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Sample records for detect protein aggregates

  1. Sensitive detection of aggregated prion protein via proximity ligation

    PubMed Central

    Hammond, Maria; Wik, Lotta; Deslys, Jean-Philippe; Comoy, Emmanuel; Linné, Tommy; Landegren, Ulf; Kamali-Moghaddam, Masood

    2014-01-01

    The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 107-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases. PMID:25482604

  2. Sensitive detection of aggregated prion protein via proximity ligation.

    PubMed

    Hammond, Maria; Wik, Lotta; Deslys, Jean-Philippe; Comoy, Emmanuel; Linné, Tommy; Landegren, Ulf; Kamali-Moghaddam, Masood

    2014-01-01

    The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10(7)-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.

  3. Holographic characterization of protein aggregates

    NASA Astrophysics Data System (ADS)

    Wang, Chen; Zhong, Xiao; Ruffner, David; Stutt, Alexandra; Philips, Laura; Ward, Michael; Grier, David

    Holographic characterization directly measures the size distribution of subvisible protein aggregates in suspension and offers insights into their morphology. Based on holographic video microscopy, this analytical technique records and interprets holograms of individual aggregates in protein solutions as they flow down a microfluidic channel, without requiring labeling or other exceptional sample preparation. The hologram of an individual protein aggregate is analyzed in real time with the Lorenz-Mie theory of light scattering to measure that aggregate's size and optical properties. Detecting, counting and characterizing subvisible aggregates proceeds fast enough for time-resolved studies, and lends itself to tracking trends in protein aggregation arising from changing environmental factors. No other analytical technique provides such a wealth of particle-resolved characterization data in situ. Holographic characterization promises accelerated development of therapeutic protein formulations, improved process control during manufacturing, and streamlined quality assurance during storage and at the point of use. Mrsec and MRI program of the NSF, Spheryx Inc.

  4. Thermodynamics of Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

    Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

  5. Detection of Protein Aggregates in Brain and Cerebrospinal Fluid Derived from Multiple Sclerosis Patients

    PubMed Central

    David, Monique Antoinette; Tayebi, Mourad

    2014-01-01

    Studies of the properties of soluble oligomer species of amyloidogenic proteins, derived from different proteins with little sequence homology, have indicated that they share a common structure and may share similar pathogenic mechanisms. Amyloid β, tau protein, as well as amyloid precursor protein normally associated with Alzheimer’s disease and Parkinson’s disease were found in lesions and plaques of multiple sclerosis patients. The objective of the study is to investigate whether brain and cerebrospinal fluid (CSF) samples derived from multiple sclerosis patients demonstrate the presence of soluble oligomers normally associated with protein-misfolding diseases such as Alzheimer’s disease. We have used anti-oligomer monoclonal antibodies to immunodetect soluble oligomers in CSF and brain tissues derived from multiple sclerosis patients. In this report, we describe the presence of soluble oligomers in the brain tissue and cerebral spinal fluid of multiple sclerosis patients detected with our monoclonal anti-oligomer antibodies with Western blot and Sandwich enzyme-linked immunosorbent assay (sELISA). These results might suggest that protein aggregation plays a role in multiple sclerosis pathogenesis although further and more refined studies are needed to confirm the role of soluble aggregates in multiple sclerosis. PMID:25520699

  6. Protein aggregation and prionopathies.

    PubMed

    Renner, M; Melki, R

    2014-06-01

    Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are "transmissible" between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob's disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  7. Protein Colloidal Aggregation Project

    NASA Technical Reports Server (NTRS)

    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.

  8. dRHP-PseRA: detecting remote homology proteins using profile-based pseudo protein sequence and rank aggregation.

    PubMed

    Chen, Junjie; Long, Ren; Wang, Xiao-Long; Liu, Bin; Chou, Kuo-Chen

    2016-09-01

    Protein remote homology detection is an important task in computational proteomics. Some computational methods have been proposed, which detect remote homology proteins based on different features and algorithms. As noted in previous studies, their predictive results are complementary to each other. Therefore, it is intriguing to explore whether these methods can be combined into one package so as to further enhance the performance power and application convenience. In view of this, we introduced a protein representation called profile-based pseudo protein sequence to extract the evolutionary information from the relevant profiles. Based on the concept of pseudo proteins, a new predictor, called "dRHP-PseRA", was developed by combining four state-of-the-art predictors (PSI-BLAST, HHblits, Hmmer, and Coma) via the rank aggregation approach. Cross-validation tests on a SCOP benchmark dataset have demonstrated that the new predictor has remarkably outperformed any of the existing methods for the same purpose on ROC50 scores. Accordingly, it is anticipated that dRHP-PseRA holds very high potential to become a useful high throughput tool for detecting remote homology proteins. For the convenience of most experimental scientists, a web-server for dRHP-PseRA has been established at http://bioinformatics.hitsz.edu.cn/dRHP-PseRA/.

