ~7. The average size was significantly correlated with aggregation of bovine serum albumin, BSA (mDP: r = -0.916; amw: r = -0.925; p<0.01; df = 27, and gelatin (mDP: r = -0.961; amw: r = -0.981; p<0.01; df = 12. The procyanidin/prodelphinidin and cis-/trans-flavan-3-ol ratios gave no significant correlations. Tryptophan fluorescence quenching indicated that procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface and in the hydrophobic binding pocket (r = 0.677; p<0.05; df = 9 and r = 0.887; p<0.01; df = 9, respectively. Circular dichroism revealed that higher proportions of prodelphinidins decreased the apparent α-helix content (r = -0.941; p<0.01; df = 5 and increased the apparent β-sheet content (r = 0.916; p<0.05; df = 5 of BSA.

  8. Aggregation of gluten proteins in model dough after fibre polysaccharide addition.

    Science.gov (United States)

    Nawrocka, Agnieszka; Szymańska-Chargot, Monika; Miś, Antoni; Wilczewska, Agnieszka Z; Markiewicz, Karolina H

    2017-09-15

    FT-Raman spectroscopy, thermogravimetry and differential scanning calorimetry were used to study changes in structure of gluten proteins and their thermal properties influenced by four dietary fibre polysaccharides (microcrystalline cellulose, inulin, apple pectin and citrus pectin) during development of a model dough. The flour reconstituted from wheat starch and wheat gluten was mixed with the polysaccharides in five concentrations: 3%, 6%, 9%, 12% and 18%. The obtained results showed that all polysaccharides induced similar changes in secondary structure of gluten proteins concerning formation of aggregates (1604cm -1 ), H-bonded parallel- and antiparallel-β-sheets (1690cm -1 ) and H-bonded β-turns (1664cm -1 ). These changes concerned mainly glutenins since β-structures are characteristic for them. The observed structural changes confirmed hypothesis about partial dehydration of gluten network after polysaccharides addition. The gluten aggregation and dehydration processes were also reflected in the DSC results, while the TGA ones showed that gluten network remained thermally stable after polysaccharides addition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. 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)

  10. 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.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    Rui Li

    2016-03-01

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

  13. The Copper Metabolism MURR1 domain protein 1 (COMMD1 modulates the aggregation of misfolded protein species in a client-specific manner.

    Directory of Open Access Journals (Sweden)

    Willianne I M Vonk

    Full Text Available The Copper Metabolism MURR1 domain protein 1 (COMMD1 is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α. Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1 and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS, cause misfolding and aggregation of the mutant SOD1 (mSOD1 protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

  14. Grb2 is regulated by foxd3 and has roles in preventing accumulation and aggregation of mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Shounak Baksi

    Full Text Available Growth factor receptor protein binding protein 2 (Grb2 is known to be associated with intracellular growth and proliferation related signaling cascades. Huntingtin (Htt, a ubiquitously expressed protein, when mutated, forms toxic intracellular aggregates - the hallmark of Huntington's disease (HD. We observed an elevated expression of Grb2 in neuronal cells in animal and cell models of HD. Grb2 overexpression was predominantly regulated by the transcription factor Forkhead Box D3 (Foxd3. Exogenous expression of Grb2 also reduced aggregation of mutant Htt in Neuro2A cells. Grb2 is also known to interact with Htt, depending on epidermal growth factor receptor (EGFR activation. Grb2- mutant Htt interaction in the contrary, took place in vesicular structures, independent of EGFR activation that eventually merged with autophagosomes and activated the autophagy machinery helping in autophagosome and lysosome fusion. Grb2, with its emerging dual role, holds promise for a survival mechanism for HD.

  15. Creation of model proteins to investigate the mechanism of aggregation of expanded-polyglutamine proteins. Insertion of polyglutamine tracts into the ß-lactamase BlaP

    OpenAIRE

    Scarafone, Natacha

    2012-01-01

    Polyglutamine (polyQ) diseases are characterized by the formation of intranuclear amyloid-like aggregates by proteins containing an expansion of a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis of the diseases. The only known common point between the causative proteins is the expanded polyQ tract, suggesting that their aggregation critically depends on the expansion of the polyQ tract abov...

  16. Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome.

    Science.gov (United States)

    Bonanomi, Marcella; Mazzucchelli, Serena; D'Urzo, Annalisa; Nardini, Marco; Konarev, Petr V; Invernizzi, Gaetano; Svergun, Dmitri I; Vanoni, Marco; Regonesi, Maria Elena; Tortora, Paolo

    2014-06-01

    Ataxin-3 (AT3) is the protein that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. It cleaves isopeptide bonds between ubiquitin monomers, an event involved in protein quality control mechanisms. AT3 has been implicated in the pathway that sorts aggregated protein to aggresomes via microtubules, in which dynein and histone deacetylase 6 (HDAC6) also seem to be involved. By taking advantage of small angle X-ray scattering (SAXS) and surface plasmon resonance (SPR), we have investigated the interaction of AT3 with tubulin and HDAC6. Based on SAXS results, the AT3 oligomer, consisting of 6-7 subunits, tightly binds to the tubulin hexameric oligomer in a "parallel" fashion. By SPR analysis we have demonstrated that AT3 binds to tubulin dimer with a 50nM affinity. Binding fits with a Langmuir 1:1 model and involves a single binding interface. Nevertheless, the interaction surface consists of three distinct, discontinuous tubulin-binding regions (TBR), one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. In the absence of any of the three TBRs, the affinity is drastically reduced. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, with affinity in the range 0.1-1μM. These results shed light on the interactions among the components of the transport machinery that sorts aggregate protein to the aggresome, and pave the way to in vivo studies aimed at further clarifying their roles. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Docosahexaenoic acid-mediated protein aggregates may reduce proteasome activity and delay myotube degradation during muscle atrophy in vitro.

    Science.gov (United States)

    Shin, Seung Kyun; Kim, Ji Hyeon; Lee, Jung Hoon; Son, Young Hoon; Lee, Min Wook; Kim, Hak Joong; Noh, Sue Ah; Kim, Kwang Pyo; Kim, In-Gyu; Lee, Min Jae

    2017-01-20

    Proteasomes are the primary degradation machinery for oxidatively damaged proteins that compose a class of misfolded protein substrates. Cellular levels of reactive oxygen species increase with age and this cellular propensity is particularly harmful when combined with the age-associated development of various human disorders including cancer, neurodegenerative disease and muscle atrophy. Proteasome activity is reportedly downregulated in these disease conditions. Herein, we report that docosahexaenoic acid (DHA), a major dietary omega-3 polyunsaturated fatty acid, mediates intermolecular protein cross-linkages through oxidation, and the resulting protein aggregates potently reduce proteasomal activity both in vitro and in cultured cells. Cellular models overexpressing aggregation-prone proteins such as tau showed significantly elevated levels of tau aggregates and total ubiquitin conjugates in the presence of DHA, thereby reflecting suppressed proteasome activity. Strong synergetic cytotoxicity was observed when the cells overexpressing tau were simultaneously treated with DHA. Antioxidant N-acetyl cysteine significantly desensitized the cells to DHA-induced oxidative stress. DHA significantly delayed the proteasomal degradation of muscle proteins in a cellular atrophy model. Thus, the results of our study identified DHA as a potent inducer of cellular protein aggregates that inhibit proteasome activity and potentially delay systemic muscle protein degradation in certain pathologic conditions.

  18. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions.

    Directory of Open Access Journals (Sweden)

    Andrzej Stanisław Cieplak

    Full Text Available Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer's amyloid β and tau, Parkinson's α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and

  19. 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

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

    Directory of Open Access Journals (Sweden)

    Zhao Wang

    2018-02-01

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

  1. Markovian and non-Markovian protein sequence evolution: aggregated Markov process models.

    Science.gov (United States)

    Kosiol, Carolin; Goldman, Nick

    2011-08-26

    Over the years, there have been claims that evolution proceeds according to systematically different processes over different timescales and that protein evolution behaves in a non-Markovian manner. On the other hand, Markov models are fundamental to many applications in evolutionary studies. Apparent non-Markovian or time-dependent behavior has been attributed to influence of the genetic code at short timescales and dominance of physicochemical properties of the amino acids at long timescales. However, any long time period is simply the accumulation of many short time periods, and it remains unclear why evolution should appear to act systematically differently across the range of timescales studied. We show that the observed time-dependent behavior can be explained qualitatively by modeling protein sequence evolution as an aggregated Markov process (AMP): a time-homogeneous Markovian substitution model observed only at the level of the amino acids encoded by the protein-coding DNA sequence. The study of AMPs sheds new light on the relationship between amino acid-level and codon-level models of sequence evolution, and our results suggest that protein evolution should be modeled at the codon level rather than using amino acid substitution models. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. The physical relationship between infectivity and prion protein aggregates is strain-dependent.

    Directory of Open Access Journals (Sweden)

    Philippe Tixador

    2010-04-01

    Full Text Available Prions are unconventional infectious agents thought to be primarily composed of PrP(Sc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP(C. They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP(Sc conformation could encode this 'strain' diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP(Sc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP(Sc aggregates from PrP(C. The distribution of PrP(Sc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP(Sc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12-30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP(Sc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.

  3. NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses.

    Directory of Open Access Journals (Sweden)

    Jie Zhou

    Full Text Available Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.

  4. Wine protein haze: mechanisms of formation and advances in prevention.

    Science.gov (United States)

    Van Sluyter, Steven C; McRae, Jacqui M; Falconer, Robert J; Smith, Paul A; Bacic, Antony; Waters, Elizabeth J; Marangon, Matteo

    2015-04-29

    Protein haze is an aesthetic problem in white wines that can be prevented by removing the grape proteins that have survived the winemaking process. The haze-forming proteins are grape pathogenesis-related proteins that are highly stable during winemaking, but some of them precipitate over time and with elevated temperatures. Protein removal is currently achieved by bentonite addition, an inefficient process that can lead to higher costs and quality losses in winemaking. The development of more efficient processes for protein removal and haze prevention requires understanding the mechanisms such as the main drivers of protein instability and the impacts of various wine matrix components on haze formation. This review covers recent developments in wine protein instability and removal and proposes a revised mechanism of protein haze formation.

  5. 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.

  6. Analysis of Conformational Stability of Abnormal Prion Protein Aggregates across the Spectrum of Creutzfeldt-Jakob Disease Prions

    Science.gov (United States)

    Cescatti, Maura; Saverioni, Daniela; Capellari, Sabina; Tagliavini, Fabrizio; Kitamoto, Tetsuyuki; Ironside, James; Giese, Armin

    2016-01-01

    ABSTRACT The wide phenotypic variability of prion diseases is thought to depend on the interaction of a host genotype with prion strains that have self-perpetuating biological properties enciphered in distinct conformations of the misfolded prion protein PrPSc. This concept is largely based on indirect approaches studying the effect of proteases or denaturing agents on the physicochemical properties of PrPSc aggregates. Furthermore, most data come from studies on rodent-adapted prion strains, making current understanding of the molecular basis of strains and phenotypic variability in naturally occurring diseases, especially in humans, more limited. To fill this gap, we studied the effects of guanidine hydrochloride (GdnHCl) and heating on PrPSc aggregates extracted from 60 sporadic Creutzfeldt-Jakob disease (CJD) and 6 variant CJD brains. While denaturation curves obtained after exposure of PrPSc to increasing GdnHCl concentrations showed similar profiles among the 7 CJD types analyzed, PrPSc exposure to increasing temperature revealed significantly different and type-specific responses. In particular, MM1 and VV2, the most prevalent and fast-replicating CJD types, showed stable and highly resistant PrPSc aggregates, whereas VV1, a rare and slowly propagating type, revealed unstable aggregates that easily dissolved at low temperature. Taken together, our results indicate that the molecular interactions mediating the aggregation state of PrPSc, possibly enciphering strain diversity, are differently targeted by GdnHCl, temperature, and proteases. Furthermore, the detected positive correlation between the thermostability of PrPSc aggregates and disease transmission efficiency makes inconsistent the proposed hypothesis that a decrease in conformational stability of prions results in an increase in their replication efficiency. IMPORTANCE Prion strains are defined as infectious isolates propagating distinctive phenotypic traits after transmission to syngeneic hosts

  7. Stability of white wine proteins: combined effect of pH, ionic strength, and temperature on their aggregation.

    Science.gov (United States)

    Dufrechou, Marie; Poncet-Legrand, Céline; Sauvage, François-Xavier; Vernhet, Aude

    2012-02-08

    Protein haze development in white wines is an unacceptable visual defect attributed to slow protein unfolding and aggregation. It is favored by wine exposure to excessive temperatures but can also develop in properly stored wines. In this study, the combined impact of pH (2.5-4.0), ionic strength (0.02-0.15 M), and temperature (25, 40, and 70 °C) on wine protein stability was investigated. The results showed three classes of proteins with low conformational stability involved in aggregation at room temperature: β-glucanases, chitinases, and some thaumatin-like protein isoforms (22-24 kDa). Unexpectedly, at 25 °C, maximum instability was observed at the lower pH, far from the protein isoelectric point. Increasing temperatures led to a shift of the maximum haze at higher pH. These different behaviors could be explained by the opposite impact of pH on intramolecular (conformational stability) and intermolecular (colloidal stability) electrostatic interactions. The present results highlight that wine pH and ionic strength play a determinant part in aggregation mechanisms, aggregate characteristics, and final haze.

  8. CRISPR-Cas9 Mediated Telomere Removal Leads to Mitochondrial Stress and Protein Aggregation

    Directory of Open Access Journals (Sweden)

    Hyojung Kim

    2017-10-01

    Full Text Available Aging is considered the major risk factor for neurodegenerative diseases including Parkinson’s disease (PD. Telomere shortening is associated with cellular senescence. In this regard, pharmacological or genetic inhibition of telomerase activity has been used to model cellular aging. Here, we employed CRISPR-Cas9 technology to instantly remove the telomere to induce aging in a neuroblastoma cell line. Expression of both Cas9 and guide RNA targeting telomere repeats ablated the telomere, leading to retardation of cell proliferation. Instant deletion of telomere in SH-SY5Y cells impaired mitochondrial function with diminished mitochondrial respiration and cell viability. Supporting the pathological relevance of cell aging by CRISPR-Cas9 mediated telomere removal, alterations were observed in the levels of PD-associated proteins including PTEN-induced putative kinase 1, peroxisome proliferator-activated receptor γ coactivator 1-α, nuclear respiratory factor 1, parkin, and aminoacyl tRNA synthetase complex interacting multifunctional protein 2. Significantly, α-synuclein expression in the background of telomere removal led to the enhancement of protein aggregation, suggesting positive feed-forward interaction between aging and PD pathogenesis. Collectively, our results demonstrate that CRISPR-Cas9 can be used to efficiently model cellular aging and PD.

  9. Curcumin Prevents Formation of Polyglutamine Aggregates by Inhibiting Vps36, a Component of the ESCRT-II Complex

    Science.gov (United States)

    Verma, Meenakshi; Sharma, Abhishek; Naidu, Swarna; Bhadra, Ankan Kumar; Kukreti, Ritushree; Taneja, Vibha

    2012-01-01

    Small molecules with antioxidative properties have been implicated in amyloid disorders. Curcumin is the active ingredient present in turmeric and known for several biological and medicinal effects. Adequate evidence substantiates the importance of curcumin in Alzheimer's disease and recent evidence suggests its role in Prion and Parkinson's disease. However, contradictory effects have been suggested for Huntington's disease. This difference provided a compelling reason to investigate the effect of curcumin on glutamine-rich (Q-rich) and non-glutamine-rich (non Q-rich) amyloid aggregates in the well established yeast model system. Curcumin significantly inhibited the formation of htt72Q-GFP (a Q-rich) and Het-s-GFP (a non Q-rich) aggregates in yeast. We show that curcumin prevents htt72Q-GFP aggregation by down regulating Vps36, a component of the ESCRT-II (Endosomal sorting complex required for transport). Moreover, curcumin disrupted the htt72Q-GFP aggregates that were pre-formed in yeast and cured the yeast prion, [PSI +]. PMID:22880132

  10. Nonlinear Surface Dilatational Rheology and Foaming Behavior of Protein and Protein Fibrillar Aggregates in the Presence of Natural Surfactant.

    Science.gov (United States)

    Wan, Zhili; Yang, Xiaoquan; Sagis, Leonard M C

    2016-04-19

    The surface and foaming properties of native soy glycinin (11S) and its heat-induced fibrillar aggregates, in the presence of natural surfactant steviol glycoside (STE), were investigated and compared at pH 7.0 to determine the impact of protein structure modification on protein-surfactant interfacial interactions. The adsorption at, and nonlinear dilatational rheological behavior of, the air-water interface were studied by combining drop shape analysis tensiometry, ellipsometry, and large-amplitude oscillatory dilatational rheology. Lissajous plots of surface pressure versus deformation were used to analyze the surface rheological response in terms of interfacial microstructure. The heat treatment generates a mixture of long fibrils and unconverted peptides. The presence of small peptides in 11S fibril samples resulted in a faster adsorption kinetics than that of native 11S. The addition of STE affected the adsorption of 11S significantly, whereas no apparent effect on the adsorption of the 11S fibril-peptide system was observed. The rheological response of interfaces stabilized by 11S-STE mixtures also differed significantly from the response for 11S fibril-peptide-STE mixtures. For 11S, the STE reduces the degree of strain hardening in extension and increases strain hardening in compression, suggesting the interfacial structure may change from a surface gel to a mixed phase of protein patches and STE domains. The foams generated from the mixtures displayed comparable foam stability to that of pure 11S. For 11S fibril-peptide mixtures STE only significantly affects the response in extension, where the degree of strain softening is decreased compared to the pure fibril-peptide system. The foam stability of the fibril-peptide system was significantly reduced by STE. These findings indicate that fibrillization of globular proteins could be a potential strategy to modify the complex surface and foaming behaviors of protein-surfactant mixtures.

  11. Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography.

    Science.gov (United States)

    Pan, Hai; Chen, Ken; Pulisic, Matt; Apostol, Izydor; Huang, Gang

    2009-05-15

    Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.

  12. 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).

  13. Molecular crime and cellular punishment: active detoxification of misfolded and aggregated proteins in the cell by the chaperone and protease networks.

    Science.gov (United States)

    Hinault, Marie-Pierre; Goloubinoff, Pierre

    2007-01-01

    Labile or mutation-sensitised proteins may spontaneously convert into aggregation-prone conformations that may be toxic and infectious. This hazardous behavior, which can be described as a form of "molecular criminality", can be actively counteracted in the cell by a network of molecular chaperone and proteases. Similar to law enforcement agents, molecular chaperones and proteases can specifically identify, apprehend, unfold and thus neutralize "criminal" protein conformers, allowing them to subsequently refold into harmless functional proteins. Irreversibly damaged polypeptides that have lost the ability to natively refold are preferentially degraded by highly controlled ATP-consuming proteases. Damaged proteins that escape proteasomal degradation can also be "incarcerated" into dense amyloids, "evicted" from the cell, or internally "exiled" to the lysosome to be hydrolysed and recycled. Thus, remarkable parallels exist between molecular and human forms of criminality, as well as in the cellular and social responses to various forms of crime. Yet, differences also exist: whereas programmed death is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom chosen by lawless societies. Significantly, there is no cellular equivalent for the role of familial care and of education in general, which is so crucial to the proper shaping of functional persons in the society. Unlike in the cell, humanism introduces a bias against radical solutions such as capital punishment, favouring crime prevention, reeducation and social reinsertion of criminals.

  14. Molecular Tweezers for Lysine and Arginine – Powerful Inhibitors of Pathologic Protein Aggregation

    Science.gov (United States)

    Schrader, Thomas; Bitan, Gal; Klärner, Frank-Gerrit

    2016-01-01

    Molecular tweezers represent the first class of artificial receptor molecules that made the way from a supramolecular host to a drug candidate with promising results in animal tests. Due to their unique structure, only lysine and arginine are well complexed with exquisite selectivity by a threading mechanism, which unites electrostatic, hydrophobic and dispersive attraction. However, tweezer design must avoid self-dimerization, self-inclusion and external guest binding. Moderate affinities of the molecular tweezers towards sterically well accessible basic amino acids with fast on and off rates protect normal proteins from a potential interference with their biological function. However, the early stages of abnormal Aβ, α-synuclein, and TTR assembly are redirected upon tweezer binding towards the generation of amorphous non-toxic material that can be degraded by the intracellular and extracellular clearance mechanisms. Thus, specific host–guest chemistry between aggregation-prone proteins and lysine/arginine binders rescues cell viability and restores animal health in models of AD, PD, and TTR amyloidosis. PMID:27546596

  15. Molecular tweezers for lysine and arginine - powerful inhibitors of pathologic protein aggregation.

    Science.gov (United States)

    Schrader, Thomas; Bitan, Gal; Klärner, Frank-Gerrit

    2016-10-15

    Molecular tweezers represent the first class of artificial receptor molecules that have made the way from a supramolecular host to a drug candidate with promising results in animal tests. Due to their unique structure, only lysine and arginine are well complexed with exquisite selectivity by a threading mechanism, which unites electrostatic, hydrophobic and dispersive attraction. However, tweezer design must avoid self-dimerization, self-inclusion and external guest binding. Moderate affinities of molecular tweezers towards sterically well accessible basic amino acids with fast on and off rates protect normal proteins from potential interference with their biological function. However, the early stages of abnormal Aβ, α-synuclein, and TTR assembly are redirected upon tweezer binding towards the generation of amorphous non-toxic materials that can be degraded by the intracellular and extracellular clearance mechanisms. Thus, specific host-guest chemistry between aggregation-prone proteins and lysine/arginine binders rescues cell viability and restores animal health in models of AD, PD, and TTR amyloidosis.

  16. Acupuncture promotes mTOR-independent autophagic clearance of aggregation-prone proteins in mouse brain.

    Science.gov (United States)

    Tian, Tian; Sun, Yanhong; Wu, Huangan; Pei, Jian; Zhang, Jing; Zhang, Yi; Wang, Lu; Li, Bin; Wang, Lihua; Shi, Jiye; Hu, Jun; Fan, Chunhai

    2016-01-21

    Acupuncture has historically been practiced to treat medical disorders by mechanically stimulating specific acupoints with fine needles. Despite its well-documented efficacy, its biological basis remains largely elusive. In this study, we found that mechanical stimulation at the acupoint of Yanglingquan (GB34) promoted the autophagic clearance of α-synuclein (α-syn), a well known aggregation-prone protein closely related to Parkinson's disease (PD), in the substantia nigra par compacta (SNpc) of the brain in a PD mouse model. We found the protein clearance arose from the activation of the autophagy-lysosome pathway (ALP) in a mammalian target of rapamycin (mTOR)-independent approach. Further, we observed the recovery in the activity of dopaminergic neurons in SNpc, and improvement in the motor function at the behavior level of PD mice. Whereas acupuncture and rapamycin, a chemical mTOR inhibitor, show comparable α-syn clearance and therapeutic effects in the PD mouse model, the latter adopts a distinctly different, mTOR-dependent, autophagy induction process. Due to this fundamental difference, acupuncture may circumvent adverse effects of the rapamycin treatment. The newly discovered connection between acupuncture and autophagy not only provides a new route to understanding the molecular mechanism of acupuncture but also sheds new light on cost-effective and safe therapy of neurodegenerative diseases.

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

    Science.gov (United States)

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

    2017-08-15

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

  18. Psychological stress as a determinant of protein levels and salivary-induced aggregation of Streptococcus gordonii in human whole saliva

    NARCIS (Netherlands)

    Bosch, J.A.; Brand, H.S.; Ligtenberg, T.J.M.; Bermond, B.; Hoogstraten, J.; Nieuw Amerongen, A.V.

    1996-01-01

    Several pathologies of the oral cavity have been associated with stress, so we investigated salivary-induced aggregation during psychological stress. In addition, salivary total protein, alpha-amylase, and secretory immunoglobulin A (s-IgA) were assessed. In this longitudinal study, 28 dental

  19. Escherichia coli EDA is a novel fusion expression partner to improve solubility of aggregation-prone heterologous proteins.

    Science.gov (United States)

    Kang, Yoon-Sik; Song, Jong-Am; Han, Kyung-Yeon; Lee, Jeewon

    2015-01-20

    Since the use of solubility enhancer proteins is one of the effective methods to produce active recombinant proteins within Escherichia coli, the development of a novel fusion expression partner that can be applied to various aggregation-prone proteins is of crucial importance. In our previous work, two-dimensional electrophoresis (2-DE) was employed to systematically analyze the E. coli BL21 (DE3) proteome profile in response to heat treatment, and KDPG aldolase (EDA) was identified as a heat-responsive and aggregation-resistant protein. When used as fusion expression partner, EDA significantly increased the solubility of seven aggregation-prone heterologous proteins in the E. coli cytoplasm. The efficacy of EDA as a fusion expression partner was evaluated through the analysis of bioactivity or secondary structure of several target proteins: EDA-fusion expression resulted in the synthesis of bioactive human ferritin light chain and bacterial arginine deiminase and the formation of correct secondary structure of human granulocyte colony stimulation factor. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. 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.

  1. 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

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

    Directory of Open Access Journals (Sweden)

    Arun K Upadhyay

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

  3. A monoclonal antibody prevents aggregation of the NBD1 domain of the cystic fibrosis transmembrane conductance regulator.

    Science.gov (United States)

    Lovato, V; Roesli, C; Ahlskog, J; Scheuermann, J; Neri, D

    2007-12-01

    The homozygous deletion of the phenylalanine at position 508 (DeltaPhe508) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF-causing genetic defect. It has been proposed that the propensity of NBD1 to aggregate may lead to a lower display of the CFTR chloride channel to the cell membrane and to the disease, thus opening an avenue for the pharmacological development of CFTR folding correctors. Here, we show that a human monoclonal antibody fragment specific to the folded conformation of NBD1 inhibits the aggregation of NBD1 in vitro. However, in contrast to the previously published observations, we proved experimentally that NBD1 of wild-type and DeltaPhe508 version of CFTR display comparable propensities to aggregate in vitro and that the corresponding full-length CFTR protein reaches the cell membrane with comparable efficiency in mammalian cell expression systems. On the basis of our results, the 'folding defect' hypothesis seems unlikely to represent the causal mechanism for the pathogenesis of CF. A solid understanding of how the DeltaPhe508 deletion leads to the disease represents an absolute requirement for the development of effective drugs against CF.

  4. 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.

  5. Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates

    Directory of Open Access Journals (Sweden)

    Mahony Timothy J

    2011-07-01

    Full Text Available Abstract Background Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB. Results The expression of a truncated form of BVDV-E2 protein (E2-T1 in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0 containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies. Conclusion We have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit

  6. Kinetics, aggregation behavior and optimization of the fractionation of whey protein isolate with hydrochloric acid

    Science.gov (United States)

    Concentrated WPI solutions (10% (w/w)) containing approximately 30% alpha-lactalbumin (alpha-LA) and 60% beta-lactoglobulin (beta-LG) were fractionated with HCl at acidic pH and moderate temperatures to denature alpha-LA and recover the alpha-LA aggregates via centrifugation. Aggregation behavior an...

  7. EGCG in Green Tea Induces Aggregation of HMGB1 Protein through Large Conformational Changes with Polarized Charge Redistribution

    Science.gov (United States)

    Meng, Xuan-Yu; Li, Baoyu; Liu, Shengtang; Kang, Hongsuk; Zhao, Lin; Zhou, Ruhong

    2016-02-01

    As a major effective component in green tea, (-)-epigallocatechin-3-gallate (EGCG)’s potential benefits to human health have been widely investigated. Recent experimental evidences indicate that EGCG can induce the aggregation of HMGB1 protein, a late mediator of inflammation, which subsequently stimulates the autophagic degradation and thus provides protection from lethal endotoxemia and sepsis. In this study, we use molecular dynamics (MD) simulations to explore the underlying molecular mechanism of this aggregation of HMGB1 facilitated by EGCG. Our simulation results reveal that EGCG firmly binds to HMGB1 near Cys106, which supports previous preliminary experimental evidence. A large HMGB1 conformational change is observed, where Box A and Box B, two homogenous domains of HMGB1, are repositioned and packed together by EGCG. This new HMGB1 conformation has large molecular polarity and distinctive electrostatic potential surface. We suggest that the highly polarized charge distribution leads to the aggregation of HMGB1, which differs from the previous hypothesis that two HMGB1 monomers are linked by the dimer of EGCG. Possible aggregating modes have also been investigated with potential of mean force (PMF) calculations. Finally, we conclude that the conformation induced by EGCG is more aggregation-prone with higher binding free energies as compared to those without EGCG.

  8. Tau Protein Hyperphosphorylation and Aggregation in Alzheimer’s Disease and Other Tauopathies, and Possible Neuroprotective Strategies

    Science.gov (United States)

    Šimić, Goran; Babić Leko, Mirjana; Wray, Selina; Harrington, Charles; Delalle, Ivana; Jovanov-Milošević, Nataša; Bažadona, Danira; Buée, Luc; de Silva, Rohan; Di Giovanni, Giuseppe; Wischik, Claude; Hof, Patrick R.

    2016-01-01

    Abnormal deposition of misprocessed and aggregated proteins is a common final pathway of most neurodegenerative diseases, including Alzheimer’s disease (AD). AD is characterized by the extraneuronal deposition of the amyloid β (Aβ) protein in the form of plaques and the intraneuronal aggregation of the microtubule-associated protein tau in the form of filaments. Based on the biochemically diverse range of pathological tau proteins, a number of approaches have been proposed to develop new potential therapeutics. Here we discuss some of the most promising ones: inhibition of tau phosphorylation, proteolysis and aggregation, promotion of intra- and extracellular tau clearance, and stabilization of microtubules. We also emphasize the need to achieve a full understanding of the biological roles and post-translational modifications of normal tau, as well as the molecular events responsible for selective neuronal vulnerability to tau pathology and its propagation. It is concluded that answering key questions on the relationship between Aβ and tau pathology should lead to a better understanding of the nature of secondary tauopathies, especially AD, and open new therapeutic targets and strategies. PMID:26751493

  9. Interactions between L-arginine/L-arginine derivatives and lysozyme and implications to their inhibition effects on protein aggregation.

    Science.gov (United States)

    Gao, Ming-Tao; Dong, Xiao-Yan; Sun, Yan

    2013-01-01

    L-arginine (Arg), L-homoarginine (HArg), L-arginine ethylester (ArgEE), and L-arginine methylester (ArgME) were found effective in inhibiting protein aggregation, but the molecular mechanisms are not clear. Herein, stopped-flow fluorescence spectroscopy, isothermal titration calorimetry, and mass spectroscopy were used to investigate the folding kinetics of lysozyme and the interactions of the additives with lysozyme. It was found that the interactions of ArgME and ArgEE with lysozyme were similar to that of guanidine hydrochloride and were much stronger than those of Arg and HArg. The binding forces were all mainly hydrogen bonding and cation-π interaction from the guanidinium group, but their differences in molecular states led to the significantly different binding strengths. The additives formed molecular clusters in an increasing order of ArgEE, ArgME, HArg, and Arg. Arg and HArg mainly formed annular clusters with head-to-tail bonding, while ArgME and ArgEE formed linear clusters with guanidinium groups stacking. The interactions between the additives and lysozyme were positively related to the monomer contents. That is, the monomers were the primary species that participated in the direct interactions due to their intact guanidinium groups and small sizes, while the clusters performed as barriers to crowd out the protein-protein interactions for aggregation. Thus, it is concluded that the effects of Arg and its derivatives on protein aggregation stemmed from the direct interactions by the monomers and the crowding effects by the clusters. Interplay of the two effects led to the differences in their inhibition effects on protein aggregation. © 2013 American Institute of Chemical Engineers.

  10. Analyses of Protease Resistance and Aggregation State of Abnormal Prion Protein across the Spectrum of Human Prions*

    Science.gov (United States)

    Saverioni, Daniela; Notari, Silvio; Capellari, Sabina; Poggiolini, Ilaria; Giese, Armin; Kretzschmar, Hans A.; Parchi, Piero

    2013-01-01

    Prion diseases are characterized by tissue accumulation of a misfolded, β-sheet-enriched isoform (scrapie prion protein (PrPSc)) of the cellular prion protein (PrPC). At variance with PrPC, PrPSc shows a partial resistance to protease digestion and forms highly aggregated and detergent-insoluble polymers, two properties that have been consistently used to distinguish the two proteins. In recent years, however, the idea that PrPSc itself comprises heterogeneous species has grown. Most importantly, a putative proteinase K (PK)-sensitive form of PrPSc (sPrPSc) is being increasingly investigated for its possible role in prion infectivity, neurotoxicity, and strain variability. The study of sPrPSc, however, remains technically challenging because of the need of separating it from PrPC without using proteases. In this study, we have systematically analyzed both PK resistance and the aggregation state of purified PrPSc across the whole spectrum of the currently characterized human prion strains. The results show that PrPSc isolates manifest significant strain-specific differences in their PK digestion profile that are only partially explained by differences in the size of aggregates, suggesting that other factors, likely acting on PrPSc aggregate stability, determine its resistance to proteolysis. Fully protease-sensitive low molecular weight aggregates were detected in all isolates but in a limited proportion of the overall PrPSc (i.e. PrPSc in the biogenesis of prion strains. Finally, we highlight the limitations of current operational definitions of sPrPSc and of the quantitative analytical measurements that are not based on the isolation of a fully PK-sensitive PrPSc form. PMID:23897825

  11. 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.

  12. Dispersible amyloid β-protein oligomers, protofibrils, and fibrils represent diffusible but not soluble aggregates: their role in neurodegeneration in amyloid precursor protein (APP) transgenic mice.

    Science.gov (United States)

    Rijal Upadhaya, Ajeet; Capetillo-Zarate, Estibaliz; Kosterin, Irina; Abramowski, Dorothee; Kumar, Sathish; Yamaguchi, Haruyasu; Walter, Jochen; Fändrich, Marcus; Staufenbiel, Matthias; Thal, Dietmar Rudolf

    2012-11-01

    Soluble amyloid β-protein (Aβ) aggregates have been identified in the Alzheimer's disease (AD) brain. Dispersed Aβ aggregates in the brain parenchyma are different from soluble, membrane-associated and plaque-associated solid aggregates. They are in mixture with the extra- or intracellular fluid but can be separated from soluble proteins by ultracentrifugation. To clarify the role of dispersible Aβ aggregates for neurodegeneration we analyzed 2 different amyloid precursor protein (APP)-transgenic mouse models. APP23 mice overexpress human mutant APP with the Swedish mutation. APP51/16 mice express high levels of human wild type APP. Both mice develop Aβ-plaques. Dendritic degeneration, neuron loss, and loss of asymmetric synapses were seen in APP23 but not in APP51/16 mice. The soluble and dispersible fractions not separated from one another were received as supernatant after centrifugation of native forebrain homogenates at 14,000 × g. Subsequent ultracentrifugation separated the soluble, i.e., the supernatant, from the dispersible fraction, i.e., the resuspended pellet. The major biochemical difference between APP23 and APP51/16 mice was that APP23 mice exhibited higher levels of dispersible Aβ oligomers, protofibrils and fibrils precipitated with oligomer (A11) and protofibril/fibril (B10AP) specific antibodies than APP51/16 mice. These differences, rather than soluble Aβ and Aβ plaque pathology were associated with dendritic degeneration, neuron, and synapse loss in APP23 mice in comparison with APP51/16 mice. Immunoprecipitation of dispersible Aβ oligomers, protofibrils, and fibrils revealed that they were associated with APP C-terminal fragments (APP-CTFs). These results indicate that dispersible Aβ oligomers, protofibrils, and fibrils represent an important pool of Aβ aggregates in the brain that critically interact with membrane-associated APP C-terminal fragments. The concentration of dispersible Aβ aggregates, thereby, presumably determines

  13. 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.

  14. Glycolipid-based nanostructures with thermal-phase transition behavior functioning as solubilizers and refolding accelerators for protein aggregates.

    Science.gov (United States)

    Kameta, N; Matsuzawa, T; Yaoi, K; Fukuda, J; Masuda, M

    2017-05-03

    The self-assembly of synthetic glycolipids produced nanostructures such as vesicles and nanotubes consisting of bilayer membranes, which underwent a gel-to-liquid crystalline thermal phase transition. Vesicles formed at temperatures above the thermal phase transition temperatures (T g-l ) could solubilize aggregates of denatured proteins by trapping them in the fluid bilayer membranes. Cooling to temperatures below T g-l caused a morphological transformation into nanotubes that accompanied the thermal phase transition from the fluid to the solid state. This phenomenon allowed the trapped proteins to be quickly released into the bulk solution and simultaneously facilitated the refolding of the proteins. The refolding efficiency strongly depended on the electrostatic attraction between the bilayer membranes of the nanostructures and the proteins. Because of the long shape (>400 nm) of the nanotubes, simple membrane filtration through a pore size of 200 nm led to complete separation and recovery of the refolded proteins (3-9 nm sizes).

  15. Characterization of flows in micro contractions using micro PIV and CFD to study the protein aggregation process

    Science.gov (United States)

    Tovar-Lopez, Francisco J.; Mitchell, Arnan; Rosengarten, Gary

    2007-12-01

    Protein aggregation is arguably the most common and troubling manifestation of protein instability, encountered in almost all stages of protein drug development. The production process in the pharmaceutical industry can induce flows with shear and extensional components and high strain rates which can affect the stability of proteins. We use a microfluidic platform to produce accurately controlled strain regions in order to systematically study the main parameters of the flow involved in the protein aggregation. This work presents a characterization of the pressure driven flow encountered in arrays of micro channels. The micro channels were fabricated in polydimethyl siloxane (PDMS) using standard soft-lithography techniques with a photolithographically patterned KMPR mold. We present a relationship of the main geometrical variables of the micro channels and its impact on the extensional strain rate along the center line, for different cross sectional shapes and over a range of strain rates typically encountered in protein processing. Computational Fluid Dynamics (CFD) simulations have been carried out to gain more detailed local flow information, and the results have been validated with experiments. We show good agreement between the CFD and experiments and demonstrate the use of microfluidics in the production of a large range of controllable shear and extensional rates that can mimic large scale processing conditions.

  16. Iron oxide nanoparticles may damage to the neural tissue through iron accumulation, oxidative stress, and protein aggregation.

    Science.gov (United States)

    Yarjanli, Zahra; Ghaedi, Kamran; Esmaeili, Abolghasem; Rahgozar, Soheila; Zarrabi, Ali

    2017-06-26

    In the recent decade, iron oxide nanoparticles (IONPs) have been proposed for several applications in the central nervous system (CNS), including targeting amyloid beta (Aβ) in the arteries, inhibiting the microglial cells, delivering drugs, and increasing contrast in magnetic resonance imaging. Conversely, a notable number of studies have reported the role of iron in neurodegenerative diseases. Therefore, this study has reviewed the recent studies to determine whether IONPs iron can threaten the cellular viability same as iron. Iron contributes in Fenton's reaction and produces reactive oxygen species (ROS). ROS cause to damage the macromolecules and organelles of the cell via oxidative stress. Iron accumulation and oxidative stress are able to aggregate some proteins, including Aβ and α-synuclein, which play a critical role in Alzheimer's and Parkinson's diseases, respectively. Iron accumulation, oxidative stress, and protein aggregation make a positive feedback loop, which can be toxic for the cell. The release of iron ions from IONPs may result in iron accumulation in the targeted tissue, and thus, activate the positive feedback loop. However, the levels of IONPs induced toxicity depend on the size, concentration, surface charge, and the type of coating and functional groups of IONPs. IONPs depending on their properties can lead to iron accumulation, oxidative stress and protein aggregation in the neural cells. Therefore, in order to apply IONPs in the CNS, the consideration of IONPs properties is crucial.

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

    Directory of Open Access Journals (Sweden)

    Maxime Saffon

    2013-09-01

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

  18. Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca²⁺-ATPase.

    Science.gov (United States)

    Li, Chenge; Kumar, Saroj; Montigny, Cédric; le Maire, Marc; Barth, Andreas

    2014-09-07

    Infrared spectroscopy was used to characterise recombinant sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). In the amide I region, its spectrum differed from that of Ca(2+)-ATPase prepared from rabbit fast twitch muscle below 1650 cm(-1). A band at 1642 cm(-1) is reduced in the spectrum of the recombinant protein and a band at 1631 cm(-1) is more prominent. By comparison of amide I band areas with the known secondary structure content of the protein, we assigned the 1642 cm(-1) band to β-sheet structure. Further investigation revealed that the 1642 cm(-1) band decreased and the 1631 cm(-1) band increased upon storage at room temperature and upon repeated washing of a protein film with water. Also protein aggregates obtained after solubilisation of the rabbit muscle enzyme showed a prominent band at 1631 cm(-1), whereas the spectrum of solubilised ATPase resembled that of the membrane bound protein. The spectral position of the 1631 cm(-1) band is similar to that of a band observed for inclusion bodies of other proteins. The findings show that the absence of the 1642 cm(-1) band and the presence of a prominent band at 1631 cm(-1) indicate protein aggregation and can be used as a quality marker for the optimisation of recombinant protein production. We conclude that recombinant production of SERCA1a, storage at room temperature, repeated washing and aggregation after solubilisation all modify existing β-sheets in the cytosolic domains so that they become similar to those found in inclusion bodies of other proteins.

  19. Anti-Amyloid Aggregation Activity of Black Sesame Pigment: Toward a Novel Alzheimer’s Disease Preventive Agent

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    Lucia Panzella

    2018-03-01

    Full Text Available Black sesame pigment (BSP represents a low cost, easily accessible material of plant origin exhibiting marked antioxidant and heavy metal-binding properties with potential as a food supplement. We report herein the inhibitory properties of the potentially bioaccessible fraction of BSP following simulated gastrointestinal digestion against key enzymes involved in Alzheimer’s disease (AD. HPLC analysis indicated that BSP is transformed under the pH conditions mimicking the intestinal environment and the most abundant of the released compounds was identified as vanillic acid. More than 80% inhibition of acetylcholinesterase-induced aggregation of the β-amyloid Aβ1-40 was observed in the presence of the potentially bioaccessible fraction of BSP, which also efficiently inhibited self-induced Aβ1-42 aggregation and β-secretase (BACE-1 activity, even at high dilution. These properties open new perspectives toward the use of BSP as an ingredient of functional food or as a food supplement for the prevention of AD.

  20. Protein oxidation and aggregation in UVA-irradiated Escherichia coli cells as signs of accelerated cellular senescence.

    Science.gov (United States)

    Bosshard, Franziska; Riedel, Kathrin; Schneider, Thomas; Geiser, Carina; Bucheli, Margarete; Egli, Thomas

    2010-11-01

    Solar disinfection (SODIS) is a simple drinking water treatment method that improves microbiological water quality where other means are unavailable. It makes use of the deleterious effect of solar irradiation on pathogenic microbes and viruses. A positive impact on health has been documented in several epidemiological studies. However, the molecular mechanisms damaging cells during this simple treatment are not yet fully understood. Here we show that protein damage is crucial in the process of inactivation by sunlight. Protein damages in UVA-irradiated Escherichia coli cells have been evaluated by an immunoblot method for carbonylated proteins and an aggregation assay based on semi-quantitative proteomics. A wide spectrum of structural and enzymatic proteins within the cell is affected by carbonylation and aggregation. Vital cellular functions like the transcription and translation apparatus, transport systems, amino acid synthesis and degradation, respiration, ATP synthesis, glycolysis, the TCA cycle, chaperone functions and catalase are targeted by UVA irradiation. The protein damage pattern caused by SODIS strongly resembles the pattern caused by reactive oxygen stress. Hence, sunlight probably accelerates cellular senescence and leads to the inactivation and finally death of UVA-irradiated cells. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  1. 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.

  2. CAMELOT: A machine learning approach for coarse-grained simulations of aggregation of block-copolymeric protein sequences

    Energy Technology Data Exchange (ETDEWEB)

    Ruff, Kiersten M. [Computational and Systems Biology Program and Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, Missouri 63130-4899 (United States); Harmon, Tyler S. [Department of Physics and Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, Missouri 63130-4899 (United States); Pappu, Rohit V., E-mail: pappu@wustl.edu [Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University in St. Louis, CB 1097, St. Louis, Missouri 63130-4899 (United States)

    2015-12-28

    We report the development and deployment of a coarse-graining method that is well suited for computer simulations of aggregation and phase separation of protein sequences with block-copolymeric architectures. Our algorithm, named CAMELOT for Coarse-grained simulations Aided by MachinE Learning Optimization and Training, leverages information from converged all atom simulations that is used to determine a suitable resolution and parameterize the coarse-grained model. To parameterize a system-specific coarse-grained model, we use a combination of Boltzmann inversion, non-linear regression, and a Gaussian process Bayesian optimization approach. The accuracy of the coarse-grained model is demonstrated through direct comparisons to results from all atom simulations. We demonstrate the utility of our coarse-graining approach using the block-copolymeric sequence from the exon 1 encoded sequence of the huntingtin protein. This sequence comprises of 17 residues from the N-terminal end of huntingtin (N17) followed by a polyglutamine (polyQ) tract. Simulations based on the CAMELOT approach are used to show that the adsorption and unfolding of the wild type N17 and its sequence variants on the surface of polyQ tracts engender a patchy colloid like architecture that promotes the formation of linear aggregates. These results provide a plausible explanation for experimental observations, which show that N17 accelerates the formation of linear aggregates in block-copolymeric N17-polyQ sequences. The CAMELOT approach is versatile and is generalizable for simulating the aggregation and phase behavior of a range of block-copolymeric protein sequences.

  3. Combined Protein A and size exclusion high performance liquid chromatography for the single-step measurement of mAb, aggregates and host cell proteins.

    Science.gov (United States)

    Gjoka, Xhorxhi; Schofield, Mark; Cvetkovic, Aleksandar; Gantier, Rene

    2014-12-01

    Quantification of monoclonal antibody (mAb) monomer, mAb aggregates, and host cell proteins (HCPs) is critical for the optimization of the mAb production process. The present work describes a single high throughput analytical tool capable of tracking the concentration of mAb, mAb aggregate and HCPs in a growing cell culture batch. By combining two analytical HPLC methods, Protein A affinity and size-exclusion chromatography (SEC), it is possible to detect a relative increase or decrease in the concentration of all three entities simultaneously. A comparison of the combined Protein A-SEC assay to SEC alone was performed, demonstrating that it can be useful tool for the quantification of mAb monomer along with trending data for mAb aggregate and HCP. Furthermore, the study shows that the Protein A-SEC method is at least as accurate as other commonly used analytical methods such as ELISA and Bradford. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Maiko eTasaki

    2014-04-01

    Full Text Available 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.

  6. 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.

  7. Proteins that accumulate with age in human skeletal-muscle aggregates contribute to declines in muscle mass and function inCaenorhabditis elegans.

    Science.gov (United States)

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Suri, Pooja; Mackintosh, Samuel G; Tackett, Alan J; Sullivan, Dennis H; Shmookler Reis, Robert J; Dennis, Richard A

    2016-12-15

    Protein aggregation increases with age in normal tissues, and with pathology and age in Alzheimer's hippocampus and mouse cardiac muscle. We now ask whether human skeletal muscle accumulates aggregates with age. Detergent-insoluble protein aggregates were isolated from vastus lateralis biopsies from 5 young (23–27 years of age) and 5 older (64-80 years) adults. Aggregates, quantified after gel electrophoresis, contain 2.1-fold more protein ( P proteins identified by liquid chromatography coupled to tandem mass spectrometry, 56 (11%) were significantly more abundant in older muscle, while 21 (4%) were depleted with age (each P proteins were then targeted in C. elegans by RNA interference. Six of the seven knockdown treatments decreased protein aggregation (range 6-45%, P muscle mass (range 1.5- to 1.85-fold, P proteins, discovered as differentially abundant in aging human muscle, have orthologs that contribute functionally to aggregation and age-associated muscle loss in nematodes, and thus can be considered potential drug targets for sarcopenia in humans.

  8. Oxidation of the cysteine-rich regions of parkin perturbs its E3 ligase activity and contributes to protein aggregation

    Directory of Open Access Journals (Sweden)

    Ma Yuliang

    2011-05-01

    Full Text Available Abstract Background Accumulation of aberrant proteins to form Lewy bodies (LBs is a hallmark of Parkinson's disease (PD. Ubiquitination-mediated degradation of aberrant, misfolded proteins is critical for maintaining normal cell function. Emerging evidence suggests that oxidative/nitrosative stress compromises the precisely-regulated network of ubiquitination in PD, particularly affecting parkin E3 ligase activity, and contributes to the accumulation of toxic proteins and neuronal cell death. Results To gain insight into the mechanism whereby cell stress alters parkin-mediated ubiquitination and LB formation, we investigated the effect of oxidative stress. We found significant increases in oxidation (sulfonation and subsequent aggregation of parkin in SH-SY5Y cells exposed to the mitochondrial complex I inhibitor 1-methyl-4-phenlypyridinium (MPP+, representing an in vitro cell-based PD model. Exposure of these cells to direct oxidation via pathological doses of H2O2 induced a vicious cycle of increased followed by decreased parkin E3 ligase activity, similar to that previously reported following S-nitrosylation of parkin. Pre-incubation with catalase attenuated H2O2 accumulation, parkin sulfonation, and parkin aggregation. Mass spectrometry (MS analysis revealed that H2O2 reacted with specific cysteine residues of parkin, resulting in sulfination/sulfonation in regions of the protein similar to those affected by parkin mutations in hereditary forms of PD. Immunohistochemistry or gel electrophoresis revealed an increase in aggregated parkin in rats and primates exposed to mitochondrial complex I inhibitors, as well as in postmortem human brain from patients with PD with LBs. Conclusion These findings show that oxidative stress alters parkin E3 ligase activity, leading to dysfunction of the ubiquitin-proteasome system and potentially contributing to LB formation.

  9. SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI).

    Science.gov (United States)

    Chen, Xueping; Guan, Teng; Li, Chen; Shang, Huifang; Cui, Liying; Li, Xin-Min; Kong, Jiming

    2012-10-12

    Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration. To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI) and copper-zinc superoxide dismutase (SOD1) were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining. Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the formation of ubiquitinated-protein aggregates in cultured

  10. SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI

    Directory of Open Access Journals (Sweden)

    Chen Xueping

    2012-10-01

    Full Text Available Abstract Background Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration. Methods To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI and copper-zinc superoxide dismutase (SOD1 were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining. Results Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the

  11. Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

    Science.gov (United States)

    Pashley, Clare L; Hewitt, Eric W; Radford, Sheena E

    2016-02-13

    The mouse and human β2-microglobulin protein orthologs are 70% identical in sequence and share 88% sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Comparison of the aggregation behavior of soy and bovine whey protein hydrolysates

    NARCIS (Netherlands)

    Kuipers, B.J.H.; Alting, A.C.; Gruppen, H.