  9. Tracking protein aggregate interactions

    PubMed Central

    Bartz, Jason C; Nilsson, K Peter R

    2011-01-01

    Amyloid fibrils share a structural motif consisting of highly ordered β-sheets aligned perpendicular to the fibril axis.1, 2 At each fibril end, β-sheets provide a template for recruiting and converting monomers.3 Different amyloid fibrils often co-occur in the same individual, yet whether a protein aggregate aids or inhibits the assembly of a heterologous protein is unclear. In prion disease, diverse prion aggregate structures, known as strains, are thought to be the basis of disparate disease phenotypes in the same species expressing identical prion protein sequences.4–7 Here we explore the interactions reported to occur when two distinct prion strains occur together in the central nervous system. PMID:21597336

  10. Proteins aggregation and human diseases

    NASA Astrophysics Data System (ADS)

    Hu, Chin-Kun

    2015-04-01

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

  11. Non-Arrhenius protein aggregation.

    PubMed

    Wang, Wei; Roberts, Christopher J

    2013-07-01

    Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. Therefore, investigations of protein aggregation remain a major focus in pharmaceutical companies and academic institutions. Due to the complexity of the aggregation process and temperature-dependent conformational stability, temperature-induced protein aggregation is often non-Arrhenius over even relatively small temperature windows relevant for product development, and this makes low-temperature extrapolation difficult based simply on accelerated stability studies at high temperatures. This review discusses the non-Arrhenius nature of the temperature dependence of protein aggregation, explores possible causes, and considers inherent hurdles for accurately extrapolating aggregation rates from conventional industrial approaches for selecting accelerated conditions and from conventional or more advanced methods of analyzing the resulting rate data.

  12. Method for detecting and diagnosing disease caused by pathological protein aggregation

    SciTech Connect

    Stevens, F.J.

    1994-12-31

    A method is provided for detecting pathological macromolecules in a patient, comprising obtaining body fluid from the patient, pretreating the body fluid, subjecting the pretreated body fluid to size-exclusion chromatography to create an excluded fluid, and analyzing the excluded fluid to detect macromolecules having a predetermined molecular weight. The method also allows for comparing elution spectra with reference spectra of suspect pathologic proteins.

  13. Method for detecting and diagnosing disease caused by pathological protein aggregation

    DOEpatents

    Stevens, Fred J.; Myatt, Elizabeth A.; Solomon, Alan

    2000-01-01

    A method is provided for detecting pathological macromolecules in a patient, comprising obtaining body fluid from the patient, pretreating the body fluid, subjecting the pretreated body fluid to size-exclusion chromatography to create an excluded fluid, and analyzing the excluded fluid to detect macromolecules having a predetermined molecular weight. The method also allows for comparing elution spectra with reference spectra of suspect pathologic proteins.

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

    SciTech Connect

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

    2012-10-26

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

  15. Protein aggregation in a membrane environment.

    PubMed

    Gorbenko, Galyna; Trusova, Valeriya

    2011-01-01

    Biological membranes are featured by a remarkable ability to modulate a wide range of physiological and pathological processes. Of these, protein aggregation is currently receiving the greatest attention, as one type of the ordered protein aggregates, amyloid fibrils, proved to be involved in molecular etiology of a number of fatal diseases. It has been hypothesized that nucleation of amyloid fibrils and toxic action of their precursors is mediated by lipid-protein interactions. Lipid bilayer provides a variety of environments in which aggregated state of polypeptide chain appears to be more thermodynamically favorable than its monomeric form. The major factors responsible for the enhanced self-association propensity of membrane-bound proteins include (i) structural transition of polypeptide chain into aggregation-prone conformation; (ii) protein crowding in a lipid phase; (iii) particular aggregation-favoring orientation and bilayer embedment of the protein molecules. All these factors are considered in the present review with an emphasis being put on the role of electrostatic, hydrophobic, and hydrogen-bonding phenomena in initiating and modulating the protein aggregation on a membrane template. Likewise, we survey the advanced experimental techniques employed for detection and structural characterization of the aggregated species in membrane systems.

  16. Protein aggregation in salt solutions

    PubMed Central

    Kastelic, Miha; Kalyuzhnyi, Yurij V.; Hribar-Lee, Barbara; Dill, Ken A.; Vlachy, Vojko

    2015-01-01

    Protein aggregation is broadly important in diseases and in formulations of biological drugs. Here, we develop a theoretical model for reversible protein–protein aggregation in salt solutions. We treat proteins as hard spheres having square-well-energy binding sites, using Wertheim’s thermodynamic perturbation theory. The necessary condition required for such modeling to be realistic is that proteins in solution during the experiment remain in their compact form. Within this limitation our model gives accurate liquid–liquid coexistence curves for lysozyme and γ IIIa-crystallin solutions in respective buffers. It provides good fits to the cloud-point curves of lysozyme in buffer–salt mixtures as a function of the type and concentration of salt. It than predicts full coexistence curves, osmotic compressibilities, and second virial coefficients under such conditions. This treatment may also be relevant to protein crystallization. PMID:25964322

  17. Age-Dependent Protein Aggregation Initiates Amyloid-β Aggregation

    PubMed Central

    Groh, Nicole; Bühler, Anika; Huang, Chaolie; Li, Ka Wan; van Nierop, Pim; Smit, August B.; Fändrich, Marcus; Baumann, Frank; David, Della C.