    2007-01-01

    Abstract Soy-derived proteins (soy protein isolate, glycinin, and ß-conglycinin) and bovine whey-derived proteins (whey protein isolate, ¿-lactalbumin, ß-lactoglobulin) were hydrolyzed using subtilisin Carlsberg, chymotrypsin, trypsin, bromelain, and papain. The (in)solubility of the hydrolysates

  13. Protein and exercise in the prevention of sarcopenia and aging.

    Science.gov (United States)

    Naseeb, Manal A; Volpe, Stella L

    2017-04-01

    Aging is associated with a progressive decline in skeletal muscle mass and strength. The decline, known as sarcopenia, could lead to physical disability, poor quality of life, and death. In addition, the older population usually experiences age-related muscle changes that affect muscle mass, muscular strength, and functional abilities. The purpose of this review is to describe the role of protein and exercise in slowing the progression of sarcopenia. It will also discuss whether age-related changes can be attenuated by dietary protein and exercise in the older population. This review will also cover one of the possible mechanisms of how dietary protein and exercise are involved in sarcopenia prevention, as well as the available measurement tools. Based on the findings of this review, the adequate amount of protein required for older men and women needs to be revised and likely be higher. Moreover, studies are required to explore some inconclusive findings concerning sarcopenia in the older population. Further research is required to investigate the following: (1) the safety and effectiveness concerning the consumption of 1.4 g of protein/kg of body weight (or more) in this vulnerable population; (2) the effectiveness of amino acid supplementation in reducing progression of sarcopenia over time through longitudinal studies; (3) the preferred source and timing of protein for the older population to maintain muscular strength and attenuate sarcopenia; (4) exercise interventions, especially those of longer duration, in the attenuation of sarcopenia; (5) other types of exercise and their effects on age-related muscle changes; (6) the mechanism of how protein and exercise prevent muscle loss with aging; and (7) determine the best method to diagnose sarcopenia. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Identification of the Chromophores Involved in Aggregation-dependent Energy Quenching of the Monomeric Photosystem II Antenna Protein Lhcb5*

    Science.gov (United States)

    Ballottari, Matteo; Girardon, Julien; Betterle, Nico; Morosinotto, Tomas; Bassi, Roberto

    2010-01-01

    Non-photochemical quenching (NPQ) of excess absorbed light energy is a fundamental process that regulates photosynthetic light harvesting in higher plants. Among several proposed NPQ mechanisms, aggregation-dependent quenching (ADQ) and charge transfer quenching have received the most attention. In vitro spectroscopic features of both mechanisms correlate with very similar signals detected in more intact systems and in vivo, where full NPQ can be observed. A major difference between the models is the proposed quenching site, which is predominantly the major trimeric light-harvesting complex II in ADQ and exclusively monomeric Lhcb proteins in charge transfer quenching. Here, we studied ADQ in both monomeric and trimeric Lhcb proteins, investigating the activities of each antenna subunit and their dependence on zeaxanthin, a major modulator of NPQ in vivo. We found that monomeric Lhcb proteins undergo stronger quenching than light-harvesting complex II during aggregation and that this is enhanced by binding to zeaxanthin, as occurs during NPQ in vivo. Finally, the analysis of Lhcb5 mutants showed that chlorophyll 612 and 613, in close contact with lutein bound at site L1, are important facilitators of ADQ. PMID:20584907

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

    Directory of Open Access Journals (Sweden)

    Krishna Madhuri Manda

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

  16. An approach to prevent aggregation during the purification and crystallization of wild type acyl coenzyme A : Isopenicillin N acyltransferase from Penicillium chrysogenum

    NARCIS (Netherlands)

    Yoshida, Hiromi; Hensgens, Charles M.H.; Laan, Jan Metske van der; Sutherland, John D.; Hart, Darren J.; Dijkstra, Bauke W.

    2005-01-01

    Acyl coenzyme A: isopenicillin N acyltransferase (AT) from Penicillium chrysogenum is an enzyme of interest for the biosynthesis of β-lactam antibiotics. Severe aggregation problems with wild type AT have, however, prevented significant progress in the structure–function analysis of this enzyme for

  17. Atick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation

    Czech Academy of Sciences Publication Activity Database

    Chmelař, Jindřich; Oliveira, C. J.; Řezáčová, Pavlína; Francischetti, I.M.B.; Kovářová, Zuzana; Pejler, G.; Kopáček, Petr; Ribeiro, J.M.C.; Mareš, Michael; Kopecký, Jan; Kotsyfakis, Michalis

    2011-01-01

    Roč. 117, č. 2 (2011), s. 736-744 ISSN 0006-4971 R&D Projects: GA AV ČR IAA600960811; GA MŠk(CZ) LC06009; GA ČR(CZ) GAP207/10/2183 Institutional research plan: CEZ:AV0Z60220518; CEZ:AV0Z40550506 Keywords : parasite serpin * IRS-2 * tick * Ixodes ricinus * platelet aggregation * inflammation * cathepsin G * chymase Subject RIV: EC - Immunology Impact factor: 9.898, year: 2011

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-01

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

  19. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington's disease.

    Science.gov (United States)

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington's disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington's disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson's and Alzheimer's diseases. To identify potential neuroprotective molecules for Huntington's disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington's disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington's disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results.

  20. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Science.gov (United States)

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington’s disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results. PMID:27110099

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-09-23

    ABSTRACT

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

  2. Arginine as an eluent for automated on-line Protein A/size exclusion chromatographic analysis of monoclonal antibody aggregates in cell culture.

    Science.gov (United States)

    Wang, Sean; Raghani, Anil

    2014-01-15

    An automated two-dimensional method using Protein A chromatography followed by size exclusion HPLC was developed for analysis of aggregates of monoclonal antibodies in mammalian cell culture samples. The method development was intended to address the analysis of IgG2 monoclonal antibody products that are particularly prone to aggregation at pHProtein A chromatography. In addition, the arginine solution is a compatible mobile phase for the analysis of these samples by size exclusion HPLC, separating aggregates from the monomer of the monoclonal antibody. The effect of arginine concentration in the eluent on parameters such as protein recovery from Protein A chromatography and resolution of aggregates from the monomer are reported. The developed method was shown to provide accuracy of reported aggregates greater than 98%, and intermediate precision of 4.4% RSD. The method limit of quantitation for aggregates was determined to be 0.1%. Application of the method is demonstrated for analysis of aggregates in cell culture samples to aid in the development of cell culture conditions for the production of antibodies. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. 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

    OpenAIRE

    Haina Qin; Liangzhong Lim; Yuanyuan Wei; Garvita Gupta; Jianxing Song

    2014-01-01

    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 spe...

  4. Impact of high cholesterol in a Parkinson's disease model: Prevention of lysosomal leakage versus stimulation of α-synuclein aggregation.

    Science.gov (United States)

    Eriksson, Ida; Nath, Sangeeta; Bornefall, Per; Giraldo, Ana Maria Villamil; Öllinger, Karin

    2017-03-01

    Parkinson's disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP + ), we found that MPP + -induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP + -induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinson's disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation. Copyright © 2017 Elsevier GmbH. All rights reserved.

  5. The Achilles' Heel of "Ultrastable" Hyperthermophile Proteins: Submillimolar Concentrations of SDS Stimulate Rapid Conformational Change, Aggregation, and Amyloid Formation in Proteins Carrying Overall Positive Charge.

    Science.gov (United States)

    Khan, Javed M; Sharma, Prerna; Arora, Kanika; Kishor, Nitin; Kaila, Pallavi; Guptasarma, Purnananda

    2016-07-19

    Low concentrations (SDS) have been shown to induce the formation of amyloid fibers in more than 20 different mesophile-derived proteins in the cationic state. It is not known whether SDS has similar effects on hyperthermophile-derived proteins, which are otherwise thought to be "ultrastable" and inordinately resistant to structural perturbations at room temperature. Here, we show that low (SDS rapidly induce the formation of aggregates and amyloid fibers in five different ultrastable Pyrococcus furiosus proteins in the cationic state. We also show that amyloid formation is accompanied by the development of a characteristic, negative circular dichroism band at ∼230 nm. These effects are not seen if the proteins have a net negative charge or when higher concentrations of SDS are used (which induce helix formation instead). Our results appear to reveal a potential weakness or "Achilles' heel" in ultrastable proteins from hyperthermophiles. They also provide very strong support for the view that SDS initially interacts with proteins through electrostatic interactions, and not hydrophobic interactions, eliciting similar effects entirely regardless of protein molecular weight, or structural features such as quaternary structure or tertiary structural stability.

  6. The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds

    DEFF Research Database (Denmark)

    Roher, N; Miró, F; Boldyreff, B

    2001-01-01

    The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment...... with dithiothreitol. Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation. This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar...

  7. 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

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

    Science.gov (United States)

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

    2016-10-15

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

  9. Protein aggregation containing beta-amyloid, alpha-synuclein and hyperphosphorylated tau in cultured cells of hippocampus, substantia nigra and locus coeruleus after rotenone exposure

    Directory of Open Access Journals (Sweden)

    Martins Stephanie A

    2010-11-01

    Full Text Available Abstract Background Protein aggregates containing alpha-synuclein, beta-amyloid and hyperphosphorylated tau are commonly found during neurodegenerative processes which is often accompanied by the impairment of mitochondrial complex I respiratory chain and dysfunction of cellular systems of protein degradation. In view of this, we aimed to develop an in vitro model to study protein aggregation associated to neurodegenerative diseases using cultured cells from hippocampus, locus coeruleus and substantia nigra of newborn Lewis rats exposed to 0.5, 1, 10 and 25 nM of rotenone, which is an agricultural pesticide, for 48 hours. Results We demonstrated that the proportion of cells in culture is approximately the same as found in the brain nuclei they were extracted from. Rotenone at 0.5 nM was able to induce alpha-synuclein and beta amyloid aggregation, as well as increased hyperphosphorylation of tau, although high concentrations of this pesticide (over 1 nM lead cells to death before protein aggregation. We also demonstrated that the 14kDa isoform of alpha-synuclein is not present in newborn Lewis rats. Conclusion Rotenone exposure may lead to constitutive protein aggregation in vitro, which may be of relevance to study the mechanisms involved in idiopathic neurodegeneration.

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

    Directory of Open Access Journals (Sweden)

    Lina Leinartaitė

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

  11. Predictions of RNA-binding ability and aggregation propensity of proteins

    OpenAIRE

    Agostini, Federico, 1985-

    2014-01-01

    RNA-binding proteins (RBPs) control the fate of a multitude of coding and non-coding transcripts. Formation of ribonucleoprotein (RNP) complexes fine-tunes regulation of post-transcriptional events and influences gene expression. Recently, it has been observed that non-canonical proteins with RNA-binding ability are enriched in structurally disordered and low-complexity regions that are generally involved in functional and dysfunctional associations. Therefore, it is possible that interaction...

  12. Heavy Metals and Metalloids As a Cause for Protein Misfolding and Aggregation

    OpenAIRE

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

    2014-01-01

    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...

  13. Sea star tenacity mediated by a protein that fragments, then aggregates.

    Science.gov (United States)

    Hennebert, Elise; Wattiez, Ruddy; Demeuldre, Mélanie; Ladurner, Peter; Hwang, Dong Soo; Waite, J Herbert; Flammang, Patrick

    2014-04-29

    Sea stars adhere firmly but temporarily to various substrata as a result of underwater efficient adhesive secretions released by their tube feet. Previous studies showed that this material is mainly made up of proteins, which play a key role in its adhesiveness and cohesiveness. Recently, we solubilized the majority of these proteins and obtained 43 de novo-generated peptide sequences by tandem MS. Here, one of these sequences served to recover the full-length sequence of Sea star footprint protein 1 (Sfp1), by RT-PCR and tube foot transcriptome analysis. Sfp1, a large protein of 3,853 aa, is the second most abundant constituent of the secreted adhesive. By using MS and Western blot analyses, we showed that Sfp1 is translated from a single mRNA and then cleaved into four subunits linked together by disulphide bridges in tube foot adhesive cells. The four subunits display specific protein-, carbohydrate-, and metal-binding domains. Immunohistochemistry and immunocytochemistry located Sfp1 in granules stockpiled by one of the two types of adhesive cells responsible for the secretion of the adhesive material. We also demonstrated that Sfp1 makes up the structural scaffold of the adhesive footprint that remains on the substratum after tube foot detachment. Taken together, the results suggest that Sfp1 is a major structural protein involved in footprint cohesion and possibly in adhesive interactions with the tube foot surface. In recombinant form, it could be used for the design of novel sea star-inspired biomaterials.

  14. HIV-1 Tat Induces Unfolded Protein Response and Endoplasmic Reticulum Stress in Astrocytes and Causes Neurotoxicity through Glial Fibrillary Acidic Protein (GFAP) Activation and Aggregation.

    Science.gov (United States)

    Fan, Yan; He, Johnny J

    2016-10-21

    HIV-1 Tat is a major culprit for HIV/neuroAIDS. One of the consistent hallmarks of HIV/neuroAIDS is reactive astrocytes or astrocytosis, characterized by increased cytoplasmic accumulation of the intermediate filament glial fibrillary acidic protein (GFAP). We have shown that that Tat induces GFAP expression in astrocytes and that GFAP activation is indispensable for astrocyte-mediated Tat neurotoxicity. However, the underlying molecular mechanisms are not known. In this study, we showed that Tat expression or GFAP expression led to formation of GFAP aggregates and induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in astrocytes. In addition, we demonstrated that GFAP up-regulation and aggregation in astrocytes were necessary but also sufficient for UPR/ER stress induction in Tat-expressing astrocytes and for astrocyte-mediated Tat neurotoxicity. Importantly, we demonstrated that inhibition of Tat- or GFAP-induced UPR/ER stress by the chemical chaperone 4-phenylbutyrate significantly alleviated astrocyte-mediated Tat neurotoxicity in vitro and in the brain of Tat-expressing mice. Taken together, these results show that HIV-1 Tat expression leads to UPR/ER stress in astrocytes, which in turn contributes to astrocyte-mediated Tat neurotoxicity, and raise the possibility of developing HIV/neuroAIDS therapeutics targeted at UPR/ER stress. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. 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 of this select......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...... 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...... years, significantly higher than in other primates and rodents, although these paradoxically generally evolve much faster. The high evolutionary rate was partly due to relaxation of some selection pressures and partly to distinct positive selection of SOD1 in great apes. We then show that higher...

  16. 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...... 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...... years, significantly higher than in other primates and rodents, although these paradoxically generally evolve much faster. The high evolutionary rate was partly due to relaxation of some selection pressures and partly to distinct positive selection of SOD1 in great apes. We then show that higher...

  17. 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.

  18. De Novo Design of Supercharged, Unfolded Protein Polymers, and Their Assembly into Supramolecular Aggregates

    NARCIS (Netherlands)

    Kolbe, Anke; Mercato, Loretta L. del; Abbasi, Azhar Z.; Rivera Gil, Pilar; Gorzini, Sekineh J.; Huibers, Willem; Poolman, Bert; Parak, Wolfgang J.; Herrmann, Andreas

    2011-01-01

    Here we report for the first time the design and expression of highly charged, unfolded protein polymers based on elastin-like peptides (ELPs). Positively and negatively charged variants were achieved by introducing lysine and glutamic acid residues, respectively, within the repetitive pentapeptide

  19. Extrusion of misfolded and aggregated proteins--a protective strategy of aging neurons?

    Science.gov (United States)

    Doehner, Jana; Genoud, Christel; Imhof, Claudine; Krstic, Dimitrije; Knuesel, Irene

    2012-06-01

    Cellular senescence is the consequence of repetitive exposures to oxidative stress, perturbed energy homeostasis, accumulation of damaged proteins and lesions in their nucleic acids. Whereas mitotic cells are equipped with efficient cell replacement strategies; postmitotic neurons have--with a few exceptions--no mechanism to substitute dysfunctional cells within a complex neuronal network. Here we propose a potential strategy by which aging neurons contend against abnormal accumulation of damaged/misfolded proteins. The suggested mechanism involves the formation of 'budding-like' extrusions and their subsequent clearance by glia. This hypothesis emerged from our previous investigations of the aged hippocampus revealing layer-specific accumulations of Reelin, a glycoprotein with fundamental roles during brain development and adult synaptic plasticity. We showed that Reelin deposits constitute a conserved neuropathological feature of aging, which is significantly accelerated in adult wild-type mice prenatally exposed to a viral-like infection. Here, we employed two- and three-dimensional immunoelectron microscopy to elucidate their morphological properties, localization and origin in immune challenged vs. control mice. In controls, Reelin-positive deposits were dispersed in the neuropil, some being engulfed by glia. In immune challenged mice, however, significantly more Reelin-immunoreactive deposits were associated with neuritic swellings containing mitochondria, vacuoles and cellular debris, pointing to their intracellular origin and suggesting that 'budding-like' neuronal extrusions of misfolded proteins and glial clearance may represent a protective strategy to counteract aging-associated impairments in proteosomal/lysosomal degradation. Neurons exposed to chronic neuroinflammation with increased levels of misfolded/damaged proteins, however, may fail to combat intraneuronal protein accumulations, a process probably underlying neuronal dysfunction and

  20. 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

  1. Probing amyloid protein aggregation with optical superresolution methods: from the test tube to models of disease.

    Science.gov (United States)

    Kaminski, Clemens F; Kaminski Schierle, Gabriele S

    2016-10-01

    The misfolding and self-assembly of intrinsically disordered proteins into insoluble amyloid structures are central to many neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Optical imaging of this self-assembly process in vitro and in cells is revolutionizing our understanding of the molecular mechanisms behind these devastating conditions. In contrast to conventional biophysical methods, optical imaging and, in particular, optical superresolution imaging, permits the dynamic investigation of the molecular self-assembly process in vitro and in cells, at molecular-level resolution. In this article, current state-of-the-art imaging methods are reviewed and discussed in the context of research into neurodegeneration.

  2. ESI-IMS–MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules

    Science.gov (United States)

    Young, Lydia M.; Saunders, Janet C.; Mahood, Rachel A.; Revill, Charlotte H.; Foster, Richard J.; Ashcroft, Alison E.; Radford, Sheena E.

    2016-01-01

    Electrospray ionisation-ion mobility spectrometry–mass spectrometry (ESI-IMS–MS) is a powerful method for the study of conformational changes in protein complexes, including oligomeric species populated during protein self-aggregation into amyloid fibrils. Information on the mass, stability, cross-sectional area and ligand binding capability of each transiently populated intermediate, present in the heterogeneous mixture of assembling species, can be determined individually in a single experiment in real-time. Determining the structural characterisation of oligomeric species and alterations in self-assembly pathways observed in the presence of small molecule inhibitors is of great importance, given the urgent demand for effective therapeutics. Recent studies have demonstrated the capability of ESI-IMS–MS to identify small molecule modulators of amyloid assembly and to determine the mechanism by which they interact (positive, negative, non-specific binding, or colloidal) in a high-throughput format. Here, we demonstrate these advances using self-assembly of Aβ40 as an example, and reveal two new inhibitors of Aβ40 fibrillation. PMID:26007606

  3. ESI-IMS-MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules.

    Science.gov (United States)

    Young, Lydia M; Saunders, Janet C; Mahood, Rachel A; Revill, Charlotte H; Foster, Richard J; Ashcroft, Alison E; Radford, Sheena E

    2016-02-15

    Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is a powerful method for the study of conformational changes in protein complexes, including oligomeric species populated during protein self-aggregation into amyloid fibrils. Information on the mass, stability, cross-sectional area and ligand binding capability of each transiently populated intermediate, present in the heterogeneous mixture of assembling species, can be determined individually in a single experiment in real-time. Determining the structural characterisation of oligomeric species and alterations in self-assembly pathways observed in the presence of small molecule inhibitors is of great importance, given the urgent demand for effective therapeutics. Recent studies have demonstrated the capability of ESI-IMS-MS to identify small molecule modulators of amyloid assembly and to determine the mechanism by which they interact (positive, negative, non-specific binding, or colloidal) in a high-throughput format. Here, we demonstrate these advances using self-assembly of Aβ40 as an example, and reveal two new inhibitors of Aβ40 fibrillation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Sealing ability of mineral trioxide aggregate and Portland cement for furcal perforation repair: a protein leakage study.

    Science.gov (United States)

    Shahi, Shahriar; Rahimi, Saeed; Hasan, Maryam; Shiezadeh, Vahab; Abdolrahimi, Majid

    2009-12-01

    The purpose of this study was to compare the sealing ability of gray mineral trioxide aggregate (GMTA), white MTA (WMTA), and both white and gray Portland cement as furcation perforation repair materials. A total of 120 human mandibular first molars were used. After root canal obturation and preparation of furcal perforations the specimens were randomly divided into four groups of 25 teeth each. In groups A, B, C, and D furcation perforations were filled with WMTA, GMTA, white Portland cement, and type II Portland cement, respectively. Ten teeth were used as positive controls with no filling materials in the perforations and 10 teeth with complete coverage with two layers of nail varnish were used as negative controls. A protein leakage model utilizing 22% bovine serum albumin (BSA) was used for evaluation. Leakage was noted when color conversion of the protein reagent was observed. The controls behaved as expected. Leakage was found in the samples from group A (WMTA), group B (GMTA), and in the two other groups (white and gray Portland cement). There were no statistically significant differences between GMTA and WMTA or white and gray Portland cement, but significant differences were observed between the MTA groups and the Portland cement groups. It was concluded that Portland cements have better sealing ability than MTA, and can be recommended for repair of furcation perforation if the present results are supported by other in vivo and in vitro studies.

  5. Protein Misfolding Diseases.

    Science.gov (United States)

    Hartl, F Ulrich

    2017-06-20

    The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.

  6. Understanding the mechanisms of secondary nucleation for protein aggregation: an analytical approach

    Science.gov (United States)

    Michaels, Thomas; Knowles, Tuomas P. J.

    2013-03-01

    Filamentous protein self-assembly is a general type of behaviour accessible to a wide range of different polypeptide sequences. This phenomenon underlies key molecular events both in normal and aberrant biology, but a general theory of the crucial nucleation steps that govern this process has remained elusive. In this talk we discuss our attempts to provide a general description of secondary nucleation in filamentous protein assembly based on the Becker-Döring kinetic scheme to describe cluster-catalytic effects. This systematic procedure allows extracting low-dimensional systems of equations out of the full kinetic model, in a master equation formalism typically consisting of infinitely many coupled non-linear equations. Using this procedure, we propose and discuss various mechanisms that can underlie the secondary nucleation process. Using data curve-fitting and analysis we show that the addition of a monomer to heterogeneous nuclei is effectively irreversible and discuss the implications of our framework for the more general understanding of the physics of multi-step nucleation phenomena in nature.

  7. Immunogenicity of self-associated aggregates and chemically cross-linked conjugates of the 42 kDa Plasmodium falciparum merozoite surface protein-1.

    Science.gov (United States)

    Qian, Feng; Reiter, Karine; Zhang, Yanling; Shimp, Richard L; Nguyen, Vu; Aebig, Joan A; Rausch, Kelly M; Zhu, Daming; Lambert, Lynn; Mullen, Gregory E D; Martin, Laura B; Long, Carole A; Miller, Louis H; Narum, David L

    2012-01-01

    Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP1(42)) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP1(42) conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP1(42) self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP1(42) specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis.

  8. Immunogenicity of self-associated aggregates and chemically cross-linked conjugates of the 42 kDa Plasmodium falciparum merozoite surface protein-1.

    Directory of Open Access Journals (Sweden)

    Feng Qian

    Full Text Available Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25, was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP1(42 was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP1(42 conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP1(42 self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP1(42 specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis.

  9. Discrimination between silicone oil droplets and protein aggregates in biopharmaceuticals: a novel multiparametric image filter for sub-visible particles in microflow imaging analysis.

    Science.gov (United States)

    Strehl, René; Rombach-Riegraf, Verena; Diez, Manuel; Egodage, Kamal; Bluemel, Markus; Jeschke, Margit; Koulov, Atanas V

    2012-02-01

    Accurate monitoring of the sub-visible particle load in protein biopharmaceuticals is increasingly important to drug development. Manufacturers are expected to characterize and control sub-visible protein particles in their products due to their potential immunogenicity. Light obscuration, the most commonly used analytical tool to count microscopic particles, does not allow discrimination between potentially harmful protein aggregates and harmless pharmaceutical components, e.g. silicone oil, commonly present in drug products. Microscopic image analysis in flow-microscopy techniques allows not only counting, but also classification of sub-visible particles based on morphology. We present a novel approach to define software filters for analysis of particle morphology in flow-microscopic images enhancing the capabilities of flow-microscopy. Image morphology analysis was applied to analyze flow-microscopy data from experimental test sets of protein aggregates and silicone oil suspensions. A combination of four image morphology parameters was found to provide a reliable basis for automatic distinction between silicone oil droplets and protein aggregates in protein biopharmaceuticals resulting in low misclassification errors. A novel, custom-made software filter for discrimination between proteinaceous particles and silicone oil droplets in flow-microscopy imaging analysis was successfully developed.

  10. 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.

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

    NARCIS (Netherlands)

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

    2017-01-01

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

  12. Protein glycation and aggregation inhibitory potency of biomolecules from black gram milled by-product.

    Science.gov (United States)

    Girish, Talakatta K; Prasada Rao, Ummiti Js

    2016-12-01

    Persistent hyperglycaemia causes increased advanced glycation end products (AGEs), which contribute to the pathogenesis of diabetic complication. Therefore, effect of black gram milled by-product (BGBP) extract on inhibition of AGE formation in a bovine serum albumin (BSA)/glucose system was investigated. BGBP extract had a total polyphenol content of 82 mg GAE g -1 and flavonoid content of 46 mg CE g -1 . Ferulic acid, protocatechuic acid, gallic acid, gentisic acid, isovitexin, vitexin and epicatechin were the major bioactives in the extract. BGBP extract exhibited an effective Fe 2+ chelating activity. Size exclusion-high-performance liquid chromatographic studies indicated that upon BSA-AGE formation the BSA monomer content was 38%; however, in the presence of BGBP extract at 50 and 100 µg levels, the monomer content increased and it was found to be 48% and 73%, respectively. BGBP extract at 50 and 100 µg levels decreased the protein carbonyl and fructosamine contents, and quenched the fluorescence intensity of glycated BSA in a dose-dependent manner. Further, fluorescence and transmission electron microscopic studies confirmed the decrease in formation of AGEs by BGBP extract. As BGBP extract inhibited the formation of AGEs, the extract can be used as a nutraceutical or it can be incorporated into food products to obtain functional foods. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  13. Whey protein isolate modified by transglutaminase aggregation and emulsion gel properties

    Science.gov (United States)

    Qi, Weiwei; Chen, Chong; Liu, Mujun; Yu, Guoping; Cai, Xinghang; Guo, Peipei; Yao, Yuxiu; Mei, Sijie

    2015-07-01

    Whey protein isolate and commercial soybean salad oil were used to produce the WPI emulsion dispersions. The properties of TG-catalyzed emulsion gelation produced from WPI emulsion dispersions were investigated by the amount of TG, temperature, pH and reaction time. Specifically, the texture properties (hardness and springiness), water-holding capacity and rheological properties (G' and G") were assessed. The result of Orthogonal tests showed WPI emulsion can form better hardness and springiness gel when the ratio of TG and WPI was 20U/g, pH 7.5, treatment temperature and time were 50°C and 3 h, respectively. The microstructure of TG emulsion gels was more compact, gel pore is smaller, distribution more uniform, the oil droplets size smaller compared with untreated emulsion gels. Compared to the control of rheological properties, G' and G" were significantly increased and G' > G", results showed that the gel was solid state, and TG speeded up the process of gelation.

  14. Kinetic model for the coupling between allosteric transitions in GroEL and substrate protein folding and aggregation.

    Science.gov (United States)

    Tehver, Riina; Thirumalai, D

    2008-04-04

    The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (TR-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the

  15. 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

  16. Cloning of subunits of convulxin, a collagen-like platelet-aggregating protein from Crotalus durissus terrificus venom.

    Science.gov (United States)

    Leduc, M; Bon, C

    1998-07-15

    Convulxin (CVX) is a potent platelet-aggregating glycoprotein from the venom of the snake Crotalus durissus terrificus. It consists of two subunits, alpha and beta, joined by disulphide bridges in a hexameric structure. A cDNA library from venom gland was constructed in the vector pT3T7. The cloned cDNAs encoding the two chains of CVX were sequenced. Both are preceded by an identical 23-amino acid peptide signal sequence and encode sequences of 135 amino acids for the alpha chain and 125 amino acids for the beta chain. These polypeptides include a carbohydrate-recognition domain (CRD) in which some of the specific amino acids required for binding Ca2+ and galactose or mannose are absent. The presence of such a domain means that CVX can be included in the family of C-type lectins along with other snake venom proteins, although it is not a true lectin. Assuming that the localization of intracatenary disulphide bridges of each CVX chain is similar to that of the CRD and that an intercatenary bridge between the alpha and beta chains is similar to that of the C-type lectin botrocetin, we postulate the existence of an additional intercatenary bridge, which explains the tridimeric structure (alphabeta)3 of CVX.

  17. Confined laminar flow on a super-hydrophobic surface drives the initial stages of tau protein aggregation

    KAUST Repository

    Moretti, Manola

    2018-02-01

    Super-hydrophobic micro-patterned surfaces are ideal substrates for the controlled self-assembly and substrate-free characterization of biological molecules. In this device, the tailored surface supports a micro-volume drop containing the molecules of interest. While the quasi-spherical drop is evaporating under controlled conditions, its de-wetting direction is guided by the pillared microstructure on top of the device, leading to the formation of threads between the neighboring pillars. This effect has been exploited here to elucidate the mechanism triggering the formation of amyloid fibers and oligomers in tau related neurodegenerative diseases. By using Raman spectroscopy, we demonstrate that the fiber bridging the pillars contains β-sheets, a characteristic feature of amyloid aggregation. We propose that the combination of laminar flow, shear stress and molecular crowding taking place while the drop is evaporating on the SHMS, induces the reorganization of the tau protein secondary structure and we suggest that this effect could in fact closely mimic the actual mechanism occurring in the human brain environment. Such a straightforward technique opens up new possibilities in the field of self-assembly of biomolecules and their characterization by different methods (SEM, AFM, Raman spectroscopy, TEM), in a single device.

  18. Proteolytic stress causes heat shock protein induction, tau ubiquitination, and the recruitment of ubiquitin to tau-positive aggregates in oligodendrocytes in culture.

    Science.gov (United States)

    Goldbaum, Olaf; Richter-Landsberg, Christiane

    2004-06-23

    Molecular chaperones and the ubiquitin-proteasome system are participants in the defense against unfolded proteins and provide an effective protein quality control system that is essential for cellular functions and survival. Ubiquitinated tau-positive inclusion bodies containing the small heat shock protein alphaB-crystallin in oligodendrocytes are consistent features of a variety of neurodegenerative diseases, and defects in the proteasome system might contribute to the aggregation process. Oligodendrocytes, the myelin-forming cells of the CNS, are specifically sensitive to stress situations. Here we can show that in cultured rat brain oligodendrocytes proteasomal inhibition by MG-132 or lactacystin caused apoptotic cell death and the induction of heat shock proteins in a time- and concentration-dependent manner. Specifically, alphaB-crystallin was upregulated, and ubiquitinated proteins accumulated. After incubation with MG-132 the tau was dephosphorylated, which enhanced its microtubule-binding capacity. Proteasomal inhibition led to ubiquitination of tau and its association with alphaB-crystallin and to the occurrence of thioflavine S-positive aggregates in the oligodendroglial cytoplasm. These aggregates were positive for tau and also contained ubiquitin and alphaB-crystallin; hence they resembled the glial cytoplasmic inclusions observed in white matter disease and frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17). In summary, the data underscore the specific sensitivity of oligodendrocytes to stress situations and point to a causal relationship of proteasomal impairment and inclusion body formation.

  19. Bcl-2 Decreases the Affinity of SQSTM1/p62 to Poly-Ubiquitin Chains and Suppresses the Aggregation of Misfolded Protein in Neurodegenerative Disease.

    Science.gov (United States)

    Zhou, Liang; Wang, Hongfeng; Ren, Haigang; Hu, Qingsong; Ying, Zheng; Wang, Guanghui

    2015-12-01

    Poly-ubiquitinated protein aggregate formation is the most striking hallmark of various neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion disease. Mutations of many ubiquitin-associated proteins involved in the regulation of protein aggregation, such as SQSTM1/p62 (p62), parkin, and VCP, are closely linked to neurodegeneration. B-cell lymphoma 2 (Bcl-2) is a key regulator in autophagy, apoptosis, and mitochondria quality control in many cell types including neurons, and it plays important roles in the pathogenesis of neurodegenerative diseases mentioned above. Our previous work showed that Bcl-2 can directly bind to p62, and here we report that Bcl-2 directly interacts with the N-terminus of p62, but not the C-terminus (UBA domain). Interestingly and importantly, Bcl-2 affects the affinity of p62 to poly-ubiquitin chains and suppresses the aggregation of poly-ubiquitinated proteins such as mutant huntingtin associated with Huntington's disease. Our study reveals a role of Bcl-2 that involves in the regulation of misfolded proteins.

  20. 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.

  1. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Directory of Open Access Journals (Sweden)

    Zeng YX

    2016-04-01

    stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington’s disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results. Keywords: Huntington’s disease, mutant Huntingtin, xyloketal derivatives, Caenorhabditis elegans, protein misfolding, molecular target

  2. Interaction between holo transferrin and HSA-PPIX complex in the presence of lomefloxacin: An evaluation of PPIX aggregation in protein-protein interactions

    Science.gov (United States)

    Sattar, Zohreh; Iranfar, Hediye; Asoodeh, Ahmad; Saberi, Mohammad Reza; Mazhari, Mahboobeh; Chamani, Jamshidkhan

    2012-11-01

    Human serum albumin (HSA) and holo transferrin (TF) are two serum carrier proteins that are able to interact with each other, thereby altering their binding behavior toward their ligands. During the course of this study, the interaction between HSA-PPIX and TF, in the presence and absence of lomefloxacin (LMF), was for the first time investigated using different spectroscopic and molecular modeling techniques. Fluorescence spectroscopy experiments were performed in order to study conformational changes of proteins. The RLS technique was utilized to investigate the effect of LMF on J-aggregation of PPIX, which is the first report of its kind. Our findings present clear-cut evidence for the alteration of interactions between HSA and TF in the presence of PPIX and changes in drug-binding to HSA and HSA-PPIX complex upon interaction with TF. Moreover, molecular modeling studies suggested that the binding site for LMF became switched in the presence of PPIX, and that LMF bound to the site IIA of HSA. The obtained results should give new insight into research in this field and may cast some light on the dynamics of drugs in biological systems.

  3. The small heat shock protein B8 (HSPB8) efficiently removes aggregating species of dipeptides produced in C9ORF72-related neurodegenerative diseases.

    Science.gov (United States)

    Cristofani, Riccardo; Crippa, Valeria; Vezzoli, Giulia; Rusmini, Paola; Galbiati, Mariarita; Cicardi, Maria Elena; Meroni, Marco; Ferrari, Veronica; Tedesco, Barbara; Piccolella, Margherita; Messi, Elio; Carra, Serena; Poletti, Angelo

    2018-01-01

    Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases in which similar pathogenic mechanisms are involved. Both diseases associate to the high propensity of specific misfolded proteins, like TDP-43 or FUS, to mislocalize and aggregate. This is partly due to their intrinsic biophysical properties and partly as a consequence of failure of the neuronal protein quality control (PQC) system. Several familial ALS/FTD cases are linked to an expansion of a repeated G4C2 hexanucleotide sequence present in the C9ORF72 gene. The G4C2, which localizes in an untranslated region of the C9ORF72 transcript, drives an unconventional repeat-associated ATG-independent translation. This leads to the synthesis of five different dipeptide repeat proteins (DPRs), which are not "classical" misfolded proteins, but generate aberrant aggregation-prone unfolded conformations poorly removed by the PQC system. The DPRs accumulate into p62/SQSTM1 and ubiquitin positive inclusions. Here, we analyzed the biochemical behavior of the five DPRs in immortalized motoneurons. Our data suggest that while the DPRs are mainly processed via autophagy, this system is unable to fully clear their aggregated forms, and thus they tend to accumulate in basal conditions. Overexpression of the small heat shock protein B8 (HSPB8), which facilitates the autophagy-mediated disposal of a large variety of classical misfolded aggregation-prone proteins, significantly decreased the accumulation of most DPR insoluble species. Thus, the induction of HSPB8 might represent a valid approach to decrease DPR-mediated toxicity and maintain motoneuron viability.

  4. 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.

  5. 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.

  6. Prevention of aggregation and renaturation of carbonic anhydrase via weak association with octadecyl- or azobenzene-modified poly(acrylate) derivatives.

    Science.gov (United States)

    Martin, Nicolas; Ruchmann, Juliette; Tribet, Christophe

    2015-01-01

    The prevention of aggregation during renaturation of urea-denatured carbonic anhydrase B (CAB) via hydrophobic and Coulomb association with anionic polymers was studied in mixed solutions of CAB and amphiphilic poly(acrylate) copolymers. The polymers were derivatives of a parent poly(acrylic acid) randomly grafted with hydrophobic side groups (either 3 mol % octadecyl group, or 1-5 mol % alkylamidoazobenzene photoresponsive groups). CAB:polymer complexes were characterized by light scattering and fluorescence correlation spectroscopy in aqueous buffers (pH 7.75 or 5.9). Circular dichroism and enzyme activity assays enabled us to study the kinetics of renaturation. All copolymers, including the hydrophilic PAA parent chain, provided a remarkable protective effect against CAB aggregation during renaturation, and most of them (but not the octadecyl-modified one) markedly enhanced the regain of activity as compared to CAB alone. The significant role of Coulomb binding in renaturation and comparatively the lack of efficacy of hydrophobic association was highlighted by measurements of activity regain before and after in situ dissociation of hydrophobic complexes (achieved by phototriggering the polarity of azobenzene-modified polymers under exposure to UV light). In the presence of polymers (CAB:polymer of 1:1 w/w ratio) at concentration ∼0.6 g L(-1), the radii of the largest complexes were similar to the radii of the copolymers alone, suggesting that the binding of CAB involves one or a few polymer chain(s). These complexes dissociated by dilution (0.01 g L(-1)). It is concluded that prevention of irreversible aggregation and activity recovery were achieved when marginally stable complexes are formed. Reaching a balanced stability of the complex plays the main role in CAB renaturation, irrespective of the nature of the binding (by Coulomb association, with or without contribution of hydrophobic association).

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

    Directory of Open Access Journals (Sweden)

    Haina Qin

    2014-07-01

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

  8. From The Cover: Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

    Science.gov (United States)

    Nollen, Ellen A. A.; Garcia, Susana M.; van Haaften, Gijs; Kim, Soojin; Chavez, Alejandro; Morimoto, Richard I.; Plasterk, Ronald H. A.

    2004-04-01

    Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation. protein misfolding | neurodegenerative diseases

  9. Protein Colloidal Aggregation

    Data.gov (United States)

    National Aeronautics and Space Administration — Finding an optimal treatment for any disease is impossible until we fully understand its cause. We believe the central problem in obtaining this understanding is...

  10. Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

    Science.gov (United States)

    Simões, Ana T; Gonçalves, Nélio; Koeppen, Arnulf; Déglon, Nicole; Kügler, Sebastian; Duarte, Carlos Bandeira; Pereira de Almeida, Luís

    2012-08-01

    Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

  11. Aggregates of scrapie-associated prion protein induce the cell-free conversion of protease-sensitive prion protein to the protease-resistant state.

    Science.gov (United States)

    Caughey, B; Kocisko, D A; Raymond, G J; Lansbury, P T

    1995-12-01

    Scrapie infection instigates the in vivo conversion of normal, protease-sensitive prion protein (PrPC) into a protease-resistant form (PrPSc) by an unknown mechanism. In vitro studies have indicated that PrPSc can induce this conversion, consistent with proposals that PrPSc itself might be the infectious scrapie agent. Using this cell-free model of the PrPC to PrPSc conversion, we have studied the dependence of conversion on reactant concentration, and the properties of the PrPSc-derived species that has converting activity. The cell-free conversion of 35S PrPC to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrPSc (above an apparent minimum threshold concentration) and 35S PrPC. Analysis of the physical size of the converting activity indicated that detectable converting activity was associated only with aggregates. Under mildly chaotropic conditions, which partially disaggregated PrPSc and enhanced the converting activity, the active species were heterogeneous in size, but larger than either effectively solubilized PrP or molecular weight standards of approximately 2000 kDa. The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrPSc. These results are consistent with a nucleated polymerization mechanism of PrPSc formation and inconsistent with a heterodimer mechanism.

  12. The G46S-hPAH mutant protein: a model to study the rescue of aggregation-prone PKU mutations by chaperones.

    Science.gov (United States)

    Leandro, João; Saraste, Jaakko; Leandro, Paula; Flatmark, Torgeir

    2011-01-01

    Phenylketonuria (PKU), the most common inborn error of metabolism, is caused by dysfunction of the liver enzyme phenylalanine hydroxylase (PAH), with more than 550 PAH gene mutations identified to date. A large number of these mutations result in mutant forms of the enzyme displaying reduced stability, increased propensity to aggregate, and accelerated in cellulo degradation. Loss or reduction of human PAH activity results in hyperphenylalaninemia (HPA) which, if untreated, results in severe mental retardation and impaired cognitive development. Until now, strict low phenylalanine diet has been the most effective therapy, but as a protein misfolding disease PKU is a good candidate for treatment by natural/chemical/pharmacological chaperones. The natural cofactor of human PAH, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), has already been approved for oral treatment of HPA, giving a positive response in mild forms of the disease showing considerable residual enzymatic activity. In the case of the most severe forms of PKU, ongoing studies with chemical and pharmacological chaperones to rescue misfolded mutant proteins from aggregation and degradation are providing promising results. The PKU mutation G46S is associated with a severe form of the disease, resulting in an aggregation-prone protein. The human PAH mutant G46S is rapidly degraded in the cellular environment and, in vitro (upon removal of its stabilizing fusion partner maltose binding protein (MBP)) self-associates to form higher-order oligomers/fibrils. Here, we present an in vitro experimental model system to study the modulation of G46S aggregation by chemical/pharmacological chaperones, which may represent a useful approach to study the rescue of other severe PKU mutations by chemical/pharmacological chaperones. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. 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.

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

    DEFF Research Database (Denmark)

    Tastesen, Hanne Sørup

    Background: Obesity and related co‐morbidities are increasing problems worldwide and nutritional approaches to prevent and alleviate these diseases are thus of great interest. High‐protein diets have been shown to prevent and alleviate obesity and co‐morbidities in rodents and humans through...... protein, was found to be negligible in development of obesity and co‐morbidities in mice. Seafood protein with high endogenous taurine and glycine contents was found to prevent diet‐induced adiposity and dyslipidemia, both in ad libitum and pair‐fed settings. The ability of seafood proteins to prevent...... these metabolic disturbances was found to associate with the high endogenous taurine and glycine concentrations and to concur with increased energy expenditure and a tendency towards increased voluntary locomotor activity. Consumption of a seafood protein‐mixture prevented diet‐induced development of obesity...

  15. An approach for optimal allocation of safety resources: using the knapsack problem to take aggregated cost-efficient preventive measures.

    Science.gov (United States)

    Reniers, Genserik L L; Sörensen, Kenneth

    2013-11-01

    On the basis of the combination of the well-known knapsack problem and a widely used risk management technique in organizations (that is, the risk matrix), an approach was developed to carry out a cost-benefits analysis to efficiently take prevention investment decisions. Using the knapsack problem as a model and combining it with a well-known technique to solve this problem, bundles of prevention measures are prioritized based on their costs and benefits within a predefined prevention budget. Those bundles showing the highest efficiencies, and within a given budget, are identified from a wide variety of possible alternatives. Hence, the approach allows for an optimal allocation of safety resources, does not require any highly specialized information, and can therefore easily be applied by any organization using the risk matrix as a risk ranking tool. © 2013 Society for Risk Analysis.

  16. The N-domain of Escherichia coli phosphoglycerate kinase is a novel fusion partner to express aggregation-prone heterologous proteins.

    Science.gov (United States)

    Song, Jong-Am; Lee, Dae-Sung; Park, Jin-Seung; Han, Kyung-Yeon; Lee, Jeewon

    2012-02-01

    As a fusion partner to express aggregation-prone heterologous proteins, we investigated the efficacy of Escherichia coli phosphoglycerate kinase (ePGK) that consists of two functional domains (N- and C-domain) and reportedly has a high structural stability. When the full-length ePGK (F-ePGK) was used as a fusion partner, the solubility of the heterologous proteins increased, but some of them still had a large fraction of insoluble aggregates. Surprisingly, the fusion expression using the N-domain of ePGK (N-ePGK) made the insoluble fraction significantly reduce to less than 10% for all the heterologous fusion proteins tested. Also, we evaluated the efficacy of N-ePGK in making the target proteins be expressed with their own native function or structure. It was found that of human ferritin light chain, bacterial arginine deiminase, human granulocyte colony stimulating factor were synthesized evidently with the self-assembly function, L-arginine-degrading activity, and the correct secondary structure, respectively, through the fusion expression using N-ePGK. These results indicate that N-ePGK is a highly potent fusion partner that can be widely used for the synthesis of a variety of heterologous proteins in E. coli. Copyright © 2011 Wiley Periodicals, Inc.

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

    DEFF Research Database (Denmark)

    Tastesen, Hanne Sørup

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

  18. Protein Corona Prevents TiO2 Phototoxicity.

    Directory of Open Access Journals (Sweden)

    Maja Garvas

    Full Text Available TiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.Fluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles' surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes' surface.These results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired - as for efficient photodynamic cancer therapy.

  19. ALS-linked mutant SOD1 proteins promote Aβ aggregates in ALS through direct interaction with Aβ.

    Science.gov (United States)

    Jang, Ja-Young; Cho, Hyungmin; Park, Hye-Yoon; Rhim, Hyangshuk; Kang, Seongman

    2017-11-04

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons. Aggregation of ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) is a hallmark of a subset of familial ALS (fALS). Recently, intracellular amyloid-β (Aβ) is detected in motor neurons of both sporadic and familial ALS. We have previously shown that intracellular Aβ specifically interacts with G93A, an ALS-linked SOD1 mutant. However, little is known about the pathological and biological effect of this interaction in neurons. In this study, we have demonstrated that the Aβ-binding region is exposed on the SOD1 surface through the conformational changes due to misfolding of SOD1. Interestingly, we found that the intracellular aggregation of Aβ is enhanced through the direct interaction of Aβ with the Aβ-binding region exposed to misfolded SOD1. Ultimately, increased Aβ aggregation by this interaction promotes neuronal cell death. Consistent with this result, Aβ aggregates was three-fold higher in the brains of G93A transgenic mice than those of non Tg. Our study provides the first direct evidence that Aβ, an AD-linked factor, is associated to the pathogenesis of ALS and provides molecular clues to understand common aggregation mechanisms in the pathogenesis of neurodegenerative diseases. Furthermore, it will provide new insights into the development of therapeutic approaches for ALS. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Predicting protein aggregation during storage in lyophilized solids using solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS).

    Science.gov (United States)

    Moorthy, Balakrishnan S; Schultz, Steven G; Kim, Sherry G; Topp, Elizabeth M

    2014-06-02

    Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products.

  1. Nanoparticle Surface Specific Adsorption of Zein and Its Self-assembled Behavior of Nanocubes Formation in Relation to On-Off SERS: Understanding Morphology Control of Protein Aggregates.

    Science.gov (United States)

    Navdeep; Banipal, Tarlok Singh; Kaur, Gurinder; Bakshi, Mandeep Singh

    2016-01-27

    Zein, an industrially important protein, is characterized in terms of its food and pharmaceutical coating applications by using surface enhanced Raman spectroscopy (SERS) on Au, Ag, and PbS nanoparticles (NPs). Its specific surface adsorption behavior on Ag NPs produced self-assembled zein nanocubes which demonstrated on and off SERS activity. Both SERS characterization as well as nanocube formation of zein helped us to understand the complex protein aggregation behavior in shape controlled morphologies, a process with significant ramifications in protein crystallization to achieve ordered morphologies. Interestingly, nanocube formation was promoted in the presence of Ag rather than Au or PbS NPs under in situ synthesis and discussed in terms of specific adsorption. Zein fingerprinting was much more clear and enhanced on Au surface in comparison to Ag while PbS did not demonstrate SERS due to its semiconducting nature.

  2. External electric field: An effective way to prevent aggregation of Mg atoms on γ-graphyne for high hydrogen storage capacity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ping-Ping [College of Physical Science and Technology, Sichuan University, Chengdu 610065 (China); Zhang, Hong, E-mail: hongzhang@scu.edu.cn [College of Physical Science and Technology, Sichuan University, Chengdu 610065 (China); Key Laboratory of High Energy Density Physics and Technology of Ministry of Education, Sichuan University, Chengdu 610064 (China); Cheng, Xin-Lu, E-mail: chengxl@scu.edu.cn [Key Laboratory of High Energy Density Physics and Technology of Ministry of Education, Sichuan University, Chengdu 610064 (China); Tang, Yong-Jian, E-mail: tangyongjian2000@sina.com [Science and Technology on Plasma Physics Laboratory, Research Center of Laser Fusion, China Academy of Engineering Physics, Mianyang 621900 (China)

    2016-05-15

    Highlights: • Due to large pores in the sheet of γ-graphyne, it should be a potential materials for energy storage applications. Our calculations might motivate active experimental efforts in designing high-efficiency hydrogen storage media. • For the first time, we use an applied external electric field to prevent Mg atoms from clustering using density functional theory (DFT) calculations. • The results demonstrate that, for Mg-G after electric field (F = 0.05 V/nm) treatment, ten H{sub 2} molecules per Mg atom can be adsorbed and the hydrogen storage capacities reach to 10.64 wt%, with the average binding energies of 0.28 eV/H{sub 2}. - Abstract: In this article, we investigate the hydrogen storage capacity of Mg-decorated γ-graphyne (Mg-G) based on DFT calculations. Our results indicate that an external electric field can effectively prevent Mg atoms aggregating on γ-graphyne sheet. The Mg-G, after electric field (F = 0.05 V/nm) treatment, can store up to ten H{sub 2} molecules and the hydrogen storage capacity is 10.64 wt%, with the average adsorption energy of 0.28 eV/H{sub 2}. Our calculations demonstrate that Mg-G is a potential material for hydrogen storage with high capacity and might motivate active experimental efforts in designing hydrogen storage media.