    2017-01-01

    Aging is the most important risk factor for neurodegenerative diseases associated with pathological protein aggregation such as Alzheimer’s disease. Although aging is an important player, it remains unknown which molecular changes are relevant for disease initiation. Recently, it has become apparent that widespread protein aggregation is a common feature of aging. Indeed, several studies demonstrate that 100s of proteins become highly insoluble with age, in the absence of obvious disease processes. Yet it remains unclear how these misfolded proteins aggregating with age affect neurodegenerative diseases. Importantly, several of these aggregation-prone proteins are found as minor components in disease-associated hallmark aggregates such as amyloid-β plaques or neurofibrillary tangles. This co-localization raises the possibility that age-dependent protein aggregation directly contributes to pathological aggregation. Here, we show for the first time that highly insoluble proteins from aged Caenorhabditis elegans or aged mouse brains, but not from young individuals, can initiate amyloid-β aggregation in vitro. We tested the seeding potential at four different ages across the adult lifespan of C. elegans. Significantly, protein aggregates formed during the early stages of aging did not act as seeds for amyloid-β aggregation. Instead, we found that changes in protein aggregation occurring during middle-age initiated amyloid-β aggregation. Mass spectrometry analysis revealed several late-aggregating proteins that were previously identified as minor components of amyloid-β plaques and neurofibrillary tangles such as 14-3-3, Ubiquitin-like modifier-activating enzyme 1 and Lamin A/C, highlighting these as strong candidates for cross-seeding. Overall, we demonstrate that widespread protein misfolding and aggregation with age could be critical for the initiation of pathogenesis, and thus should be targeted by therapeutic strategies to alleviate neurodegenerative

  18. Protein aggregation: From background to inhibition strategies.

    PubMed

    Alam, Parvez; Siddiqi, Khursheed; Chturvedi, Sumit Kumar; Khan, Rizwan Hasan

    2017-10-01

    The aggregation of specific proteins is hypothesized to cause several pathological conditions, which are collectively known as amyloid disorders. The aggregation of peptides and proteins is mainly associated with the perturbation of cellular function, ageing and various human disorders. Mounting evidence suggests that protein aggregation is often caused by mutation, environmental stress or the cellular response to an imbalanced protein homeostasis. This review summarizes the background information on the protein folding, misfolding, cellular strategies against protein aggregation, factors affecting protein aggregation and mechanism of protein aggregation. We then focus on various inhibitors for protein aggregation both in vitro and in vivo. We conclude with a perspective that better therapeutics could be developed by using cocktail of small molecule inhibitors for the treatment of amyloid diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Detergent-mediated protein aggregation.

    PubMed

    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.

  20. Detergent-mediated protein aggregation

    PubMed Central

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

    2016-01-01

    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. PMID:23466535

  1. Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation.

    PubMed

    Kiese, Sylvia; Pappenberger, Astrid; Friess, Wolfgang; Mahler, Hanns-Christian

    2010-02-01

    Shaking or heat stress may induce protein aggregates. Aggregation behavior of an IgG1 stressed by shaking or heat following static storage at 5 and 25 degrees C was investigated to determine whether protein aggregates exist in equilibrium. Aggregates were detected using different analytical methods including visual inspection, turbidity, light obscuration, size exclusion chromatography, and dynamic light scattering. Significant differences were evident between shaken and heated samples upon storage. Visible and subvisible particles (insoluble aggregates), turbidity and z-average diameter decreased whilst soluble aggregate content increased in shaken samples over time. Insoluble aggregates were considered to be reversible and dissociate into soluble aggregates and both aggregate types existed in equilibrium. Heat-induced aggregates had a denatured protein structure and upon static storage, no significant change in insoluble aggregates content was shown, whilst changes in soluble aggregates content occurred. This suggested that heat-induced insoluble aggregates were irreversible and not in equilibrium with soluble aggregates. Additionally, the aggregation behavior of unstressed IgG1 after spiking with heavily aggregated material (shaken or heat stressed) was studied. The aggregation behavior was not significantly altered, independent of the spiking concentration over time. Thus, neither mechanically stressed native nor temperature-induced denatured aggregates were involved in nucleating or propagating aggregation.

  2. Investigating the mechanisms leading to protein aggregation

    NASA Astrophysics Data System (ADS)

    McNamara, Ruth; McManus, Jennifer J.

    2014-03-01

    The formation of protein aggregates is a feature of several diseases and is a problem during the manufacture of biopharmaceutical and protein based food products. During processing, stability may become compromised leading to the condensation of proteins to form non-native aggregates. The aim of this work is to induce aggregation on model proteins by the imposition of a particular stress to evaluate the extent of aggregation and to assess the degree of structural change to the protein. Aggregation of two proteins, lysozyme and bovine serum albumin has been induced by several mechanisms. Using various techniques (electrophoresis, HPLC, spectroscopic analysis, and microscopic techniques) both the level of aggregation extent of protein unfolding has been investigated for a range of solution conditions. Our results show that the amount of aggregation depends strongly on the mechanism by which non-native aggregation proceeds, and within each mechanism, solution conditions are an important factor. With the exception of aggregation by self-association (which is concentration dependent), the appearance of aggregation is driven by structural changes induced by the applied stress (heat, chemical denaturant, oxidation or contact with a surface). Author would like to acknowledge support from Science Foundation Ireland (SFI), National University of Maynooth John and Pat Hume Scholarship.