  3. Role of the p38 pathway in mineral trioxide aggregate-induced cell viability and angiogenesis-related proteins of dental pulp cell in vitro.

    Science.gov (United States)

    Huang, S-C; Wu, B-C; Kao, C-T; Huang, T-H; Hung, C-J; Shie, M-Y

    2015-03-01

    To investigate the influence of mineral trioxide aggregate (MTA) on angiogenesis of primary human dental pulp cells (hDPCs) via the MAPK pathway, in particular p38. Human dental pulp cells were cultured with MTA to angiogenesis, after which cell viability, ion concentration, osmolality, NO secretion, the von Willebrand factor (vWF) and angiopoietin-1 (Ang-1) protein expression were examined. PrestoBlue(®) was used for evaluating the proliferation of hDPCs. An enzyme-linked immunosorbent assay was employed to determine vWF and Ang-1 protein secretion in hDPCs cultured on MTA and the control. Cells cultured on the tissue culture plate without the cement were used as the control. The t-test was used to evaluate the significance of the differences between the mean values. Mineral trioxide aggregate elicited a significant (P MTA consumed calcium and phosphate ions, and released more Si ions in the medium. MTA significantly (P MTA. Expression levels for Ang-1 and vWF in hDPCs on MTA were higher than those of the MTA + p38 inhibitor (SB203580) group (P Mineral trioxide aggregate was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si increased the osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signalling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion decreased. These findings verify that the p38 pathway plays a key role in regulating the angiogenic behaviour of hDPCs cultured on MTA. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  4. Ability of silybin and its derivatives to prevent protein oxidation in different model systems

    DEFF Research Database (Denmark)

    Purchartová, K.; Baron, C.P.; Křen, V.

    2013-01-01

    to prevent activation of hemoglobin (Hb) to highly reactive hypervalent heme protein species (ferrylHb and perferrylHb) was examined. Indeed, Hb cytotoxicity has been associated with the generation of protein radicals, which are formed when the ferric iron of Hb (Fe3+) is oxidised by H2O2 to (Fe4+) to form...... perferrylHb and ferrylHb, with the later also bearing a radical on its protein. The relationship between the structural properties of silybin and its derivatives and their ability to prevent oxidation of Hb was investigated in model system in the presence or the absence of lipids. The antioxidant activities...... of silybin, dehydrosilybin, 23-O-butanoyl and 23-O-palmitoyl silybin derivatives were correlated with their interaction with Hb species. Results are discussed in relation to the potential of dehydrosilybin, silybin and C4 and C16 derivates to prevent activation of Hb to hypevalent heme protein species....

  5. Generation of chimeric minipigs by aggregating 4- to 8-cell-stage blastomeres from somatic cell nuclear transfer with the tracing of enhanced green fluorescent protein.

    Science.gov (United States)

    Ji, Huili; Long, Chuan; Feng, Chong; Shi, Ningning; Jiang, Yingdi; Zeng, Guomin; Li, Xirui; Wu, Jingjing; Lu, Lin; Lu, Shengsheng; Pan, Dengke

    2017-05-01

    Blastocyst complementation is an important technique for generating chimeric organs in organ-deficient pigs, which holds great promise for solving the problem of a shortage of organs for human transplantation procedures. Porcine chimeras have been generated using embryonic germ cells, embryonic stem cells, and induced pluripotent stem cells; however, there are no authentic pluripotent stem cells for pigs. In previous studies, blastomeres from 4- to 8-cell-stage parthenogenetic embryos were able to generate chimeric fetuses efficiently, but the resulting fetuses did not produce live-born young. Here, we used early-stage embryos from somatic cell nuclear transfer (SCNT) to generate chimeric piglets by the aggregation method. Then, the distribution of chimerism in various tissues and organs was observed through the expression of enhanced green fluorescent protein (EGFP). Initially, we determined whether 4- to 8- or 8- to 16-cell-stage embryos were more suitable to generate chimeric piglets. Chimeras were produced by aggregating two EGFP-tagged Wuzhishan minipig (WZSP) SCNT embryos and two Bama minipig (BMP) SCNT embryos. The chimeric piglets were identified by coat color and microsatellite and swine leukocyte antigen analyses. Moreover, the distribution of chimerism in various tissues and organs of the piglets was evaluated by EGFP expression. We found that more aggregated embryos were produced using 4- to 8-cell-stage embryos (157/657, 23.9%) than 8- to 16-cell-stage embryos (100/499, 20.0%). Thus, 4- to 8-cell-stage embryos were used for the generation of chimeras. The rate of blastocysts development after aggregating WZSP with BMP embryos was 50.6%. Transfer of 391 blastocysts developed from 4- to 8-cell-stage embryos to five recipients gave rise to 18 piglets, of which two (11.1%) were confirmed to be chimeric by their coat color and microsatellite examination of the skin. One of the chimeric piglets died at 35 days and was subsequently autopsied, whereas the

  6. Worksite Tobacco Prevention: A Randomized, Controlled Trial of Adoption, Dissemination Strategies, and Aggregated Health-Related Outcomes across Companies.

    Science.gov (United States)

    Friedrich, Verena; Brügger, Adrian; Bauer, Georg F

    2015-01-01

    Evidence based public health requires knowledge about successful dissemination of public health measures. This study analyses (a) the changes in worksite tobacco prevention (TP) in the Canton of Zurich, Switzerland, between 2007 and 2009; (b1) the results of a multistep versus a "brochure only" dissemination strategy; (b2) the results of a monothematic versus a comprehensive dissemination strategy that aim to get companies to adopt TP measures; and (c) whether worksite TP is associated with health-related outcomes. A longitudinal design with randomized control groups was applied. Data on worksite TP and health-related outcomes were gathered by a written questionnaire (baseline n = 1627; follow-up n = 1452) and analysed using descriptive statistics, nonparametric procedures, and ordinal regression models. TP measures at worksites improved slightly between 2007 and 2009. The multistep dissemination was superior to the "brochure only" condition. No significant differences between the monothematic and the comprehensive dissemination strategies were observed. However, improvements in TP measures at worksites were associated with improvements in health-related outcomes. Although dissemination was approached at a mass scale, little change in the advocated adoption of TP measures was observed, suggesting the need for even more aggressive outreach or an acceptance that these channels do not seem to be sufficiently effective.

  7. A Specific Population of Abnormal Prion Protein Aggregates Is Preferentially Taken Up by Cells and Disaggregated in a Strain-Dependent Manner

    Science.gov (United States)

    Choi, Young Pyo

    2013-01-01

    Prion diseases are characterized by the conversion of the soluble protease-sensitive host-encoded prion protein (PrPC) into its aggregated, protease-resistant, and infectious isoform (PrPSc). One of the earliest events occurring in cells following exposure to an exogenous source of prions is the cellular uptake of PrPSc. It is unclear how the biochemical properties of PrPSc influence its uptake, although aggregate size is thought to be important. Here we show that for two different strains of mouse prions, one that infects cells (22L) and one that does not (87V), a fraction of PrPSc associated with distinct sedimentation properties is preferentially taken up by the cells. However, while the fraction of PrPSc and the kinetics of uptake were similar for both strains, PrPSc derived from the 87V strain was disaggregated more rapidly than that derived from 22L. The increased rate of PrPSc disaggregation did not correlate with either the conformational or aggregate stability of 87V PrPSc, both of which were greater than those of 22L PrPSc. Our data suggest that the kinetics of disaggregation of PrPSc following cellular uptake is independent of PrPSc stability but may be dependent upon some component of the PrPSc aggregate other than PrP. Rapid disaggregation of 87V PrPSc by the cell may contribute, at least in part, to the inability of 87V to infect cells in vitro. PMID:23966386

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

    Directory of Open Access Journals (Sweden)

    Sukhdeep Kumar

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

  9. Rydberg aggregates

    Science.gov (United States)

    Wüster, S.; Rost, J.-M.

    2018-02-01

    We review Rydberg aggregates, assemblies of a few Rydberg atoms exhibiting energy transport through collective eigenstates, considering isolated atoms or assemblies embedded within clouds of cold ground-state atoms. We classify Rydberg aggregates, and provide an overview of their possible applications as quantum simulators for phenomena from chemical or biological physics. Our main focus is on flexible Rydberg aggregates, in which atomic motion is an essential feature. In these, simultaneous control over Rydberg–Rydberg interactions, external trapping and electronic energies, allows Born–Oppenheimer surfaces for the motion of the entire aggregate to be tailored as desired. This is illustrated with theory proposals towards the demonstration of joint motion and excitation transport, conical intersections and non-adiabatic effects. Additional flexibility for quantum simulations is enabled by the use of dressed dipole–dipole interactions or the embedding of the aggregate in a cold gas or Bose–Einstein condensate environment. Finally we provide some guidance regarding the parameter regimes that are most suitable for the realization of either static or flexible Rydberg aggregates based on Li or Rb atoms. The current status of experimental progress towards enabling Rydberg aggregates is also reviewed.

  10. Modulating the aggregation behaviour to restore the mechanical response of acid induced mixed gels of sodium caseinate and soy proteins

    NARCIS (Netherlands)

    Martin, Anneke H.; Los Reyes Jiménez, De Marta L.; Pouvreau, Laurice

    2016-01-01

    Partial replacement of milk proteins with plant proteins is a challenge due to the reported negative effect on physical and sensory properties. Understanding of how the mechanical properties of acidified milk gels can be restored when 30% casein is replaced with soy proteins is therefore

  11. Modulating the aggregation behaviour to restore the mechanical response of acid induced mixed gels of sodium caseinate and soy proteins

    NARCIS (Netherlands)

    Martin, A.H.; Reyes Jiménez, M. L. de los; Pouvreau, L.

    2016-01-01

    Partial replacement of milk proteins with plant proteins is a challenge due to the reported negative effect on physical and sensory properties. Understanding of how the mechanical properties of acidified milk gels can be restored when 30% casein is replaced with soy proteins is therefore explored.

  12. Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: inhibition of the plasmodial chaperone PfHsp70-1.

    Science.gov (United States)

    Cockburn, Ingrid L; Pesce, Eva-Rachele; Pryzborski, Jude M; Davies-Coleman, Michael T; Clark, Peter G K; Keyzers, Robert A; Stephens, Linda L; Blatch, Gregory L

    2011-05-01

    Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC(50) values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.

  13. Cloning of subunits of convulxin, a collagen-like platelet-aggregating protein from Crotalus durissus terrificus venom.

    OpenAIRE

    Leduc, M; Bon, C

    1998-01-01

    Convulxin (CVX) is a potent platelet-aggregating glycoprotein from the venom of the snake Crotalus durissus terrificus. It consists of two subunits, alpha and beta, joined by disulphide bridges in a hexameric structure. A cDNA library from venom gland was constructed in the vector pT3T7. The cloned cDNAs encoding the two chains of CVX were sequenced. Both are preceded by an identical 23-amino acid peptide signal sequence and encode sequences of 135 amino acids for the alpha chain and 125 amin...

  14. Construction aggregates

    Science.gov (United States)

    Tepordei, V.V.

    1994-01-01

    Part of a special section on industrial minerals in 1993. The 1993 production of construction aggregates increased 6.3 percent over the 1992 figure, to reach 2.01 Gt. This represents the highest estimated annual production of combined crushed stone and construction sand and gravel ever recorded in the U.S. The outlook for construction aggregates and the issues facing the industry are discussed.

  15. PLGA microspheres containing bee venom proteins for preventive immunotherapy.

    Science.gov (United States)

    Trindade, Reginaldo A; Kiyohara, Pedro K; de Araujo, Pedro S; Bueno da Costa, Maria H

    2012-02-14

    Bee venom (BV) allergy is potentially dangerous for allergic individuals because a single bee sting may induce an anaphylactic reaction, eventually leading to death. Currently, venom immunotherapy (VIT) is the only treatment with long-lasting effect for this kind of allergy and its efficiency has been recognized worldwide. This therapy consists of subcutaneous injections of gradually increasing doses of the allergen. This causes patient lack of compliance due to a long time of treatment with a total of 30-80 injections administered over years. In this article we deal with the characterization of different MS-PLGA formulations containing BV proteins for VIT. The PLGA microspheres containing BV represent a strategy to replace the multiple injections, because they can control the solute release. Physical and biochemical methods were used to analyze and characterize their preparation. Microspheres with encapsulation efficiencies of 49-75% were obtained with a BV triphasic release profile. Among them, the MS-PLGA 34kDa-COOH showed to be best for VIT because they presented a low initial burst (20%) and a slow BV release during lag phase. Furthermore, few conformational changes were observed in the released BV. Above all, the BV remained immunologically recognizable, which means that they could continuously stimulate the immune system. Those microspheres containing BV could replace sequential injections of traditional VIT with the remarkable advantage of reduced number of injections. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Prokaryotic ubiquitin-like ThiS fusion enhances the heterologous protein overexpression and aggregation in Escherichia coli.

    Science.gov (United States)

    Yuan, Sujuan; Xu, Jian; Ge, Ying; Yan, Zheng; Du, Guohua; Wang, Nan

    2013-01-01

    Fusion tags are commonly employed to enhance target protein expression, improve their folding and solubility, and reduce protein degradation in expression of recombinant proteins. Ubiquitin (Ub) and SUMO are highly conserved small proteins in eukaryotes, and frequently used as fusion tags in prokaryotic expression. ThiS, a smaller sulfur-carrier protein involved in thiamin synthesis, is conserved among most prokaryotic species. The structural similarity between ThiS and Ub provoked us into expecting that the former could be used as a fusion tag. Hence, ThiS was fused to insulin A and B chains, murine Ribonuclease Inhibitor (mRI) and EGFP, respectively. When induced in Escherichia coli, ThiS-fused insulin A and B chains were overexpressed in inclusion bodies, and to higher levels in comparison to the same proteins fused with Ub. On the contrast, ThiS fusion of mRI, an unstable protein, resulted in enhanced degradation that was not alleviated in protease-deficient strains. While the degradation of Ub- and SUMO-fused mRI was less and seemed protease-dependent. Enhanced degradation of mRI did not occur for the fusions with half-molecules of ThiS. When ThiS-tag was fused to the C-terminus of EGFP, higher expression, predominantly in inclusion bodies, was observed again. It was further found that ThiS fusion of EGFP significantly retarded its refolding process. These results indicated that prokaryotic ThiS is able to promote the expression of target proteins in E. coli, but enhanced degradation may occur in case of unstable targets. Unlike eukaryotic Ub-based tags usually increase the solubility and folding of proteins, ThiS fusion enhances the expression by augmenting the formation of inclusion bodies, probably through retardation of the folding of target proteins.

  17. Silencing the Odorant Binding Protein RferOBP1768 Reduces the Strong Preference of Palm Weevil for the Major Aggregation Pheromone Compound Ferrugineol

    Directory of Open Access Journals (Sweden)

    Binu Antony

    2018-03-01

    Full Text Available In insects, perception of the environment—food, mates, and prey—is mainly guided by chemical signals. The dynamic process of signal perception involves transport to odorant receptors (ORs by soluble secretory proteins, odorant binding proteins (OBPs, which form the first stage in the process of olfactory recognition and are analogous to lipocalin family proteins in vertebrates. Although OBPs involved in the transport of pheromones to ORs have been functionally identified in insects, there is to date no report for Coleoptera. Furthermore, there is a lack of information on olfactory perception and the molecular mechanism by which OBPs participate in the transport of aggregation pheromones. We focus on the red palm weevil (RPW Rhynchophorus ferrugineus, the most devastating quarantine pest of palm trees worldwide. In this work, we constructed libraries of all OBPs and selected antenna-specific and highly expressed OBPs for silencing through RNA interference. Aggregation pheromone compounds, 4-methyl-5-nonanol (ferrugineol and 4-methyl-5-nonanone (ferruginone, and a kairomone, ethyl acetate, were then sequentially presented to individual RPWs. The results showed that antenna-specific RferOBP1768 aids in the capture and transport of ferrugineol to ORs. Silencing of RferOBP1768, which is responsible for pheromone binding, significantly disrupted pheromone communication. Study of odorant perception in palm weevil is important because the availability of literature regarding the nature and role of olfactory signaling in this insect may reveal likely candidates representative of animal olfaction and, more generally, of molecular recognition. Knowledge of OBPs recognizing the specific pheromone ferrugineol will allow for designing biosensors for the detection of this key compound in weevil monitoring in date palm fields.

  18. The thermodynamics of protein aggregation reactions may underpin the enhanced metabolic efficiency associated with heterosis, some balancing selection, and the evolution of ploidy levels.

    Science.gov (United States)

    Ginn, B R

    2017-07-01

    Identifying the physical basis of heterosis (or "hybrid vigor") has remained elusive despite over a hundred years of research on the subject. The three main theories of heterosis are dominance theory, overdominance theory, and epistasis theory. Kacser and Burns (1981) identified the molecular basis of dominance, which has greatly enhanced our understanding of its importance to heterosis. This paper aims to explain how overdominance, and some features of epistasis, can similarly emerge from the molecular dynamics of proteins. Possessing multiple alleles at a gene locus results in the synthesis of different allozymes at reduced concentrations. This in turn reduces the rate at which each allozyme forms soluble oligomers, which are toxic and must be degraded, because allozymes co-aggregate at low efficiencies. The model developed in this paper can explain how heterozygosity impacts the metabolic efficiency of an organism. It can also explain why the viabilities of some inbred lines seem to decline rapidly at high inbreeding coefficients (F > 0.5), which may provide a physical basis for truncation selection for heterozygosity. Finally, the model has implications for the ploidy level of organisms. It can explain why polyploids are frequently found in environments where severe physical stresses promote the formation of soluble oligomers. The model can also explain why complex organisms, which need to synthesize aggregation-prone proteins that contain intrinsically unstructured regions (IURs) and multiple domains because they facilitate complex protein interaction networks (PINs), tend to be diploid while haploidy tends to be restricted to relatively simple organisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Silencing the Odorant Binding ProteinRferOBP1768Reduces the Strong Preference of Palm Weevil for the Major Aggregation Pheromone Compound Ferrugineol.

    Science.gov (United States)

    Antony, Binu; Johny, Jibin; Aldosari, Saleh A

    2018-01-01

    In insects, perception of the environment-food, mates, and prey-is mainly guided by chemical signals. The dynamic process of signal perception involves transport to odorant receptors (ORs) by soluble secretory proteins, odorant binding proteins (OBPs), which form the first stage in the process of olfactory recognition and are analogous to lipocalin family proteins in vertebrates. Although OBPs involved in the transport of pheromones to ORs have been functionally identified in insects, there is to date no report for Coleoptera. Furthermore, there is a lack of information on olfactory perception and the molecular mechanism by which OBPs participate in the transport of aggregation pheromones. We focus on the red palm weevil (RPW) Rhynchophorus ferrugineus , the most devastating quarantine pest of palm trees worldwide. In this work, we constructed libraries of all OBPs and selected antenna-specific and highly expressed OBPs for silencing through RNA interference. Aggregation pheromone compounds, 4-methyl-5-nonanol (ferrugineol) and 4-methyl-5-nonanone (ferruginone), and a kairomone, ethyl acetate, were then sequentially presented to individual RPWs. The results showed that antenna-specific RferOBP1768 aids in the capture and transport of ferrugineol to ORs. Silencing of RferOBP1768 , which is responsible for pheromone binding, significantly disrupted pheromone communication. Study of odorant perception in palm weevil is important because the availability of literature regarding the nature and role of olfactory signaling in this insect may reveal likely candidates representative of animal olfaction and, more generally, of molecular recognition. Knowledge of OBPs recognizing the specific pheromone ferrugineol will allow for designing biosensors for the detection of this key compound in weevil monitoring in date palm fields.

  20. Non-immunospecific association of immunoglobulin G with chromatin during elution from protein A inflates host contamination, aggregate content, and antibody loss.

    Science.gov (United States)

    Gagnon, Pete; Nian, Rui; Yang, Yuansheng; Yang, Qiaohui; Lim, Chiew Ling

    2015-08-21

    Monoclonal IgG at pH 3.5 expressed a tendency to self-associate and associate non-specifically with surfaces, including the surfaces of precipitated chromatin heteroaggregates. The tendency was elevated with protein A-eluted IgG still in elution buffer (100mM acetate, pH 3.5). Association of IgG with chromatin elements under protein A elution conditions amplified host protein contamination of the elution fraction about 15-fold, caused formation of aggregates that persisted after pH neutralization, and imposed an approximate 5% loss on IgG recovery. Neutralization released eluted IgG from its low pH associations with chromatin and caused heteroaggregate remnants to associate into large particles easily removed by microfiltration. Most effective host contaminant clearance was achieved by filtration after neutralization to pH 5.5. All chromatin-mediated liabilities were suspended by extraction of chromatin heteroaggregates in advance of protein A. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. HSV usurps eukaryotic initiation factor 3 subunit M for viral protein translation: novel prevention target.

    Directory of Open Access Journals (Sweden)

    Natalia Cheshenko

    2010-07-01

    Full Text Available Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by approximately 90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m. Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease.

  2. Effective Rho-associated protein kinase inhibitor treatment to dissociate human iPS cells for suspension culture to form embryoid body-like cell aggregates.

    Science.gov (United States)

    Horiguchi, Ayumi; Yazaki, Koyuki; Aoyagi, Mami; Ohnuki, Yoshitsugu; Kurosawa, Hiroshi

    2014-11-01

    Treatment conditions using Y-27632 in the preparation of cell suspension of dissociated human pluripotent stem cells (hiPSCs) were investigated in the context of embryoid body (EB)-like cell aggregates. The effectiveness of a pretreatment with Y-27632 before cell dissociation and that of a Y-27632 treatment during cell dissociation were investigated from the viewpoint of simplicity and robustness. The duration of Y-27632 treatment in the preparation process affected the circularity and agglomeration of dissociated hiPSCs. A single application of pretreatment failed to prevent the onset of blebbing. However, a pretreatment promoted the agglomeration of dissociated hiPSCs when combined with the addition of Y-27632 to cell suspension. Our results indicate that pretreatment enhances the agglomeration potential of dissociated hiPSCs. When cell dissociation was performed in the presence of Y-27632, dissociated hiPSCs possessed the highest circularity and significant agglomerating property. It was shown that treatment with Y-27632 during cell dissociation is a simple and robust method to prepare dissociated hiPSCs for suspension culture to form EB-like cell aggregates. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates

    NARCIS (Netherlands)

    Teuling, Eva; Ahmed, Suaad; Haasdijk, Elize; Demmers, Jeroen; Steinmetz, Michel O; Akhmanova, Anna; Jaarsma, Dick; Hoogenraad, Casper C

    2007-01-01

    The vesicle-associated membrane protein-associated proteins (VAPs) VAPA and VAPB interact with lipid-binding proteins carrying a short motif containing two phenylalanines in an acidic tract (FFAT motif) and targets them to the cytosolic surface of the endoplasmic reticulum (ER). A genetic mutation

  4. Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner.

    Science.gov (United States)

    Polanco, Juan Carlos; Scicluna, Benjamin James; Hill, Andrew Francis; Götz, Jürgen

    2016-06-10

    The microtubule-associated protein tau has a critical role in Alzheimer disease and related tauopathies. There is accumulating evidence that tau aggregates spread and replicate in a prion-like manner, with the uptake of pathological tau seeds causing misfolding and aggregation of monomeric tau in recipient cells. Here we focused on small extracellular vesicles enriched for exosomes that were isolated from the brains of tau transgenic rTg4510 and control mice. We found that these extracellular vesicles contained tau, although the levels were significantly higher in transgenic mice that have a pronounced tau pathology. Tau in the vesicles was differentially phosphorylated, although to a lower degree than in the brain cells from which they were derived. Several phospho-epitopes (AT8, AT100, and AT180) thought to be critical for tau pathology were undetected in extracellular vesicles. Despite this, when assayed with FRET tau biosensor cells, extracellular vesicles derived from transgenic mice were capable of seeding tau aggregation in a threshold-dependent manner. We also observed that the dye used to label extracellular vesicle membranes was still present during nucleation and formation of tau inclusions, suggesting either a role for membranes in the seeding or in the process of degradation. Together, we clearly demonstrate that extracellular vesicles can transmit tau pathology. This indicates a role for extracellular vesicles in the transmission and spreading of tau pathology. The characteristics of tau in extracellular vesicles and the seeding threshold we identified may explain why tau pathology develops very slowly in neurodegenerative diseases such as Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells.

    Science.gov (United States)

    Tsunezawa, W; Sano, H; Sohma, H; McCormack, F X; Voelker, D R; Kuroki, Y

    1998-09-08

    Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.

  6. Novel aspects of platelet aggregation

    Directory of Open Access Journals (Sweden)

    Roka-Moya Y. M.

    2014-01-01

    Full Text Available The platelet aggregation is an important process, which is critical for the hemostatic plug formation and thrombosis. Recent studies have shown that the platelet aggregation is more complex and dynamic than it was previously thought. There are several mechanisms that can initiate the platelet aggregation and each of them operates under specific conditions in vivo. At the same time, the influence of certain plasma proteins on this process should be considered. This review intends to summarize the recent data concerning the adhesive molecules and their receptors, which provide the platelet aggregation under different conditions.

  7. 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.

  8. Physiochemical, texture properties, and the microstructure of set yogurt using whey protein-sodium tripolyphosphate aggregates as thickening agents.

    Science.gov (United States)

    Cheng, Jianjun; Xie, Siyu; Yin, Yuan; Feng, Xianmin; Wang, Shuai; Guo, Mingruo; Ni, Chunlei

    2017-07-01

    Polymerized whey protein-sodium tripolyphosphate can be induced to gel in an acidic environment provided during fermentation. The variety of thickening agent has an influence on texture that is an essential aspect of yogurt quality affecting consumer preference. Similar to polysaccharide stabilizers, the cold gelation properties of whey proteins can improve the body texture of yogurt products. Polymerized whey protein-sodium tripolyphosphate could be a favorable and interesting thickening agent for making set yogurt. The effects of whey protein isolate (WPI), heat-treated whey protein-sodium tripolyphosphate (WPI-STPP), heat-treated WPI and pectin on the storage properties and microstructure of yogurt were investigated. All samples were analyzed for syneresis, pH, titratable acidity, viscosity, texture profile and microstructure during storage. The results showed that incorporating heat-treated WPI-STPP had a significant impact on syneresis (32.22 ± 0.60), viscosity (10 956.67 ± 962.1) and hardness (209.24 ± 12.48) (p Yogurt fermented with modified WPI-STPP had higher levels of protein and better hardness compared with yogurt using pectin. The microstructure was observed to be a uniform and denser, complicated network. Heat-treated WPI-STPP may be useful for improving yogurt texture. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  9. A novel tetravalent formulation combining the four aggregated domain III-capsid proteins from dengue viruses induces a functional immune response in mice and monkeys.

    Science.gov (United States)

    Suzarte, Edith; Gil, Lázaro; Valdés, Iris; Marcos, Ernesto; Lazo, Laura; Izquierdo, Alienys; García, Angélica; López, Lázaro; Álvarez, Maylin; Pérez, Yusleydis; Castro, Jorge; Romero, Yaremis; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset

    2015-08-01

    Our group developed a subunit vaccine candidate against dengue virus based on two different viral regions: the domain III of the envelope protein and the capsid protein. The novel chimeric protein from dengue-2 virus [domain III-capsid (DIIIC-2)], when presented as aggregated incorporating oligodeoxynucleotides, induced anti-viral and neutralizing antibodies, a cellular immune response and conferred significant protection to mice and monkeys. The remaining constructs were already obtained and properly characterized. Based on this evidence, this work was aimed at assessing the immune response in mice of the chimeric proteins DIIIC of each serotype, as monovalent and tetravalent formulations. Here, we demonstrated the immunogenicity of each protein in terms of humoral and cell-mediated immunity, without antigen competition on the mixture forming the formulation tetra DIIIC. Accordingly, significant protection was afforded as measured by the limited viral load in the mouse encephalitis model. The assessment of the tetravalent formulation in non-human primates was also conducted. In this animal model, it was demonstrated that the formulation induced neutralizing antibodies and memory cell-mediated immune response with IFN-γ-secreting and cytotoxic capacity, regardless the route of immunization used. Taken together, we can assert that the tetravalent formulation of DIIIC proteins constitutes a promising vaccine candidate against dengue virus, and propose it for further efficacy experiments in monkeys or in the dengue human infection model, as it has been recently proposed. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Mfa4, an Accessory Protein of Mfa1 Fimbriae, Modulates Fimbrial Biogenesis, Cell Auto-Aggregation, and Biofilm Formation in Porphyromonas gingivalis.

    Science.gov (United States)

    Ikai, Ryota; Hasegawa, Yoshiaki; Izumigawa, Masashi; Nagano, Keiji; Yoshida, Yasuo; Kitai, Noriyuki; Lamont, Richard J; Yoshimura, Fuminobu; Murakami, Yukitaka

    2015-01-01

    Porphyromonas gingivalis, a gram-negative obligate anaerobic bacterium, is considered to be a key pathogen in periodontal disease. The bacterium expresses Mfa1 fimbriae, which are composed of polymers of Mfa1. The minor accessory components Mfa3, Mfa4, and Mfa5 are incorporated into these fimbriae. In this study, we characterized Mfa4 using genetically modified strains. Deficiency in the mfa4 gene decreased, but did not eliminate, expression of Mfa1 fimbriae. However, Mfa3 and Mfa5 were not incorporated because of defects in posttranslational processing and leakage into the culture supernatant, respectively. Furthermore, the mfa4-deficient mutant had an increased tendency to auto-aggregate and form biofilms, reminiscent of a mutant completely lacking Mfa1. Notably, complementation of mfa4 restored expression of structurally intact and functional Mfa1 fimbriae. Taken together, these results indicate that the accessory proteins Mfa3, Mfa4, and Mfa5 are necessary for assembly of Mfa1 fimbriae and regulation of auto-aggregation and biofilm formation of P. gingivalis. In addition, we found that Mfa3 and Mfa4 are processed to maturity by the same RgpA/B protease that processes Mfa1 subunits prior to polymerization.

  11. The role of tears in preventing protein deposition on contact lenses

    NARCIS (Netherlands)

    Boot, N.; Kok, J.; Kijlstra, A.

    1989-01-01

    Recently the presence of a coating inhibitory factor was described in human tears which can prevent the binding of proteins to a solid phase. In these earlier studies depositions of lactoferrin and IgG onto plastic was studied. In the study described here, peroxidase conjugated albumin was used as a

  12. Preventing protein adsorption from a range of surfaces using an aqueous fish protein extract

    DEFF Research Database (Denmark)

    Pillai, Saju; Arpanaei, Ayyoob; Meyer, Rikke L.

    2009-01-01

    We utilize an aqueous extract of fish proteins (FPs) as a coating for minimizing the adsorption of fibrinogen (Fg) and human serum albumin (HSA). The surfaces include stainless steel (SS), gold (Au), silicon dioxide (SiO2), and poly(styrene) (PS). The adsorption processes (kinetics and adsorbed...

  13. Surface aggregation of urinary proteins and aspartic acid-rich peptides on the faces of calcium oxalate monohydrate investigated by in situ force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, M L; Qiu, S R; Hoyer, J R; Casey, W H; Nancollas, G H; De Yoreo, J J

    2008-05-28

    The growth of calcium oxalate monohydrate in the presence of Tamm-Horsfall protein (THP), osteopontin (OPN), and the 27-residue synthetic peptides (DDDS){sub 6}DDD and (DDDG){sub 6}DDD [where D = aspartic acid and X = S (serine) or G (glycine)] was investigated via in situ atomic force microscopy (AFM). The results show that these three growth modulators create extensive deposits on the crystal faces. Depending on the modulator and crystal face, these deposits can occur as discrete aggregates, filamentary structures, or uniform coatings. These proteinaceous films can lead to either the inhibition or increase of the step speeds (with respect to the impurity-free system) depending on a range of factors that include peptide or protein concentration, supersaturation and ionic strength. While THP and the linear peptides act, respectively, to exclusively increase and inhibit growth on the (-101) face, both exhibit dual functionality on the (010) face, inhibiting growth at low supersaturation or high modulator concentration and accelerating growth at high supersaturation or low modulator concentration. Based on analyses of growth morphologies and dependencies of step speeds on supersaturation and protein or peptide concentration, we argue for a picture of growth modulation that accounts for the observations in terms of the strength of binding to the surfaces and steps and the interplay of electrostatic and solvent-induced forces at crystal surface.

  14. Variant size- and glycoforms of the scavenger receptor cysteine-rich protein gp-340 with differential bacterial aggregation

    DEFF Research Database (Denmark)

    Eriksson, Christer; Frängsmyr, Lars; Danielsson Niemi, Liza

    2007-01-01

    bands. Purified I to IV proteins all revealed a N-terminal sequence TGGWIP upon Edman degradation. Moreover, purified gp-340 from the seven donors and lung gp-340 shared N-glycans, sialylated Galbeta1-3GalNAc and (poly)lactosamine structures. However, the larger size gp-340 grouping II/III (n = 4...

  15. Studies of the aggregation of RNase Sa

    DEFF Research Database (Denmark)

    Khasa, Harshit; Kramer, Ryan; Maddux, Nathan

    2014-01-01

    of the mutants, little correlation was seen between the time at which aggregation is initiated or the rate of aggregation and the thermal sensitivity of the mutants. It is hypothesized that the nature of contacts between protein molecules in the associated (aggregated) phase rather than structural changes...

  16. Congo red, an amyloid-inhibiting compound, alleviates various types of cellular dysfunction triggered by mutant protein kinase cγ that causes spinocerebellar ataxia type 14 (SCA14) by inhibiting oligomerization and aggregation.

    Science.gov (United States)

    Seki, Takahiro; Takahashi, Hideyuki; Yamamoto, Kazuhiro; Ogawa, Kota; Onji, Tomoya; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2010-01-01

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation that induces apoptotic cell death. Congo red is widely used as a histological dye for amyloid detection. Recent evidence has revealed that Congo red has the property to inhibit amyloid oligomers and fibril formation of misfolded proteins. In the present study, we examine whether Congo red inhibits aggregate formation and cytotoxicity of mutant γPKC. Congo red likely inhibits aggregate formation of mutant γPKC – green fluorescent protein (GFP) without affecting its expression level in SH-SY5Y cells. Congo red counteracts the insolubilization of recombinant mutant γPKC, suggesting that the dye inhibits aggregation of mutant γPKC by a direct mechanism. Congo red also inhibits aggregation and oligomerization of mutant γPKC-GFP in primary cultured cerebellar Purkinje cells. Moreover, the dye reverses the improper development of dendrites and inhibits apoptotic cell death in Purkinje cells that express mutant γPKC-GFP. These results indicate that amyloid-inhibiting compounds like Congo red may be novel therapeutics for SCA14.

  17. Identification and further characterization of the specific cell binding fragment from sponge aggregation factor

    OpenAIRE

    1986-01-01

    Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from t...

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

    OpenAIRE

    Lund, Mikael; Jönsson, Bo

    2003-01-01

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

  19. Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids

    Czech Academy of Sciences Publication Activity Database

    Olejár, Tomáš; Pajuelo-Reguera, David; Alán, Lukáš; Dlasková, Andrea; Ježek, Petr

    2015-01-01

    Roč. 12, č. 4 (2015), s. 5185-5190 ISSN 1791-2997 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : mitochondrial nucleoid * single- strand ed DNA -binding protein * photoconvertible fluorescent protein Eos Subject RIV: EA - Cell Biology Impact factor: 1.559, year: 2015

  20. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    International Nuclear Information System (INIS)

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-01-01

    Highlights: → Depletion of cellular PABP level arrests mRNA translation in HeLa cells. → PABP knock down leads to apoptotic cell death. → PABP depletion does not affect transcription. → PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  1. In vitro inhibition of platelet aggregation by peptides derived from oat (Avena sativa L.), highland barley (Hordeum vulgare Linn. var. nudum Hook. f.), and buckwheat (Fagopyrum esculentum Moench) proteins.

    Science.gov (United States)

    Yu, Guoyong; Wang, Feng; Zhang, Bolin; Fan, Junfeng

    2016-03-01

    Bioactive compounds present in foods could have beneficial effects on human health. In this study, we report the capacity of peptides released from oat, highland barley, and buckwheat proteins after enzymatic digestion to inhibit platelet aggregation in vitro. All hydrolysates showed high antiplatelet activity, with IC50 values of 0.282mg/ml (oat flour gastrointestinal hydrolysate, 6h) to 2.496mg/ml (highland barley glutelin tryptic hydrolysate, 14h) in a dose-dependent manner. Thirty-eight peptides with more than seven residues were identified in the tryptic hydrolysates of oat globulin. Results of computational modeling revealed that nine peptides, including ALPIDVLANAYR, EFLLAGNNKR, GEEFGAFTPK, QLAQIPR, LQAFEPLR, ALPVDVLANAYR, GEEFDAFTPK, QKEFLLAGNNK, and TNPNSMVSHIAGK bound the cyclooxygenase-1 active centers with low binding energy (-6.5 to -7.5kcal/mol). This is the first report to identify antiplatelet peptides from grain hydrolysates and the binding modes at the molecular level, leading to their possible use as functional food ingredients to prevent thrombosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Study of conformational changes and protein aggregation of bovine serum albumin in presence of Sb(III) and Sb(V).

    Science.gov (United States)

    Verdugo, Marcelo; Ruiz Encinar, Jorge; Costa-Fernández, José Manuel; Menendez-Miranda, Mario; Bouzas-Ramos, Diego; Bravo, Manuel; Quiroz, Waldo

    2017-01-01

    Antimony is a metalloid that affects biological functions in humans due to a mechanism still not understood. There is no doubt that the toxicity and physicochemical properties of Sb are strongly related with its chemical state. In this paper, the interaction between Sb(III) and Sb(V) with bovine serum albumin (BSA) was investigated in vitro by fluorescence spectroscopy, and circular dichroism (CD) under simulated physiological conditions. Moreover, the coupling of the separation technique, asymmetric flow field-flow fractionation, with elemental mass spectrometry to understand the interaction of Sb(V) and Sb(III) with the BSA was also used. Our results showed a different behaviour of Sb(III) vs. Sb(V) regarding their effects on the interaction with the BSA. The effects in terms of protein aggregates and conformational changes were higher in the presence of Sb(III) compared to Sb(V) which may explain the differences in toxicity between both Sb species in vivo. Obtained results demonstrated the protective effect of GSH that modifies the degree of interaction between the Sb species with BSA. Interestingly, in our experiments it was possible to detect an interaction between BSA and Sb species, which may be related with the presence of labile complex between the Sb and a protein for the first time.

  3. Generation of Soluble Advanced Glycation End Products Receptor (sRAGE)-Binding Ligands during Extensive Heat Treatment of Whey Protein/Lactose Mixtures Is Dependent on Glycation and Aggregation

    NARCIS (Netherlands)

    Liu, Fahui; Teodorowicz, Gosia; Wichers, Harry J.; Boekel, van Tiny; Hettinga, Kasper A.

    2016-01-01

    Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE

  4. Functions and possible provenance of primordial proteins--Part II: microorganism aggregation in clouds triggered by climate change.

    Science.gov (United States)

    Sommer, Andrei P; Wickramasinghe, N Chandra

    2005-01-01

    Current models predict that the elevation of the Earth's surface temperature due to global warming is accompanied by a warming of the troposphere, and a thickening cloud cover associated with longer-lasting clouds, in particular over land. These effects can have an instant impact on the vitality level of microorganisms in clouds and the spreading of airborne diseases. Microorganisms could originate from locations on the Earth, or even arrive from space. Primordial proteins in nanobacteria, only recently identified in the atmosphere, could play a significant role in clouds--accelerating the formation of cloud droplets and interconnecting nanobacteria (and possibly nanobacteria and other microorganisms), thus enhancing their chances to eventually reach the Earth.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    T.J.C. Neiva

    1997-05-01

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

  7. Targeting the AMP-activated protein kinase for cancer prevention and therapy

    Directory of Open Access Journals (Sweden)

    InYoung eKim

    2013-07-01

    Full Text Available Despite the advances in biomedical research and clinical applications, cancer remains a leading cause of death worldwide. Given the limitations of conventional chemotherapeutics, including serious toxicities and reduced quality of life for patients, the development of safe and efficacious alternatives with known mechanism of action is much needed. Prevention of cancer through dietary intervention may hold promise and has been investigated extensively in the recent years. AMP-activated protein kinase (AMPK is an energy sensor that plays a key role in the regulation of protein and lipid metabolism in response to changes in fuel availability. When activated, AMPK promotes energy-producing catabolic pathways while inhibiting anabolic pathways, such as cell growth and proliferation—thereby antagonizing carcinogenesis. Other anti-cancer effects of AMPK may include promoting autophagy and DNA repair upon UVB damage. In the last decade, interest in AMPK has grown extensively as it emerged as an attractive target molecule for cancer prevention and treatment. Among the latest developments is the activation of AMPK by naturally-occurring dietary constituents and plant products—termed phytochemicals. Owing to their efficacy and safety, phytochemicals are considered as an alternative to the conventional harmful chemotherapy. The rising popularity of using phytochemicals for cancer prevention and therapy is supported by a substantial progress in identifying the molecular pathways involved, including AMPK. In this article, we review the recent progress in this budding field that suggests AMPK as a new molecular target in the prevention and treatment of cancer by phytochemicals.

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

    Science.gov (United States)

    Estrada, L D; Soto, C

    2007-01-01

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

  9. Ascorbic acid inhibits human insulin aggregation and protects against amyloid induced cytotoxicity.

    Science.gov (United States)

    Alam, Parvez; Beg, Ayesha Zainab; Siddiqi, Mohammad Khursheed; Chaturvedi, Sumit Kumar; Rajpoot, Ravi Kant; Ajmal, Mohd Rehan; Zaman, Masihuz; Abdelhameed, Ali S; Khan, Rizwan Hasan

    2017-05-01

    Protein aggregation into oligomers and fibrils are associated with many human pathophysiologies. Compounds that modulate protein aggregation and interact with preformed fibrils and convert them to less toxic species, expect to serve as promising drug candidates and aid to the drug development efforts against aggregation diseases. In present study, the kinetics of amyloid fibril formation by human insulin (HI) and the anti-amyloidogenic activity of ascorbic acid (AA) were investigated by employing various spectroscopic, imaging and computational approaches. We demonstrate that ascorbic acid significantly inhibits the fibrillation of HI in a dose-dependent manner. Interestingly ascorbic acid destabilise the preformed amyloid fibrils and protects human neuroblastoma cell line (SH- SY5Y) against amyloid induced cytotoxicity. The present data signifies the role of ascorbic acid that can serve as potential molecule in preventing human insulin aggregation and associated pathophysiologies. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    Science.gov (United States)

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  12. Intricacies of alpha-synuclein aggregation

    NARCIS (Netherlands)

    Mucibabic, M.

    2016-01-01

    Parkinson’s disease is a neurodegenerative disease characterized by the presence of abnormal deposits of aggregated proteins in the brain tissue, known as Lewy bodies. The major components of Lewy bodies are aggregated forms of a small presynaptic protein known as α-synuclein (α-syn). In this thesis

  13. Prevention

    Science.gov (United States)

    ... Error processing SSI file About Heart Disease & Stroke Prevention Heart disease and stroke are an epidemic in ... secondhand smoke. Barriers to Effective Heart Disease & Stroke Prevention Many people with key risk factors for heart ...

  14. CONTAMINATION OF ANTI-VEGF DRUGS FOR INTRAVITREAL INJECTION: How Do Repackaging and Newly Developed Syringes Affect the Amount of Silicone Oil Droplets and Protein Aggregates?

    Science.gov (United States)

    Schargus, Marc; Werner, Benjamin P; Geerling, Gerd; Winter, Gerhard

    2017-08-21

    The particle counts and the nature of particles of three different antivascular endothelial growth factor agents (VEGF) in different containers in a laboratory setting were compared. Original prefilled ranibizumab glass syringes, original vials with aflibercept, and repacked ready-to-use plastic syringes with bevacizumab from a compounding pharmacy and a compounding company (CC) were analyzed. Particle counts and size distributions were quantified by different particle characterization methods (nephelometry, light obscuration, Micro-Flow Imaging, nanotracking analysis, resonant mass measurement). Using high-performance size-exclusion chromatography (HP-SEC), levels of protein drug monomer and soluble aggregates were determined. Nearly all samples showed similar product quality. Light obscuration and Micro-Flow Imaging showed a 4-fold to 9-fold higher total particle count in compounding company bevacizumab (other samples up to 42,000 particles/mL). Nanotracking analysis revealed highest values for compounding company bevacizumab (6,375 million particles/mL). All containers showed similar amounts of silicone oil microdroplets. Ranibizumab showed lowest particle count of all tested agents with only one monomer peak in HP-SEC. Repackaged bevacizumab from different suppliers showed varying product quality. All three tested agents are available in similar quality regarding particulate purity and silicone oil microdroplet count. Repackaging can have a major impact on the quality.

  15. Effects of laser photherapy on bone defects grafted with mineral trioxide aggregate, bone morphogenetic proteins, and guided bone regeneration: a Raman spectroscopic study.

    Science.gov (United States)

    Pinheiro, Antonio L B; Aciole, Gilberth T S; Cangussú, Maria Cristina T; Pacheco, Marcos T T; Silveira, Landulfo

    2010-12-15

    We have used Raman analysis to assess bone healing on different models. Benefits on the isolated or combined use of mineral trioxide aggregate, bone morphogenetic proteins, guided bone regeneration and laser on bone repair have been reported, but not their combination. We studied peaks of hydroxyapatite and CH groups on defects grafted with MTA, treated or not with laser, BMPs, and GBR. Ninety rats were divided in 10 groups each, subdivided into three subgroups. Laser (λ850 nm) was applied at every other day for 2 weeks. Raman readings were taken at the surface of the defect. Statistical analysis (CHA) showed significant differences between all groups (p = 0.001) and between Group II and all other (p bone is because of increased secretion of calcium hydroxyapatite (CHA) that is indicative of greater calcification and resistance of the bone. We conclude that the association of the MTA with laser phototherapy (LPT) and/or not with GBR resulted in a better bone repair. The use of the MTA associated to IR LPT resulted in a more advanced and quality bone repair. Copyright © 2010 Wiley Periodicals, Inc.

  16. Phage shock proteins B and C prevent lethal cytoplasmic membrane permeability in Yersinia enterocolitica.

    Science.gov (United States)

    Horstman, N Kaye; Darwin, Andrew J

    2012-08-01

    The bacterial phage shock protein (Psp) stress response system is activated by events affecting the cytoplasmic membrane. In response, Psp protein levels increase, including PspA, which has been implicated as the master effector of stress tolerance. Yersinia enterocolitica and related bacteria with a defective Psp system are highly sensitive to the mislocalization of pore-forming secretin proteins. However, why secretins are toxic to psp null strains, whereas some other Psp inducers are not, has not been explained. Furthermore, previous work has led to the confounding and disputable suggestion that PspA is not involved in mitigating secretin toxicity. Here we have established a correlation between the amount of secretin toxicity in a psp null strain and the extent of cytoplasmic membrane permeability to large molecules. This leads to a morphological change resembling cells undergoing plasmolysis. Furthermore, using novel strains with dis-regulated Psp proteins has allowed us to obtain unequivocal evidence that PspA is not required for secretin-stress tolerance. Together, our data suggest that the mechanism by which secretin multimers kill psp null cells is by causing a profound defect in the cytoplasmic membrane permeability barrier. This allows lethal molecular exchange with the environment, which the PspB and PspC proteins can prevent. © 2012 Blackwell Publishing Ltd.

  17. Wheel running from a juvenile age delays onset of specific motor deficits but does not alter protein aggregate density in a mouse model of Huntington's disease

    Directory of Open Access Journals (Sweden)

    Spires Tara L

    2008-04-01

    Full Text Available Abstract Background Huntington's disease (HD is a neurodegenerative disorder predominantly affecting the cerebral cortex and striatum. Transgenic mice (R6/1 line, expressing a CAG repeat encoding an expanded polyglutamine tract in the N-terminus of the huntingtin protein, closely model HD. We have previously shown that environmental enrichment of these HD mice delays the onset of motor deficits. Furthermore, wheel running initiated in adulthood ameliorates the rear-paw clasping motor sign, but not an accelerating rotarod deficit. Results We have now examined the effects of enhanced physical activity via wheel running, commenced at a juvenile age (4 weeks, with respect to the onset of various behavioral deficits and their neuropathological correlates in R6/1 HD mice. HD mice housed post-weaning with running wheels only, to enhance voluntary physical exercise, have delayed onset of a motor co-ordination deficit on the static horizontal rod, as well as rear-paw clasping, although the accelerating rotarod deficit remains unaffected. Both wheel running and environmental enrichment rescued HD-induced abnormal habituation of locomotor activity and exploratory behavior in the open field. We have found that neither environment enrichment nor wheel running ameliorates the shrinkage of the striatum and anterior cingulate cortex (ACC in HD mice, nor the overall decrease in brain weight, measured at 9 months of age. At this age, the density of ubiquitinated protein aggregates in the striatum and ACC is also not significantly ameliorated by environmental enrichment or wheel running. Conclusion These results indicate that enhanced voluntary physical activity, commenced at an early presymptomatic stage, contributes to the positive effects of environmental enrichment. However, sensory and cognitive stimulation, as well as motor stimulation not associated with running, may constitute major components of the therapeutic benefits associated with enrichment

  18. Reversal by desferrioxamine of tau protein aggregates following two days of treatment in aluminum-induced neurofibrillary degeneration in rabbit: implications for clinical trials in Alzheimer's disease.