  3. Protein Aggregation Profile of the Bacterial Cytosol

    PubMed Central

    de Groot, Natalia S.; Ventura, Salvador

    2010-01-01

    Background Protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity. Methodology/Principal Findings Here, we use the AGGRESCAN algorithm to approximate the aggregation profile of an experimental cytosolic Escherichia coli proteome. The analysis indicates that the aggregation propensity of bacterial proteins is associated with their length, conformation, location, function, and abundance. The data are consistent with the predictions of other algorithms on different theoretical proteomes. Conclusions/Significance Overall, the study suggests that the avoidance of protein aggregation in functional environments acts as a strong evolutionary constraint on polypeptide sequences in both prokaryotic and eukaryotic organisms. PMID:20195530

  4. Effects of protein aggregates: an immunologic perspective.

    PubMed

    Rosenberg, Amy S

    2006-08-04

    The capacity of protein aggregates to enhance immune responses to the monomeric form of the protein has been known for over a half-century. Despite the clear connection between protein aggregates and antibody mediated adverse events in treatment with early therapeutic protein products such as intravenous immune globulin (IVIG) and human growth hormone, surprisingly little is known about the nature of the aggregate species responsible for such effects. This review focuses on a framework for understanding how aggregate species potentially interact with the immune system to enhance immune responses, garnered from basic immunologic research. Thus, protein antigens presented in a highly arrayed structure, such as might be found in large nondenatured aggregate species, are highly potent in inducing antibody responses even in the absence of T-cell help. Their potency may relate to the ability of multivalent protein species to extensively cross-link B-cell receptor, which (1) activates B cells via Bt kinases to proliferate, and (2) targets protein to class II major histocompatibility complex (MHC)-loading compartments, efficiently eliciting T-cell help for antibody responses. The review further focuses on protein aggregates as they affect an immunogenicity risk assessment, the use of animal models and studies in uncovering effects of protein aggregates, and changes in product manufacture and packaging that may affect generation of protein aggregates.

  5. Disulphide bond formation in food protein aggregation and gelation.

    PubMed

    Visschers, Ronald W; de Jongh, Harmen H J

    2005-01-01

    In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified. The differences between heat-, high-pressure-, and denaturant-induced unfolding and aggregation are discussed. The effect of disulphide bonding between aggregates of proteins and protein mixtures on the functional macroscopic properties of space filling networks in protein gels is briefly presented.

  6. Anodic-stripping voltammetric immunoassay for ultrasensitive detection of low-abundance proteins using quantum dot aggregated hollow microspheres.

    PubMed

    Zhang, Bing; Tang, Dianping; Goryacheva, Irina Yu; Niessner, Reinhard; Knopp, Dietmar

    2013-02-11

    A new anodic-stripping voltammetric immunoassay protocol for detection of IgG1, as a model protein, was designed by using CdS quantum dot (QD) layer-by-layer assembled hollow microspheres (QDHMS) as molecular tags. Initially, monoclonal anti-human IgG1 specific antibodies were anchored on amorphous magnetic beads preferably selective to capture F(ab) of IgG1 analyte from the sample. For detection, monoclonal anti-human IgG1 (F(c)-specific) antibodies were covalently coupled to the synthesized QDHMS. In a sandwich-type immunoassay format, subsequent anodic-stripping voltammetric detection of cadmium released under acidic conditions from the coupled QDs was conducted at an in situ prepared mercury film electrode. The immunoassay combines highly efficient magnetic separation with signal amplification by the multilayered QD labels. The dynamic concentration range spanned from 1.0 fg mL(-1) to 1.0 μg mL(-1) of IgG1 with a detection limit of 0.1 fg mL(-1). The electrochemical immunoassay showed good reproducibility, selectivity, and stability. The analysis of clinical serum specimens revealed good accordance with the results obtained by an enzyme-linked immunosorbent assay method. The new immunoassay is promising for enzyme-free, and cost-effective analysis of low-abundance biomarkers. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Modeling Protein Aggregate Assembly and Structure

    NASA Astrophysics Data System (ADS)

    Guo, Jun-tao; Hall, Carol K.; Xu, Ying; Wetzel, Ronald

    One might say that "protein science" got its start in the domestic arts, built around the abilities of proteins to aggregate in response to environmental stresses such as heating (boiled eggs), heating and cooling (gelatin), and pH (cheese). Characterization of proteins in the late nineteenth century likewise focused on the ability of proteins to precipitate in response to certain salts and to aggregate in response to heating. Investigations by Chick and Martin (Chick and Martin, 1910) showed that the inactivating response of proteins to heat or solvent treatment is a two-step process involving separate denaturation and precipitation steps. Monitoring the coagulation and flocculation responses of proteins to heat and other stresses remained a major approach to understanding protein structure for decades, with solubility, or susceptibility to aggregation, serving as a kind of benchmark against which results of other methods, such as viscosity, chemical susceptibility, immune activity, crystallizability, and susceptibility to proteolysis, were compared (Mirsky and Pauling, 1936;Wu, 1931). Toward the middle of the last century, protein aggregation studies were largely left behind, as improved methods allowed elucidation of the primary sequence of proteins, reversible unfolding studies, and ultimately high-resolution structures. Curiously, the field of protein science, and in particular protein folding, is now gravitating back to a closer look at protein aggregation and protein aggregates. Unfortunately, the means developed during the second half of the twentieth century for studying native, globular proteins have not proved immediately amenable to the study of aggregate structures. Great progress is being made, however, to modify classical methods, including NMR and X-ray diffraction, as well as to develop newer techniques, that together should continue to expand our picture of aggregate structure (Kheterpal and Wetzel, 2006; Wetzel, 1999).