    Science.gov (United States)

    Savory, J; Huang, Y; Wills, M R; Herman, M M

    1998-04-01

    A clinical trial in patients with Alzheimer's disease has indicated that frequent intramuscular (i.m.) treatment with desferrioxamine (DFO) slows progression of the disease. Confirmatory trials have not been carried out, partly because of the rigors of twice daily intramuscular injections over a period of 2 years, even though the initial report gave promising results. The aim of the present study was to determine an optimal DFO treatment protocol in an animal model exhibiting Alzheimer's-like intraneuronal protein aggregates, previously shown to be partially reversed by such treatment. New Zealand white rabbits were injected intracisternally with either aluminum (Al) maltolate or with saline on day 0. Intramuscular injections of DFO were given to selected rabbits for 2 days prior to sacrifice on days 4, 6 or 8. Bielschowsky's silver impregnation demonstrated widespread neurofibrillary degeneration (NFD) in neuronal cell bodies and neurites of brain and spinal cord from Al-treated rabbits. Monoclonal antibodies Tau-2, AT8, PHF-1 and Alz-50, all of which characteristically stain neurofibrillary tangles associated with Alzheimer's disease, strongly labeled the Al-induced NFD. The number of positive neurons and staining intensities were much less in rabbits treated with Al and subsequently with DFO, than in animals only given Al. Control rabbit receiving intracisternal saline were negative for NFD. The results of quantitative immunohistochemistry using image analysis confirmed that immunostaining densities with all tau mAbs were higher in Al-treated than in Al-DFO-treated or in saline-treated controls. Furthermore, it appears that hyperphosphorylation of tau does not make this protein resistant to degradation once Al has been removed by DFO treatment. The effectiveness of only two days of DFO treatment in reversing Al-induced neurofibrillary degeneration suggests that further clinical trials of DFO for treatment of Alzheimer's disease should be attempted using much

  19. Application of Minicircle Technology of Self-Reproducing Synthetic Protein Drugs in Preventing Skin Allograft Rejection.

    Science.gov (United States)

    Lim, Sun Woo; Kim, Young Kyun; Park, Narae; Jin, Long; Jin, Jian; Doh, Kyoung Chan; Ju, Ji Hyeon; Yang, Chul Woo

    2015-07-30

    Recently, it has been reported that minicircle vectors could allow the expression of transgenes using the protein synthesis system of the host. Here, we tested a novel strategy to permit the production of synthetic biologics using minicircle technology and evaluated their feasibility as a therapeutic tool in a skin allograft model. We engineered vectors to carry cassette sequences for tocilizumab [anti-soluble interleukin-6 receptor (sIL-6R) antibody] and/or etanercept [tumor necrosis factor receptor 2 (TNFR2)-Fc fusion protein], and then isolated minicircle vectors from the parent vectors. We verified the production of proteins from minicircles and their duration in HEK293T cells and mice. We also evaluated whether these proteins were expressed at levels sufficient to ameliorate skin allograft rejection in mice. Each minicircle transfected into cells was detectable for at least 30 days. In mice, the drugs were mainly expressed in the liver and were detectable for at least 10 days after a single injection. These drugs were also detected in the blood. Treatment of mice with minicircles prolonged skin allograft survival, which was accompanied by a reduction of the number of interferon-γ+ or interleukin-17+ lymphocytes and an induction of forkhead box P3 expression. These findings suggest that blocking of sIL-6R and/or TNF-α using minicircles encoding tocilizumab and/or etanercept was functionally active and relevant for preventing acute allograft rejection. Self-reproducing synthetic protein drugs produced using minicircle technology are potentially powerful tools for preventing acute rejection in transplantation.

  20. Quantitative SUMO-1 modification of a vaccinia virus protein is required for its specific localization and prevents its self-association.

    Science.gov (United States)

    Palacios, Silvia; Perez, Laurent H; Welsch, Sonja; Schleich, Sibylle; Chmielarska, Katarzyna; Melchior, Frauke; Locker, Jacomine Krijnse

    2005-06-01

    Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.

  1. FTO genotype, dietary protein, and change in appetite: the Preventing Overweight Using Novel Dietary Strategies trial.

    Science.gov (United States)

    Huang, Tao; Qi, Qibin; Li, Yanping; Hu, Frank B; Bray, George A; Sacks, Frank M; Williamson, Donald A; Qi, Lu

    2014-05-01

    A common obesity-risk variant rs9939609 in the fat mass- and obesity-associated (FTO) gene was recently shown to affect appetite, and the gene is sensitive to the regulation of amino acids. We examined the interaction between FTO genotype and protein intake on the long-term changes in appetite in a randomized controlled trial. We genotyped FTO rs9939609 in 737 overweight adults in the 2-y Preventing Overweight Using Novel Dietary Strategies trial and assessed 4 appetite-related traits including cravings, fullness, hunger, and prospective consumption. We showed that dietary protein significantly modified genetic effects on changes in food cravings and appetite scores at 6 mo after adjustment for age, sex, ethnicity, baseline body mass index, weight change, and baseline value for respective outcomes (P-interaction = 0.027 and 0.048, respectively). The A allele was associated with a greater decrease in food cravings and appetite scores in participants with high-protein-diet intake (P = 0.027 and 0.047, respectively) but not in subjects in the low-protein-diet group (P = 0.384 and 0.078, respectively). The weight regain from 6 to 24 mo attenuated gene-protein interactions. Protein intakes did not modify FTO genotype effects on other appetite measures. Our data suggest that individuals with the FTO rs9939609 A allele might obtain more benefits in a reduction of food cravings and appetite by choosing a hypocaloric and higher-protein weight-loss diet. This trial was registered at clinicaltrials.gov as NCT00072995.

  2. Mechanism of cellular response to nanoscale aggregates of small molecules

    Science.gov (United States)

    Kuang, Yi

    This dissertation research focused on the illustration of the molecular mechanism of cellular response to nanoscale aggregates formed by small molecules. There are five chapters in this dissertation. Chapter 1 summarizes the current research on the evaluation of cell response (i.e., biocompatibility/cytotoxicity) to small molecular hydrogelators. Chapter 2 describes an interesting phenomenon that supramolecular hydrogelators consisting of N-terminated dipeptides, which exhibit selective inhibitory effects against cancer cells. This study calls for the development of a new approach for identification of protein targets of the hydrogelators. Chapter 3 describes the evaluation of interactions between cytosol proteins of a mammalian cell line and morphologically different nanoscale molecular aggregates formed by small peptidic molecules. Chapter 4 describes the research on the mechanism of a type of molecular aggregates, which cluster short microtubules to prevent the growth of microtubule. This unprecedented mechanism of "self-assembly to interfere with self-organization " contributes to inhibiting growth of cancer cells in several mammalian cell based assays and a xenograft tumor mice model. At the end, Chapter 5 reports a novel supramolecular hydrogelator, which consists of fluorene and the pentapeptide epitope (TIGYG) of potassium ion (K+) channels, to self-assemble in water to form the tunable, hierarchical nanostructures dictated by the concentration of K+. In conclusion, this dissertation research demonstrates a new approach for investigating cellular target and molecular mechanism of self-assembled aggregates formed by small peptide derivatives based hydrogelators, which will make contribution to the development of supramolecular hydrogelators as biomaterials. Moreover, the differential cytotoxicity of molecular aggregates illustrated in this research promises a new direction for developing anti-cancer drug based on interactions between molecular aggregates and

  3. Use of hydroxyethyl starch for inducing red blood cell aggregation

    NARCIS (Netherlands)

    Henkelman, Sandra; Rakhorst, Gerhard; van der Mei, Henny C.; Busscher, Henk J.

    2012-01-01

    Aggregation of human red blood cells (RBC) remains of biological and clinical interest. Replacement of plasma proteins by polymers to induce RBC aggregation may help to unravel the fundamentals of the aggregation process. Two theories exist to explain RBC aggregation mechanisms: a depletion and a

  4. Preventable and potentially preventable serious adverse reactions induced by oral protein kinase inhibitors through a database of adverse drug reaction reports.

    Science.gov (United States)

    Egron, Adeline; Olivier-Abbal, Pascale; Gouraud, Aurore; Babai, Samy; Combret, Sandrine; Montastruc, Jean-Louis; Bondon-Guitton, Emmanuelle

    2015-06-01

    Antineoplastic drugs are one of the pharmacological classes more frequently involved in occurrence of "serious" adverse drug reactions. However, few epidemiological data are available regarding the preventability of adverse drug reactions with ambulatory cancer chemotherapy. We assessed the rate and characteristics of "preventable" or "potentially preventable" "serious" adverse drug reactions induced by oral protein kinase inhibitors (PKIs). We performed a retrospective study with all "serious" adverse drug reactions (ADRs) recorded from 1 January 2008 to 31 December 2009 in the French Pharmacovigilance Database with the eight oral protein kinase inhibitors marketed in France: sorafenib, imatinib, erlotinib, sunitinib, dasatinib, lapatinib, nilotinib and everolimus (Afinitor®) using the French adverse drug reactions preventability scale. This study was carried out on 265 spontaneous notifications. Most of adverse drug reactions were "unpreventable" (63.8 %). Around one third were "unevaluable" due to notifications poorly documented (medical history, dosage, use of drugs as first or second intention, concomitant drugs). One (0.4 %) adverse drug reaction was "preventable" with dasatinib (subdural hematoma) and three (1.1 %) were "potentially preventable" (hepatic adverse drug reactions): two with imatinib and one with sorafenib. For these four cases, we identified some characteristics: incorrect dosages, drug interactions and off-label uses. An appropriate prescription could avoid the occurrence of 1.5 % "serious" adverse drug reactions with oral PKIs. This rate is low and further studies are needed to compare our results by using other preventability instruments and to improve the French ADRs Preventability Scale.

  5. 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

  6. Meiotic HORMA domain proteins prevent untimely centriole disengagement during Caenorhabditis elegans spermatocyte meiosis.

    Science.gov (United States)

    Schvarzstein, Mara; Pattabiraman, Divya; Bembenek, Joshua N; Villeneuve, Anne M

    2013-03-05

    In many species where oocytes lack centrosomes, sperm contribute both genetic material and centriole(s) to the zygote. Correct centriole organization during male meiosis is critical to guarantee a normal bipolar mitotic spindle in the zygote. During Caenorhabditis elegans male meiosis, centrioles normally undergo two rounds of duplication, resulting in haploid sperm each containing a single tightly engaged centriole pair. Here we identify an unanticipated role for C. elegans HORMA (Hop1/Rev7/Mad2) domain proteins HTP-1/2 and HIM-3 in regulating centriole disengagement during spermatocyte meiosis. In him-3 and htp-1 htp-2 mutants, centrioles separate inappropriately during meiosis II, resulting in spermatids with disengaged centrioles. Moreover, extra centrosomes are detected in a subset of zygotes. Together, these data implicate HIM-3 and HTP-1/2 in preventing centriole disengagement during meiosis II. We showed previously that HTP-1/2 prevents premature loss of sister chromatid cohesion during the meiotic divisions by inhibiting removal of meiotic cohesin complexes containing the REC-8 subunit. Worms lacking REC-8, or expressing a mutant separase protein with elevated local concentration at centrosomes and in sperm, likewise exhibit inappropriate centriole separation during spermatocyte meiosis. These observations are consistent with HIM-3 and HTP-1/2 preventing centriole disengagement by inhibiting separase-dependent cohesin removal. Our data suggest that the same specialized meiotic mechanisms that function to prevent premature release of sister chromatid cohesion during meiosis I in C. elegans also function to inhibit centriole separation at meiosis II, thereby ensuring that the zygote inherits the appropriate complement of chromosomes and centrioles.

  7. Linalool prevents oxidative stress activated protein kinases in single UVB-exposed human skin cells.

    Science.gov (United States)

    Gunaseelan, Srithar; Balupillai, Agilan; Govindasamy, Kanimozhi; Ramasamy, Karthikeyan; Muthusamy, Ganesan; Shanmugam, Mohana; Thangaiyan, Radhiga; Robert, Beaulah Mary; Prasad Nagarajan, Rajendra; Ponniresan, Veeramani Kandan; Rathinaraj, Pierson

    2017-01-01

    Ultraviolet-B radiation (285-320 nm) elicits a number of cellular signaling elements. We investigated the preventive effect of linalool, a natural monoterpene, against UVB-induced oxidative imbalance, activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling in HDFa cells. We observed that linalool treatment (30 μM) prevented acute UVB-irradiation (20 mJ/cm2) mediated loss of activities of antioxidant enzymes in HDFa cells. The comet assay results illustrate that linalool significantly prevents UVB-mediated 8-deoxy guanosine formation (oxidative DNA damage) rather than UVB-induced cyclobutane pyrimidine (CPD) formation. This might be due to its ability to prevent UVB-induced ROS formation and to restore the oxidative imbalance of cells. This has been reflected in UVB-induced overexpression of MAPK and NF-κB signaling. We observed that linalool inhibited UVB-induced phosphorylation of ERK1, JNK and p38 proteins of MAPK family. Linalool inhibited UVB-induced activation of NF-κB/p65 by activating IκBa. We further observed that UVB-induced expression of TNF-α, IL6, IL-10, MMP-2 and MMP-9 was modulated by linalool treatment in HDFa cells. Thus, linalool protects the human skin cells from the oxidative damages of UVB radiation and modulates MAPK and NF-κB signaling in HDFa cells. The present findings substantiate that linalool may act as a photoprotective agent against UVB-induced skin damages.

  8. Stainless steel modified with poly(ethylene glycol) can prevent protein adsorption but not bacterial adhesion

    DEFF Research Database (Denmark)

    Wei, Jiang; Bagge, Dorthe; Gram, Lone

    2003-01-01

    The surface of AISI 316 grade stainless steel (SS) was modified with a layer of poly(ethylene glycol) (PEG) (molecular weight 5000) with the aim of preventing protein adsorption and bacterial adhesion. Model SS substrates were first modified to introduce a very high density of reactive amine groups....... The chemical composition and uniformity of the surfaces were determined using X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) in the imaging mode. The effects of PEI concentration and different substrate pre-cleaning methods on the structure...

  9. Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Downs, S.M. (Marquette Univ., Milwaukee, WI (USA))

    1990-11-01

    The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors (CX, emetine (EM), and puromycin (PUR)) each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.

  10. Systemic Immunization with Papillomavirus L1 Protein Completely Prevents the Development of Viral Mucosal Papillomas

    Science.gov (United States)

    Suzich, Joann A.; Ghim, Shin-Je; Palmer-Hill, Frances J.; White, Wendy I.; Tamura, James K.; Bell, Judith A.; Newsome, Joseph A.; Bennett Jenson, A.; Schlegel, Richard

    1995-12-01

    Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

  11. Structure-Based Design of a Periplasmic Binding Protein Antagonist that Prevents Domain Closure

    Energy Technology Data Exchange (ETDEWEB)

    Borrok, M. Jack; Zhu, Yimin; Forest, Katrina T.; Kiessling, Laura L.; (UW)

    2009-07-31

    Many receptors undergo ligand-induced conformational changes to initiate signal transduction. Periplasmic binding proteins (PBPs) are bacterial receptors that exhibit dramatic conformational changes upon ligand binding. These proteins mediate a wide variety of fundamental processes including transport, chemotaxis, and quorum sensing. Despite the importance of these receptors, no PBP antagonists have been identified and characterized. In this study, we identify 3-O-methyl-D-glucose as an antagonist of glucose/galactose-binding protein and demonstrate that it inhibits glucose chemotaxis in E. coli. Using small-angle X-ray scattering and X-ray crystallography, we show that this antagonist acts as a wedge. It prevents the large-scale domain closure that gives rise to the active signaling state. Guided by these results and the structures of open and closed glucose/galactose-binding protein, we designed and synthesized an antagonist composed of two linked glucose residues. These findings provide a blueprint for the design of new bacterial PBP inhibitors. Given the key role of PBPs in microbial physiology, we anticipate that PBP antagonists will have widespread uses as probes and antimicrobial agents.

  12. Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure.

    Science.gov (United States)

    Figures, W R; Niewiarowski, S; Morinelli, T A; Colman, R F; Colman, R W

    1981-08-10

    Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.

  13. Ethrel-stimulated prolongation of latex flow in the rubber tree (Hevea brasiliensis Muell. Arg.): an Hev b 7-like protein acts as a universal antagonist of rubber particle aggregating factors from lutoids and C-serum.

    Science.gov (United States)

    Shi, Min-Jing; Cai, Fu-Ge; Tian, Wei-Min

    2016-02-01

    Ethrel is the most effective stimuli in prolonging the latex flow that consequently increases yield per tapping. This effect is largely ascribed to the enhanced lutoid stability, which is associated with the decreased release of initiators of rubber particle (RP) aggregation from lutoid bursting. However, the increase in both the bursting index of lutoids and the duration of latex flow after applying ethrel or ethylene gas in high concentrations suggests that a new mechanism needs to be introduced. In this study, a latex allergen Hev b 7-like protein in C-serum was identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS). In vitro analysis showed that the protein acted as a universal antagonist of RP aggregating factors from lutoids and C-serum. Ethrel treatment obviously weakened the effect of C-serum on RP aggregation, which was closely associated with the increase in the level of the Hev b 7-like protein and the decrease in the level of the 37 kDa protein, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting analysis and antibody neutralization. Thus, the increase of the Hev b 7-like protein level or the ratio of the Hev b 7-like protein to the 37 kDa protein in C-serum should be primarily ascribed to the ethrel-stimulated prolongation of latex flow duration. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  14. The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells

    Science.gov (United States)

    Jeong, Youngdan; Yang, Wonkyung; Ko, Hyunjung

    2014-01-01

    Objectives The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period. PMID:25110642

  15. The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells

    Directory of Open Access Journals (Sweden)

    Youngdan Jeong

    2014-08-01

    Full Text Available Objectives The effects of bone morphogenetic protein-2 (BMP-2 and enamel matrix derivative (EMD respectively with mineral trioxide aggregate (MTA on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply, BMP-2 (R&D Systems, EMD (Emdogain, Straumann separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich and Alizarin red (Sigma-Aldrich. The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP, osteocalcin (OCN, osteopontin (OPN and osteonectin (OSN, as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer. Results Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05. Conclusions These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

  16. Light microscopic description of the effects of laser phototherapy on bone defects grafted with mineral trioxide aggregate, bone morphogenetic proteins, and guided bone regeneration in a rodent model.

    Science.gov (United States)

    Pinheiro, Antonio L B; Soares, Luiz G P; Aciole, Gilberth T S; Correia, Neandder A; Barbosa, Artur F S; Ramalho, Luciana M P; Dos Santos, Jean N

    2011-08-01

    We carried out a histological analysis on bone defects grafted with mineral trioxide aggregate (MTA) treated or not with laser, bone morphogenetic protein (BMP), and guided bone regeneration (GBR). Benefits of the use of MTA, laser, BMPs, and GBR on bone repair are well known, but there is no report on their association with laser light. Ninety rats were divided into 10 groups each subdivided into 3. Defects on G II and I were filled with the blood clot. G II was further irradiated with LED. G III and IV were filled with MTA; G IV was further irradiated with laser. G V and VI, the defects filled with MTA and covered with a membrane (GBR). G VI was further irradiated with laser. G VII and VIII, BMPs were added to the MTA and group VIII further irradiated with laser. G IX and X, the MTA + BMP graft was covered with a membrane (GBR). G X was further irradiated with laser. Laser light (λ = 850 nm, 150 mW, 4 J/cm(2) ) was applied over the defect at 48-h intervals and repeated for 15 days. Specimens were processed, cut and stained with H&E and Sirius red and underwent histological analysis. Subjects on group X were irradiated. The results showed different tissue response on all groups during the experimental time. Major changes were seen on irradiated subjects and included marked deposition of new bone in advanced maturation. It is concluded that near infrared laser phototherapy improved the results of the use of the MTA on bone defects. Copyright © 2011 Wiley Periodicals, Inc.

  17. Amphiphilic copolymers reduce aggregation of unfolded lysozyme more effectively than polyethylene glycol

    Science.gov (United States)

    Chin, Jaemin; Mustafi, Devkumar; Poellmann, Michael J.; Lee, Raphael C.

    2017-02-01

    Certain amphiphilic block copolymers are known to prevent aggregation of unfolded proteins. To better understand the mechanism of this effect, the optical properties of heat-denatured and dithiothreitol reduced lysozyme were evaluated with respect to controls using UV-Vis spectroscopy, transmission electron microscopy (TEM) and circular dichroism (CD) measurements. Then, the effects of adding Polyethylene Glycol (8000 Da), the triblock surfactant Poloxamer 188 (P188), and the tetrablock copolymer Tetronic 1107 (T1107) to the lysozyme solution were compared. Overall, T1107 was found to be more effective than P188 in inhibiting aggregation, while PEG exhibited no efficacy. TEM imaging of heat-denatured and reduced lysozymes revealed spherical aggregates with on average 250-450 nm diameter. Using CD, more soluble lysozyme was recovered with T1107 than P188 with β-sheet secondary structure. The greater effectiveness of the larger T1107 in preventing aggregation of unfolded lysozyme than the smaller P188 and PEG points to steric hindrance at play; signifying the importance of size match between the hydrophobic region of denatured protein and that of amphiphilic copolymers. Thus, our results corroborate that certain multi-block copolymers are effective in preventing heat-induced aggregation of reduced lysozymes and future studies warrant more detailed focus on specific applications of these copolymers.

  18. 17-AAG induces cytoplasmic alpha-synuclein aggregate clearance by induction of autophagy.

    Science.gov (United States)

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

    2010-01-18

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

  19. Familial Aggregation of Insomnia.

    Science.gov (United States)

    Jarrin, Denise C; Morin, Charles M; Rochefort, Amélie; Ivers, Hans; Dauvilliers, Yves A; Savard, Josée; LeBlanc, Mélanie; Merette, Chantal

    2017-02-01

    There is little information about familial aggregation of insomnia; however, this type of information is important to (1) improve our understanding of insomnia risk factors and (2) to design more effective treatment and prevention programs. This study aimed to investigate evidence of familial aggregation of insomnia among first-degree relatives of probands with and without insomnia. Cases (n = 134) and controls (n = 145) enrolled in a larger epidemiological study were solicited to invite their first-degree relatives and spouses to complete a standardized sleep/insomnia survey. In total, 371 first-degree relatives (Mage = 51.9 years, SD = 18.0; 34.3% male) and 138 spouses (Mage = 55.5 years, SD = 12.2; 68.1% male) completed the survey assessing the nature, severity, and frequency of sleep disturbances. The dependent variable was insomnia in first-degree relatives and spouses. Familial aggregation was claimed if the risk of insomnia was significantly higher in the exposed (relatives of cases) compared to the unexposed cohort (relatives of controls). The risk of insomnia was also compared between spouses in the exposed (spouses of cases) and unexposed cohort (spouses of controls). The risk of insomnia in exposed and unexposed biological relatives was 18.6% and 10.4%, respectively, yielding a relative risk (RR) of 1.80 (p = .04) after controlling for age and sex. The risk of insomnia in exposed and unexposed spouses was 9.1% and 4.2%, respectively; however, corresponding RR of 2.13 (p = .28) did not differ significantly. Results demonstrate evidence of strong familial aggregation of insomnia. Additional research is warranted to further clarify and disentangle the relative contribution of genetic and environmental factors in insomnia. © Sleep Research Society 2016. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  20. Preventive effect of fermented Maillard reaction products from milk proteins in cardiovascular health.

    Science.gov (United States)

    Oh, N S; Kwon, H S; Lee, H A; Joung, J Y; Lee, J Y; Lee, K B; Shin, Y K; Baick, S C; Park, M R; Kim, Y; Lee, K W; Kim, S H

    2014-01-01

    The aim of this study was to determine the dual effect of Maillard reaction and fermentation on the preventive cardiovascular effects of milk proteins. Maillard reaction products (MRP) were prepared from the reaction between milk proteins, such as whey protein concentrates (WPC) and sodium caseinate (SC), and lactose. The hydrolysates of MRP were obtained from fermentation by lactic acid bacteria (LAB; i.e., Lactobacillus gasseri H10, L. gasseri H11, Lactobacillus fermentum H4, and L. fermentum H9, where human-isolated strains were designated H1 to H15), which had excellent proteolytic and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities (>20%). The antioxidant activity of MRP was greater than that of intact proteins in assays of the reaction with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and trivalent ferric ions; moreover, the effect of MRP was synergistically improved by fermentation. The Maillard reaction dramatically increased the level of antithrombotic activity and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitory effect of milk proteins, but did not change the level of activity for micellar cholesterol solubility. Furthermore, specific biological properties were enhanced by fermentation. Lactobacillus gasseri H11 demonstrated the greatest activity for thrombin and HMGR inhibition in Maillard-reacted WPC, by 42 and 33%, respectively, whereas hydrolysates of Maillard-reacted SC fermented by L. fermentum H9 demonstrated the highest reduction rate for micellar cholesterol solubility, at 52%. In addition, the small compounds that were likely released by fermentation of MRP were identified by size-exclusion chromatography. Therefore, MRP and hydrolysates of fermented MRP could be used to reduce cardiovascular risks. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  1. Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation.

    Science.gov (United States)

    Komaru, Keiichi; Ishida, Yoko; Amaya, Yoshihiro; Goseki-Sone, Masae; Orimo, Hideo; Oda, Kimimitsu

    2005-04-01

    In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).