  8. Regulated protein aggregation: stress granules and neurodegeneration

    PubMed Central

    2012-01-01

    The protein aggregation that occurs in neurodegenerative diseases is classically thought to occur as an undesirable, nonfunctional byproduct of protein misfolding. This model contrasts with the biology of RNA binding proteins, many of which are linked to neurodegenerative diseases. RNA binding proteins use protein aggregation as part of a normal regulated, physiological mechanism controlling protein synthesis. The process of regulated protein aggregation is most evident in formation of stress granules. Stress granules assemble when RNA binding proteins aggregate through their glycine rich domains. Stress granules function to sequester, silence and/or degrade RNA transcripts as part of a mechanism that adapts patterns of local RNA translation to facilitate the stress response. Aggregation of RNA binding proteins is reversible and is tightly regulated through pathways, such as phosphorylation of elongation initiation factor 2α. Microtubule associated protein tau also appears to regulate stress granule formation. Conversely, stress granule formation stimulates pathological changes associated with tau. In this review, I propose that the aggregation of many pathological, intracellular proteins, including TDP-43, FUS or tau, proceeds through the stress granule pathway. Mutations in genes coding for stress granule associated proteins or prolonged physiological stress, lead to enhanced stress granule formation, which accelerates the pathophysiology of protein aggregation in neurodegenerative diseases. Over-active stress granule formation could act to sequester functional RNA binding proteins and/or interfere with mRNA transport and translation, each of which might potentiate neurodegeneration. The reversibility of the stress granule pathway also offers novel opportunities to stimulate endogenous biochemical pathways to disaggregate these pathological stress granules, and perhaps delay the progression of disease. PMID:23164372

  9. The amyloid interactome: Exploring protein aggregation

    PubMed Central

    Mastrokalou, Chara V.; Hamodrakas, Stavros J.

    2017-01-01

    Protein-protein interactions are the quintessence of physiological activities, but also participate in pathological conditions. Amyloid formation, an abnormal protein-protein interaction process, is a widespread phenomenon in divergent proteins and peptides, resulting in a variety of aggregation disorders. The complexity of the mechanisms underlying amyloid formation/amyloidogenicity is a matter of great scientific interest, since their revelation will provide important insight on principles governing protein misfolding, self-assembly and aggregation. The implication of more than one protein in the progression of different aggregation disorders, together with the cited synergistic occurrence between amyloidogenic proteins, highlights the necessity for a more universal approach, during the study of these proteins. In an attempt to address this pivotal need we constructed and analyzed the human amyloid interactome, a protein-protein interaction network of amyloidogenic proteins and their experimentally verified interactors. This network assembled known interconnections between well-characterized amyloidogenic proteins and proteins related to amyloid fibril formation. The consecutive extended computational analysis revealed significant topological characteristics and unraveled the functional roles of all constituent elements. This study introduces a detailed protein map of amyloidogenicity that will aid immensely towards separate intervention strategies, specifically targeting sub-networks of significant nodes, in an attempt to design possible novel therapeutics for aggregation disorders. PMID:28249044

  10. Protein aggregation profile of the human kinome

    PubMed Central

    Graña-Montes, Ricardo; Sant'Anna de Oliveira, Ricardo; Ventura, Salvador

    2012-01-01

    Protein aggregation into amyloid fibrils is associated with the onset of an increasing number of human disorders, including Alzheimer's disease, diabetes, and some types of cancer. The ability to form toxic amyloids appears to be a property of most polypeptides. Accordingly, it has been proposed that reducing aggregation and its effect in cell fitness is a driving force in the evolution of proteins sequences. This control of protein solubility should be especially important for regulatory hubs in biological networks, like protein kinases. These enzymes are implicated in practically all processes in normal and abnormal cell physiology, and phosphorylation is one of the most frequent protein modifications used to control protein activity. Here, we use the AGGRESCAN algorithm to study the aggregation propensity of kinase sequences. We compared them with the rest of globular proteins to decipher whether they display differential aggregation properties. In addition, we compared the human kinase complement with the kinomes of other organisms to see if we can identify any evolutionary trend in the aggregational properties of this protein superfamily. Our analysis indicates that kinase domains display significant aggregation propensity, a property that decreases with increasing organism complexity. PMID:23181023

  11. An energy landscape approach to protein aggregation

    NASA Astrophysics Data System (ADS)

    Buell, Alexander; Knowles, Tuomas

    2012-02-01

    Protein aggregation into ordered fibrillar structures is the hallmark of a class of diseases, the most prominent examples of which are Alzheimer's and Parkinson's disease. Recent results (e.g. Baldwin et al. J. Am. Chem. Soc. 2011) suggest that the aggregated state of a protein is in many cases thermodynamically more stable than the soluble state. Therefore the solubility of proteins in a cellular context appears to be to a large extent under kinetic control. Here, we first present a conceptual framework for the description of protein aggregation ( see AK Buell et al., Phys. Rev. Lett. 2010) that is an extension to the generally accepted energy landscape model for protein folding. Then we apply this model to analyse and interpret a large set of experimental data on the kinetics of protein aggregation, acquired mainly with a novel biosensing approach (see TPJK Knowles et al, Proc. Nat. Acad. Sc. 2007). We show how for example the effect of sequence modifications on the kinetics and thermodynamics of human lysozyme aggregation can be understood and quantified (see AK Buell et al., J. Am. Chem. Soc. 2011). These results have important implications for therapeutic strategies against protein aggregation disorders, in this case lysozyme systemic amyloidosis.

  12. Influence of polysaccharides on wine protein aggregation.

    PubMed

    Jaeckels, Nadine; Meier, Miriam; Dietrich, Helmut; Will, Frank; Decker, Heinz; Fronk, Petra

    2016-06-01

    Polysaccharides are the major high-molecular weight components of wines. In contrast, proteins occur only in small amounts in wine, but contribute to haze formation. The detailed mechanism of aggregation of these proteins, especially in combination with other wine components, remains unclear. This study demonstrates the different aggregation behavior between a buffer and a model wine system by dynamic light scattering. Arabinogalactan-protein, for example, shows an increased aggregation in the model wine system, while in the buffer system a reducing effect is observed. Thus, we could show the importance to examine the behavior of wine additives under conditions close to reality, instead of simpler buffer systems. Additional experiments on melting points of wine proteins reveal that only some isoforms of thaumatin-like proteins and chitinases are involved in haze formation. We can confirm interactions between polysaccharides and proteins, but none of these polysaccharides is able to prevent haze in wine. Copyright © 2016. Published by Elsevier Ltd.

  13. Aggregated Gas Molecules: Toxic to Protein?

    PubMed Central

    Zhang, Meng; Zuo, Guanghong; Chen, Jixiu; Gao, Yi; Fang, Haiping

    2013-01-01

    The biological toxicity of high levels of breathing gases has been known for centuries, but the mechanism remains elusive. Earlier work mainly focused on the influences of dispersed gas molecules dissolved in water on biomolecules. However, recent studies confirmed the existence of aggregated gas molecules at the water-solid interface. In this paper, we have investigated the binding preference of aggregated gas molecules on proteins with molecular dynamics simulations, using nitrogen (N2) gas and the Src-homology 3 (SH3) domain as the model system. Aggregated N2 molecules were strongly bound by the active sites of the SH3 domain, which could impair the activity of the protein. In contrast, dispersed N2 molecules did not specifically interact with the SH3 domain. These observations extend our understanding of the possible toxicity of aggregates of gas molecules in the function of proteins. PMID:23588597

  14. Protein aggregation and misfolding: good or evil?

    NASA Astrophysics Data System (ADS)

    Pastore, Annalisa; Temussi, Pierandrea

    2012-06-01

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

  15. Simulations of kinetically irreversible protein aggregate structure.

    PubMed Central

    Patro, S Y; Przybycien, T M

    1994-01-01

    We have simulated the structure of kinetically irreversible protein aggregates in two-dimensional space using a lattice-based Monte-Carlo routine. Our model specifically accounts for the intermolecular interactions between hydrophobic and hydrophilic protein surfaces and a polar solvent. The simulations provide information about the aggregate density, the types of inter-monomer contacts and solvent content within the aggregates, the type and extent of solvent exposed perimeter, and the short- and long-range order all as a function of (i) the extent of monomer hydrophobic surface area and its distribution on the model protein surface and (ii) the magnitude of the hydrophobic-hydrophobic contact energy. An increase in the extent of monomer hydrophobic surface area resulted in increased aggregate densities with concomitant decreased system free energies. These effects are accompanied by increases in the number of hydrophobic-hydrophobic contacts and decreases in the solvent-exposed hydrophobic surface area of the aggregates. Grouping monomer hydrophobic surfaces in a single contiguous stretch resulted in lower aggregate densities and lower short range order. More favorable hydrophobic-hydrophobic contact energies produced structures with higher densities but the number of unfavorable protein-protein contacts was also observed to increase; greater configurational entropy produced the opposite effect. Properties predicted by our model are in good qualitative agreement with available experimental observations. Images FIGURE 6 FIGURE 13 PMID:8061184

  16. Combining Protein Dynamics and Aggregation Measurements

    NASA Astrophysics Data System (ADS)

    Meuse, Curtis

    2015-03-01

    Infrared spectroscopy has long been used to deduce concentration and structural descriptions of proteins in a variety of static and time resolved experiments. We have developed an infrared order parameter to describe protein conformation variations around the average molecular values. Here, we combine our order parameter measurements with circular dichroism, light scattering and atomic force microscopy measurements to clarify the characterization of protein structure and aggregation. By combining the information from our suite of methods, we explore the relationship between protein stability, dynamics and aggregation. Our focus is on developing new methods to compare the structure, dynamics and function of nearly identical biopharmaceutical protein ensembles. Examples include lysozyme, albumin cytochrome c and the characterization of amyloid beta during aggregation.

  17. Protein misfolding and aggregation in Parkinson's disease.

    PubMed

    Tan, Jeanne M M; Wong, Esther S P; Lim, Kah-Leong

    2009-09-01

    Protein aggregation as a result of misfolding is a common theme underlying neurodegenerative diseases. In Parkinson's disease (PD), research on protein misfolding and aggregation has taken center stage following the association of alpha-synuclein gene mutations with familial forms of the disease, and importantly, the identification of the protein as a major component of Lewy bodies, a pathological hallmark of PD. Fueling this excitement is the subsequent identification of another PD-linked gene, parkin, as a ubiquitin ligase associated with the proteasome, a major intracellular protein degradation machinery that destroys unwanted, albeit mainly soluble, proteins. Notably, a role for parkin in the clearance of insoluble protein aggregates via macroautophagy has also been implicated by more recent studies. Paradoxically, like alpha-synuclein, parkin is also prone to misfolding, especially in the presence of age-related stress. Similarly, protein misfolding can also affect the function of other key PD-linked genes such as DJ-1, PINK1, and perhaps also LRRK2. Here, we discuss the role of protein misfolding and aggregation in PD, and how impairments of the various cellular protein quality systems could precipitate these events and lead to neuronal demise. Towards the end of our discussion, we also revisited the role of Lewy body formation in PD.