  2. Recycled aggregates concrete: aggregate and mix properties

    OpenAIRE

    González-Fonteboa, B.; Martínez-Abella, F.

    2005-01-01

    This study of structural concrete made with recycled concrete aggregate focuses on two issues: 1. The characterization of such aggregate on the Spanish market. This involved conducting standard tests to determine density, water absorption, grading, shape, flakiness and hardness. The results obtained show that, despite the considerable differences with respect to density and water absorption between these and natural aggregates, on the whole recycled aggregate is apt for use in concrete produc...

  3. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    Science.gov (United States)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  4. Hydrolyzed whey protein prevents the development of food allergy to β-lactoglobulin in sensitized mice.

    Science.gov (United States)

    Gomes-Santos, Ana Cristina; Fonseca, Roberta Cristelli; Lemos, Luisa; Reis, Daniela Silva; Moreira, Thaís Garcias; Souza, Adna Luciana; Silva, Mauro Ramalho; Silvestre, Marialice Pinto Coelho; Cara, Denise Carmona; Faria, Ana Maria Caetano

    2015-01-01

    Food allergy is an adverse immune response to dietary proteins. Hydrolysates are frequently used for children with milk allergy. However, hydrolysates effects afterwards are poorly studied. The aim of this study was to investigate the immunological consequences of hydrolyzed whey protein in allergic mice. For that, we developed a novel model of food allergy in BALB/c mice sensitized with alum-adsorbed β-lactoglobulin. These mice were orally challenged with either whey protein or whey hydrolysate. Whey-challenged mice had elevated levels of specific IgE and lost weight. They also presented gut inflammation, enhanced levels of SIgA and IL-5 as well as decreased production of IL-4 and IL-10 in the intestinal mucosa. Conversely, mice challenged with hydrolyzate maintained normal levels of IgE, IL-4 and IL-5 and showed no sign of gut inflammation probably due to increased IL-12 production in the gut. Thus, consumption of hydrolysate prevented the development of clinical signs of food allergy in mice. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Characterization and Prevention of the Adsorption of Surfactant Protein D to Polypropylene

    Science.gov (United States)

    Bratcher, Preston E.; Gaggar, Amit

    2013-01-01

    Surfactant Protein D (SP-D) is a multifunctional protein present in the lung and in respiratory secretions. In the process of developing new experimental approaches to examine SP-D function, we observed that SP-D adsorbs to polypropylene tubes to a great extent, thereby depleting SP-D from the solution. Although it is well known that proteins adsorb nonspecifically to plastic, this effect is usually diminished by treatments to make the plastic “low-retention” or “low-binding”. However, these treatments actually increased the binding of SP-D to the plastic. In addition, this adsorption affected the results of several assays, including proteolytic cleavage assays. In order to block SP-D from adsorbing to polypropylene and the effects caused by this adsorption, we coated the tubes with bovine serum albumin (BSA), as is commonly performed for ELISAs. This coating greatly diminished the amount of SP-D sticking to the plastic, providing an inexpensive and effective method for preventing adsorption and the artifacts resulting from this adsorption. PMID:24039953

  6. Prevention

    Science.gov (United States)

    ... Contact Aging & Health A to Z Find a Geriatrics Healthcare Professional Medications & Older Adults Making Your Wishes ... Prevention Hearing Loss Heart Attack High Blood Pressure Nutrition Osteoporosis Shingles Skin Cancer Related News Quitting Smoking, ...

  7. Modifiers of mutant huntingtin aggregation : Functional conservation of C. elegans-modifiers of polyglutamine aggregation

    NARCIS (Netherlands)

    Teuling, Eva; Bourgonje, Annika; Veenje, Sven; Thijssen, Karen; de Boer, Jelle; van der Velde, Joeri; Swertz, Morris; Nollen, Ellen

    2011-01-01

    Protein aggregation is a common hallmark of a number of age-related neurodegenerative diseases, including Alzheimer's, Parkinson's, and polyglutamine-expansion disorders such as Huntington's disease, but how aggregation-prone proteins lead to pathology is not known. Using a genome-wide RNAi screen

  8. Detection of ubiquitinated huntingtin species in intracellular aggregates

    Directory of Open Access Journals (Sweden)

    Katrin eJuenemann

    2015-01-01

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

  9. Sequence-dependent internalization of aggregating peptides.

    Science.gov (United States)

    Couceiro, José R; Gallardo, Rodrigo; De Smet, Frederik; De Baets, Greet; Baatsen, Pieter; Annaert, Wim; Roose, Kenny; Saelens, Xavier; Schymkowitz, Joost; Rousseau, Frederic

    2015-01-02

    Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 μm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

    2013-03-01

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

  11. Modeling In-Network Aggregation in VANETs

    NARCIS (Netherlands)

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

    2011-01-01

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

  12. Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss

    OpenAIRE

    Weaver, Beth A.A.; Bonday, Zahid Q.; Putkey, Frances R.; Kops, Geert J.P.L.; Silk, Alain D.; Cleveland, Don W.

    2003-01-01

    Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in p...

  13. Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation*

    Science.gov (United States)

    Monsellier, Elodie; Redeker, Virginie; Ruiz-Arlandis, Gemma; Bousset, Luc; Melki, Ronald

    2015-01-01

    The aggregation of polyglutamine (polyQ)-containing proteins is at the origin of nine neurodegenerative diseases. Molecular chaperones prevent the aggregation of polyQ-containing proteins. The exact mechanism by which they interact with polyQ-containing, aggregation-prone proteins and interfere with their assembly is unknown. Here we dissect the mechanism of interaction between a huntingtin exon 1 fragment of increasing polyQ lengths (HttEx1Qn), the aggregation of which is tightly associated with Huntington's disease, and molecular chaperone Hsc70. We show that Hsc70, together with its Hsp40 co-chaperones, inhibits HttEx1Qn aggregation and modifies the structural, seeding, and infectious properties of the resulting fibrils in a polyQ-independent manner. We demonstrate that Hsc70 binds the 17-residue-long N-terminal flank of HttEx1Qn, and we map Hsc70-HttEx1Qn surface interfaces at the residue level. Finally, we show that this interaction competes with homotypic interactions between the N termini of different HttEx1Qn molecules that trigger the aggregation process. Our results lay the foundations of future therapeutic strategies targeting huntingtin aggregation in Huntington disease. PMID:25505179

  14. Prevention

    DEFF Research Database (Denmark)

    Halken, S; Høst, A

    2001-01-01

    , breastfeeding should be encouraged for 4-6 months. In high-risk infants a documented extensively hydrolysed formula is recommended if exclusive breastfeeding is not possible for the first 4 months of life. There is no evidence for preventive dietary intervention neither during pregnancy nor lactation...... populations. These theories remain to be documented in proper, controlled and prospective studies. Breastfeeding and the late introduction of solid foods (>4 months) is associated with a reduced risk of food allergy, atopic dermatitis, and recurrent wheezing and asthma in early childhood. In all infants....... Preventive dietary restrictions after the age of 4-6 months are not scientifically documented....

  15. 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...

  16. The protein kinase promiscuities in the cancer-preventive mechanisms of NSAIDs.

    Science.gov (United States)

    Norvaisas, Povilas; Chan, Diana; Yokoi, Kenji; Dave, Bhuvanesh; Ziemys, Arturas

    2016-01-01

    NSAIDs have been observed to have cancer-preventive properties, but the actual mechanism is elusive. We hypothesize that NSAIDs might have an effect through common pathways and targets of anticancer drugs by exploiting promiscuities of anticancer drug targets. Here, we have explored NSAIDs by their structural and pharmacophoric similarities with small anticancer molecules. In-silico analyses have shown a strong similarity between NSAIDs and protein kinase (PK) inhibitors. The calculated affinities of NSAIDs were found to be lower than the affinities of anticancer drugs, but higher than the affinities of compounds that are not specific to PKs. The competitive inhibition model suggests that PK might be inhibited by around 10%, which was confirmed by biochemical screening of some NSAIDs against PKs. NSAIDs did not affect all PKs universally, but had specificities for certain sets of PKs, which differed according to the NSAID. The study revealed potentially new features and mechanisms of NSAIDs that are useful in explaining their role in cancer prevention, which might lead to clinically significant breakthroughs in the future.

  17. Amelogenin processing by MMP-20 prevents protein occlusion inside calcite crystals.

    Science.gov (United States)

    Bromley, Keith M; Lakshminarayanan, Rajamani; Thompson, Mitchell; Lokappa, Sowmya B; Gallon, Victoria A; Cho, Kang R; Qiu, S Roger; Moradian-Oldak, Janet

    2012-10-03

    Calcite crystals were grown in the presence of full-length amelogenin and during its proteolysis by recombinant human matrix metalloproteinase 20 (rhMMP-20). Recombinant porcine amelogenin (rP172) altered the shape of calcite crystals by inhibiting the growth of steps on the {104} faces and became occluded inside the crystals. Upon co-addition of rhMMP-20, the majority of the protein was digested resulting in a truncated amelogenin lacking the C-terminal segment. In rP172-rhMMP-20 samples, the occlusion of amelogenin into the calcite crystals was drastically decreased. Truncated amelogenin (rP147) and the 25-residue C-terminal domain produced crystals with regular shape and less occluded organic material. Removal of the C-terminal diminished the affinity of amelogenin to the crystals and therefore prevented occlusion. We hypothesize that HAP and calcite interact with amelogenin in a similar manner. In the case of each material, full-length amelogenin binds most strongly, truncated amelogenin binds weakly and the C-terminus alone has the weakest interaction. Regarding enamel crystal growth, the prevention of occlusion into maturing enamel crystals might be a major benefit resulting from the selective cleavage of amelogenin at the C-terminus by MMP-20. Our data have important implications for understanding the hypomineralized enamel phenotype in cases of amelogenesis imperfecta resulting from MMP-20 mutations and will contribute to the design of enamel inspired biomaterials.

  18. Prediction of preeclampsia by placental protein 13 and background risk factors and its prevention by aspirin.

    Science.gov (United States)

    Meiri, Hamutal; Sammar, Marei; Herzog, Ayelet; Grimpel, Yael-Inna; Fihaman, Galina; Cohen, Aliza; Kivity, Vered; Sharabi-Nov, Adi; Gonen, Ron

    2014-09-01

    Evaluation of placental protein 13 (PP13) and risk factors (RFs) as markers for predicting preeclampsia (PE) and use of aspirin for PE prevention. First-trimester pregnancy screening was based on having PP13 level ≤0.4 multiple of the median (MoM) and/or at least one major risk factor (RF) for PE. Management was by routine care or combined with daily treatment with 75 mg aspirin between 14 and 35 weeks of gestation. Of 820 deliveries, 63 women developed PE (7.7%). Median PP13 levels was 0.2MoM in the PE group compared with 0.83MoM among unaffected and 1.0MoM in unaffected not treated with aspirin (Pprevention by aspirin was most effective when the risk was determined by low PP13 alone, less effective for combining low PP13 with RFs, and ineffective when determined by RFs alone. When PE risk is determined by low first trimester PP13 or by combined low PP13 and RFs, prevention with aspirin is warranted.

  19. Zinc in the prevention of Fe2initiated lipid and protein oxidation

    Directory of Open Access Journals (Sweden)

    M. PAOLA ZAGO

    2000-01-01

    Full Text Available In the present study we characterized the capacity of zinc to protect lipids and proteins from Fe2+-initiated oxidative damage. The effects of zinc on lipid oxidation were investigated in liposomes composed of brain phosphatidylcholine (PC and phosphatidylserine (PS at a molar relationship of 60:40 (PC:PS, 60:40. Lipid oxidation was evaluated as the oxidation of cis-parinaric acid or as the formation of 2-thiobarbituric acid-reactive substances (TBARS. Zinc protected liposomes from Fe2+ (2.5-50 muM-supported lipid oxidation. However, zinc (50 muM did not prevent the oxidative inactivation of glutamine synthelase and glucose 6-phosphate dehydrogenase when rat brain superntants were oxidized in the presence of 5 muM Fe2+ and 0.5 mM H2O2 .We also studied the interactions of zinc with epicatechin in the prevention of liid oxidation in liposomes. The simulaneous addition of 0.5 muM epicatechin (EC and 50 muM zinc or EC separately. Zinc (50 muM also protecte liposomes from the stimulatory effect of aluminum on Fe2+-initiated lipid oxidation. Zinc could play an important role as an antioxidant in biological systems, replacing iron and other metals with pro-oxidant activity from binding sites and interacting with other components of the oxidant defense system.

  20. Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids

    Science.gov (United States)

    Cao, Lixue; Huang, Qiao; Wu, Zhichao; Cao, Dong-dong; Ma, Zhanling; Xu, Qianghua; Hu, Peng; Fu, Yanxia; Shen, Yu; Chan, Jiulin; Zhou, Cong-zhao; Zhai, Wanying; Chen, Liangbiao

    2016-01-01

    The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP–ice interaction mechanism distinct from the known antifreeze proteins. PMID:27698404

  1. Preventive Effects of Chitosan Coacervate Whey Protein on Body Composition and Immunometabolic Aspect in Obese Mice

    Directory of Open Access Journals (Sweden)

    Gabriel Inácio de Morais Honorato de Souza

    2014-01-01

    Full Text Available Functional foods containing bioactive compounds of whey may play an important role in prevention and treatment of obesity. The aim of this study was to investigate the prospects of the biotechnological process of coacervation of whey proteins (CWP in chitosan and test its antiobesogenic potential. Methods. CWP (100 mg·kg·day was administered in mice with diet-induced obesity for 8 weeks. The animals were divided into four groups: control normocaloric diet gavage with water (C or coacervate (C-CWP, and high fat diet gavage with water (HF or coacervate (HF-CWP. Results. HF-CWP reduced weight gain and serum lipid fractions and displayed reduced adiposity and insulin. Adiponectin was significantly higher in HF-CWP group when compared to the HF. The level of LPS in HF-W group was significantly higher when compared to HF-CWP. The IL-10 showed an inverse correlation between the levels of insulin and glucose in the mesenteric adipose tissue in the HF-CWP group. CWP promoted an increase in both phosphorylation AMPK and the amount of ATGL in the mesenteric adipose tissue in HF-CWP group. Conclusion. CWP was able to modulate effects, possibly due to its high biological value of proteins. We observed a protective effect against obesity and improved the inflammatory milieu of white adipose tissue.

  2. 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)

  3. Specific protein homeostatic functions of small heat-shock proteins increase lifespan.

    Science.gov (United States)

    Vos, Michel J; Carra, Serena; Kanon, Bart; Bosveld, Floris; Klauke, Karin; Sibon, Ody C M; Kampinga, Harm H

    2016-04-01

    During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat-shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress-denatured substrates and/or to prevent aggregation of disease-associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70-dependent refolding of stress-denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70-independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  4. Investigation on the Aggregation Behaviors and Filament Morphology of Tau Protein by a Simple 90° Angle Light-Scattering Assay

    Directory of Open Access Journals (Sweden)

    Hai-Lin Liao

    2013-01-01

    Full Text Available The in vitro aggregation of tau constructs was monitored by a simple 90° angle light-scattering (LS approach which was conducted directly on fluorescence instrument. At the optimum incident wavelength (550 nm, unpolarized, the sensitivity of LS was high enough to detect tau aggregation at micromolar range. The nucleation and elongation, different events in the aggregation process of 4RMBD construct (corresponding with the four repeated units of tau Microtubule Binding Domain could be observed by this approach, as compared with ThS fluorescence assay. The validity of this technique was demonstrated over a range of tau concentrations with different tau filaments. Linear regression of scattering light against concentration yielded the x-intercept, the critical concentrations of tau constructs. The critical concentrations of 4RMBD and its S305N mutant are 5.26 μM and 4.04 μM respectively, indicating point mutation S305N, which is associated with FTDP-17, appear to enhance the heparin-induced tau aggregation in vitro. Furthermore, the slopes of concentration dependence curves, as well as the angle dependence, were discussed based on the filaments morphology examined by electron microscopy and ultrasonication experiment.

  5. Hemin as a generic and potent protein misfolding inhibitor

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-11-14

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

  6. Catalase activity prevents exercise-induced up-regulation of vasoprotective proteins in venous tissue.

    Science.gov (United States)

    Dao, Vu Thao-Vi; Floeren, Melanie; Kumpf, Stephanie; Both, Charlotte; Peter, Bärbel; Balz, Vera; Suvorava, Tatsiana; Kojda, Georg

    2011-11-01

    Physical activity induces favourable changes of arterial gene expression and protein activity, although little is known about its effect in venous tissue. Although our understanding of the initiating molecular signals is still incomplete, increased expression of endothelial nitric oxide synthase (eNOS) is considered a key event. This study sought to investigate the effects of two different training protocols on the expression of eNOS and extracellular superoxide dismutase (ecSOD) in venous and lung tissue and to evaluate the underlying molecular mechanisms. C57Bl/6 mice underwent voluntary exercise or forced physical activity. Changes of vascular mRNA and protein levels and activity of eNOS, ecSOD and catalase were determined in aorta, heart, lung and vena cava. Both training protocols similarly increased relative heart weight and resulted in up-regulation of aortic and myocardial eNOS. In striking contrast, eNOS expression in vena cava and lung remained unchanged. Likewise, exercise up-regulated ecSOD in the aorta and in left ventricular tissue but remained unchanged in lung tissue. Catalase expression in lung tissue and vena cava of exercised mice exceeded that in aorta by 6.9- and 10-fold, respectively, suggesting a lack of stimulatory effects of hydrogen peroxide. In accordance, treatment of mice with the catalase inhibitor aminotriazole for 6 weeks resulted in significant up-regulation of eNOS and ecSOD in vena cava. These data suggest that physiological venous catalase activity prevents exercise-induced up-regulation of eNOS and ecSOD. Furthermore, therapeutic inhibition of vascular catalase might improve pulmonary rehabilitation. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  7. Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin.

    Directory of Open Access Journals (Sweden)

    Vera A Borzova

    Full Text Available Thermal aggregation of bovine serum albumin (BSA has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1 of 10.3 nm. The second form (low reactive unfolded form, Ulr participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast. At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2 of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.

  8. Global epidemiology of serogroup B meningococcal disease and opportunities for prevention with novel recombinant protein vaccines.

    Science.gov (United States)

    Villena, Rodolfo; Safadi, Marco Aurelio P; Valenzuela, María Teresa; Torres, Juan P; Finn, Adam; O'Ryan, Miguel

    2018-04-18

    Meningococcal disease (MD) is a major cause of meningitis and sepsis worldwide, with a high case fatality rate and frequent sequelae. Neisseria meningitidis serogroups A, B, C, W, X and Y are responsible for most of these life-threatening infections, and its unpredictable epidemiology can cause outbreaks in communities, with significant health, social and economic impact. Currently, serogroup B is the main cause of MD in Europe and North America and one of the most prevalent serogroups in Latin America. Mass vaccination strategies using polysaccharide vaccines have been deployed since the 1970s and the use of conjugate vaccines has controlled endemic and epidemic disease caused by serogroups A, C, W and Y and more recently serogroup B using geographically-specific outer membrane vesicle based vaccines. Two novel protein-based vaccines are a significant addition to our armamentarium against N. meningitidis as they provide broad coverage against highly diverse strains in serogroup B and other groups. Early safety, effectiveness and impact data of these vaccines are encouraging. These novel serogroup B vaccines should be actively considered for individuals at increased risk of disease and to control serogroup B outbreaks occurring in institutions or specific regions, as they are likely to save lives and prevent severe sequelae. Incorporation into national programs will require thorough country-specific analysis.

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

    African Journals Online (AJOL)

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

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

    Directory of Open Access Journals (Sweden)

    Leila M Luheshi

    2010-03-01

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

  11. Rosuvastatin, inflammation, C-reactive protein, JUPITER, and primary prevention of cardiovascular disease--a perspective.

    Science.gov (United States)

    Kones, Richard

    2010-12-09

    The major public health concern worldwide is coronary heart disease, with dyslipidemia as a major risk factor. Statin drugs are recommended by several guidelines for both primary and secondary prevention. Rosuvastatin has been widely accepted because of its efficacy, potency, and superior safety profile. Inflammation is involved in all phases of atherosclerosis, with the process beginning in early youth and advancing relentlessly for decades throughout life. C-reactive protein (CRP) is a well-studied, nonspecific marker of inflammation which may reflect general health risk. Considerable evidence suggests CRP is an independent predictor of future cardiovascular events, but direct involvement in atherosclerosis remains controversial. Rosuvastatin is a synthetic, hydrophilic statin with unique stereochemistry. A large proportion of patients achieve evidence-based lipid targets while using the drug, and it slows progression and induces regression of atherosclerotic coronary lesions. Rosuvastatin lowers CRP levels significantly. The Justification for Use of statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial was designed after the observation that when both low density lipoprotein and CRP were reduced, patients fared better than when only LDL was lowered. Advocates and critics alike acknowledge that the benefits of rosuvastatin in JUPITER were real. After a review, the US Food and Drug Administration extended the indications for rosuvastatin to include asymptomatic JUPITER-eligible individuals with one additional risk factor. The American Heart Association and Centers of Disease Control and Prevention had previously recognized the use of CRP in persons with "intermediate risk" as defined by global risk scores. The Canadian Cardiovascular Society guidelines went further and recommended use of statins in persons with low LDL and high CRP levels at intermediate risk. The JUPITER study focused attention on ostensibly healthy individuals with

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

    Science.gov (United States)

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

    2016-01-15

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

  13. SHAPE CHARACTERIZATION OF CONCRETE AGGREGATE

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    Jing Hu

    2011-05-01

    Full Text Available As a composite material, the performance of concrete materials can be expected to depend on the properties of the interfaces between its two major components, aggregate and cement paste. The microstructure at the interfacial transition zone (ITZ is assumed to be different from the bulk material. In general, properties of conventional concrete have been found favoured by optimum packing density of the aggregate. Particle size is a common denominator in such studies. Size segregation in the ITZ among the binder particles in the fresh state, observed in simulation studies by concurrent algorithm-based SPACE system, additionally governs density as well as physical bonding capacity inside these shell-like zones around aggregate particles. These characteristics have been demonstrated qualitatively pertaining also after maturation of the concrete. Such properties of the ITZs have direct impact on composite properties. Despite experimental approaches revealed effects of aggregate grain shape on different features of material structure (among which density, and as a consequence on mechanical properties, it is still an underrated factor in laboratory studies, probably due to the general feeling that a suitable methodology for shape characterization is not available. A scientific argument hindering progress is the interconnected nature of size and shape. Presently, a practical problem preventing shape effects to be emphasized is the limitation of most computer simulation systems in concrete technology to spherical particles. New developments at Delft University of Technology will make it possible in the near future to generate jammed states, or other high-density fresh particle mixtures of non-spherical particles, which thereupon can be subjected to hydration algorithms. This paper will sketch the outlines of a methodological approach for shape assessment of loose (non-embedded aggregate grains, and demonstrate its use for two types of aggregate, allowing

  14. Matrix metalloproteinase-20 mediates dental enamel biomineralization by preventing protein occlusion inside apatite crystals

    Science.gov (United States)

    Prajapati, Saumya; Tao, Jinhui; Ruan, Qichao; De Yoreo, James J.; Moradian-Oldak, Janet

    2015-01-01

    Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair. PMID:26513418

  15. Matrix metalloproteinase-20 mediates dental enamel biomineralization by preventing protein occlusion inside apatite crystals.

    Science.gov (United States)

    Prajapati, Saumya; Tao, Jinhui; Ruan, Qichao; De Yoreo, James J; Moradian-Oldak, Janet

    2016-01-01

    Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Mechanistic understanding of protein-silicone oil interactions.

    Science.gov (United States)

    Li, Jinjiang; Pinnamaneni, Swathi; Quan, Yong; Jaiswal, Archana; Andersson, Fredrik I; Zhang, Xiaochun

    2012-06-01

    To investigate interactions between protein and silicone oil so that we can provide some mechanistic understanding of protein aggregation in silicone oil lubricated syringes and its prevention by formulation additives such as Polysorbate 80 and Poloxamer 188. Interfacial tension values of silicone oil/water interface of abatacept solutions with and without formulation additives were obtained under equilibrium conditions using Attension Theta optical tensiometer. Their adsorption and desorption profiles were measured using Quartz Crystal Microbalancing with Dissipation monitoring (QCM-D). The degree of aggregation of abatacept was assessed based on size exclusion measurement. Adsorption of abatacept at the oil/water interface was shown. Polysorbat 80 was more effective than Poloxamer 188 in preventing abatacept adsorption. Moreover, it was noted that some of the adsorbed abatacept molecules were not desorbed readily upon buffer rinse. Finally, no homogeneous aggregation was observed at room temperature and a slight increase of aggregation was only observed for samples measured at 40°C which can be prevented using Polysorbate 80. Interfacial adsorption of proteins is the key step and maybe responsible for the phenomenon of soluble-protein loss when contacting silicone oil and the irreversible adsorption of protein may be associated with protein denaturation/aggregation.

  17. 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

    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....... 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....

  18. Self-assembly dynamics for the transition of a globular aggregate to a fibril network of lysozyme proteins via a coarse-grained Monte Carlo simulation

    Directory of Open Access Journals (Sweden)

    R. B. Pandey

    2015-09-01

    Full Text Available The self-organizing dynamics of lysozymes (an amyloid protein with 148 residues with different numbers of protein chains, Nc = 1,5,10, and 15 (concentration