  18. Protein aggregation as an antibiotic design strategy.

    PubMed

    Bednarska, Natalia G; van Eldere, Johan; Gallardo, Rodrigo; Ganesan, Ashok; Ramakers, Meine; Vogel, Isabel; Baatsen, Pieter; Staes, An; Goethals, Marc; Hammarström, Per; Nilsson, K Peter R; Gevaert, Kris; Schymkowitz, Joost; Rousseau, Frederic

    2016-03-01

    Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases. © 2015 John Wiley & Sons Ltd.

  19. Protein Aggregation Measurement through Electrical Impedance Spectroscopy

    NASA Astrophysics Data System (ADS)

    Affanni, A.; Corazza, A.; Esposito, G.; Fogolari, F.; Polano, M.

    2013-09-01

    The paper presents a novel methodology to measure the fibril formation in protein solutions. We designed a bench consisting of a sensor having interdigitated electrodes, a PDMS hermetic reservoir and an impedance meter automatically driven by calculator. The impedance data are interpolated with a lumped elements model and their change over time can provide information on the aggregation process. Encouraging results have been obtained by testing the methodology on K-casein, a protein of milk, with and without the addition of a drug inhibiting the aggregation. The amount of sample needed to perform this measurement is by far lower than the amount needed by fluorescence analysis.

  20. The Mechanisms of Aberrant Protein Aggregation

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  1. Pathophysiological importance of aggregated damaged proteins.

    PubMed

    Höhn, Annika; Jung, Tobias; Grune, Tilman

    2014-06-01

    Reactive oxygen species (ROS) are formed continuously in the organism even under physiological conditions. If the level of ROS in cells exceeds the cellular defense capacity, components such as RNA/DNA, lipids, and proteins are damaged and modified, thus affecting the functionality of organelles as well. Proteins are especially prominent targets of various modifications such as oxidation, glycation, or conjugation with products of lipid peroxidation, leading to the alteration of their biological function, nonspecific interactions, and the production of high-molecular-weight protein aggregates. To ensure the maintenance of cellular functions, two proteolytic systems are responsible for the removal of oxidized and modified proteins, especially the proteasome and organelles, mainly the autophagy-lysosomal systems. Furthermore, increased protein oxidation and oxidation-dependent impairment of proteolytic systems lead to an accumulation of oxidized proteins and finally to the formation of nondegradable protein aggregates. Accordingly, the cellular homeostasis cannot be maintained and the cellular metabolism is negatively affected. Here we address the current knowledge of protein aggregation during oxidative stress, aging, and disease.

  2. Sequence determinants of protein aggregation: tools to increase protein solubility

    PubMed Central

    Ventura, Salvador

    2005-01-01

    Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, very often the target protein accumulates into insoluble aggregates in a misfolded and biologically inactive form. Bacterial inclusion bodies are major bottlenecks in protein production and are hampering the development of top priority research areas such structural genomics. Inclusion body formation was formerly considered to occur via non-specific association of hydrophobic surfaces in folding intermediates. Increasing evidence, however, indicates that protein aggregation in bacteria resembles to the well-studied process of amyloid fibril formation. Both processes appear to rely on the formation of specific, sequence-dependent, intermolecular interactions driving the formation of structured protein aggregates. This similarity in the mechanisms of aggregation will probably allow applying anti-aggregational strategies already tested in the amyloid context to the less explored area of protein aggregation inside bacteria. Specifically, new sequence-based approaches appear as promising tools to tune protein aggregation in biotechnological processes. PMID:15847694

  3. Protein aggregation and particle formation in prefilled glass syringes.

    PubMed

    Gerhardt, Alana; Mcgraw, Nicole R; Schwartz, Daniel K; Bee, Jared S; Carpenter, John F; Randolph, Theodore W

    2014-06-01

    The stability of therapeutic proteins formulated in prefilled syringes (PFS) may be negatively impacted by the exposure of protein molecules to silicone oil-water interfaces and air-water interfaces. In addition, agitation, such as that experienced during transportation, may increase the detrimental effects (i.e., protein aggregation and particle formation) of protein interactions with interfaces. In this study, surfactant-free formulations containing either a monoclonal antibody or lysozyme were incubated in PFS, where they were exposed to silicone oil-water interfaces (siliconized syringe walls), air-water interfaces (air bubbles), and agitation stress (occurring during end-over-end rotation). Using flow microscopy, particles (≥2 μm diameter) were detected under all conditions. The highest particle concentrations were found in agitated, siliconized syringes containing an air bubble. The particles formed in this condition consisted of silicone oil droplets and aggregated protein, as well as agglomerates of protein aggregates and silicone oil. We propose an interfacial mechanism of particle generation in PFS in which capillary forces at the three-phase (silicone oil-water-air) contact line remove silicone oil and gelled protein aggregates from the interface and transport them into the bulk. This mechanism explains the synergistic effects of silicone oil-water interfaces, air-water interfaces, and agitation in the generation of particles in protein formulations.

  4. Ageing and hypoxia cause protein aggregation in mitochondria.

    PubMed

    Kaufman, Daniel M; Wu, Xia; Scott, Barbara A; Itani, Omar A; Van Gilst, Marc R; Bruce, James E; Michael Crowder, C

    2017-10-01

    Aggregation of cytosolic proteins is a pathological finding in disease states, including ageing and neurodegenerative diseases. We have previously reported that hypoxia induces protein misfolding in Caenorhabditis elegans mitochondria, and electron micrographs suggested protein aggregates. Here, we seek to determine whether mitochondrial proteins actually aggregate after hypoxia and other cellular stresses. To enrich for mitochondrial proteins that might aggregate, we performed a proteomics analysis on purified C. elegans mitochondria to identify relatively insoluble proteins under normal conditions (110 proteins identified) or after sublethal hypoxia (65 proteins). A GFP-tagged mitochondrial protein (UCR-11 - a complex III electron transport chain protein) in the normally insoluble set was found to form widespread aggregates in mitochondria after hypoxia. Five other GFP-tagged mitochondrial proteins in the normally insoluble set similarly form hypoxia-induced aggregates. Two GFP-tagged mitochondrial proteins from the soluble set as well as a mitochondrial-targeted GFP did not form aggregates. Ageing also resulted in aggregates. The number of hypoxia-induced aggregates was regulated by the mitochondrial unfolded protein response (UPRmt) master transcriptional regulator ATFS-1, which has been shown to be hypoxia protective. An atfs-1(loss-of-function) mutant and RNAi construct reduced the number of aggregates while an atfs-1(gain-of-function) mutant increased aggregates. Our work demonstrates that mitochondrial protein aggregation occurs with hypoxic injury and ageing in C. elegans. The UPRmt regulates aggregation and may protect from hypoxia by promoting aggregation of misfolded proteins.

  5. Water Proton NMR for In Situ Detection of Insulin Aggregates.

    PubMed

    Taraban, Marc B; Truong, Huy C; Feng, Yue; Jouravleva, Elena V; Anisimov, Mikhail A; Yu, Yihua Bruce

    2015-12-01

    The need for quality control during the manufacturing and distribution of biopharmaceuticals is becoming increasingly necessary. At present, detecting drug degradation through the monitoring of active factor aggregation is accomplished through "invasive" techniques, such as size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and so on. Unfortunately, these analytical methods require sampling the drug by opening the drug container that renders the remaining drug unusable regardless of the outcome of the test. Visual inspection, the current non-invasive quality control method is qualitative and can only detect visible particulates. Thus, it will miss sub-visible protein aggregates. In this paper, human insulin preparations were used to demonstrate that the transverse relaxation rate of water protons R2 ((1) H2 O) can serve as a sensitive and reliable indicator to detect and quantify both visible and sub-visible protein aggregates. R2 ((1) H2 O) is measured using a wide-bore low-field bench-top NMR instrument with permanent magnets. Such analysis could be carried out without opening the drug container, thus saving a drug for further use. The results suggest a novel, economical, non-destructive in situ analytical technique that allows for on-the-site quantification of protein aggregation in biopharmaceutical products. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  6. Disaggregases, molecular chaperones that resolubilize protein aggregates.

    PubMed

    Mokry, David Z; Abrahão, Josielle; Ramos, Carlos H I

    2015-08-01

    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.

  7. Interfacial adsorption and aggregation of amphiphilic proteins

    NASA Astrophysics Data System (ADS)

    Cheung, David

    2012-02-01

    The adsorption and aggregation on liquid interfaces of proteins is important in many biological contexts, such as the formation of aerial structures, immune response, and catalysis. Likewise the adsorption of proteins onto interfaces has applications in food technology, drug delivery, and in personal care products. As such there has been much interest in the study of a wide range of biomolecules at liquid interfaces. One class of proteins that has attracted particular attention are hydrophobins, small, fungal proteins with a distinct, amphiphilic surface structure. This makes these proteins highly surface active and they recently attracted much interest. In order to understand their potential applications a microscopic description of their interfacial and self-assembly is necessary and molecular simulation provides a powerful tool for providing this. In this presentation I will describe some recent work using coarse-grained molecular dynamics simulations to study the interfacial and aggregation behaviour of hydrophobins. Specifically this will present the calculation of their adsorption strength at oil-water and air-water interfaces, investigate the stability of hydrophobin aggregates in solution and their interaction with surfactants.

  8. Cellular strategies for regulating functional and nonfunctional protein aggregation.

    PubMed

    Gsponer, Jörg; Babu, M Madan

    2012-11-29

    Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier's principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control.

  9. Analysis of protein aggregation kinetics.

    PubMed

    Ferrone, F

    1999-01-01

    Given a set of kinetic data, then, the preceding discussions suggest the following approach to its analysis. 1. For purposes of establishing the reaction, ignore the final stages and concentrate on the initial 10-20% of the reaction at first. A globally optimized model may be based on a faulty assumption for the initial steps. Thus, although the whole data set may look reasonably well fit, the reaction could be misrepresented, and thus the fit unhelpful if accuracy at the later stages has come at the expense of the initial phase of the reaction. 2. What is the time course of the initial reaction? (A) Is the reaction exponential? Exponential growth gives dramatic lag times (see Fig. 3), whereas nonexponential "lag times" have a visible signal from time 0 (i.e., Fig. 2). If the data set shows the abrupt appearance of signals after a period of quiescence, the chances are excellent that the time course is exponential. High sensitivity measurement of the signal at times during the lag phase should be used to confirm the exponential nature quantitatively. Exponential reactions mean a secondary pathway is operative. (a) A cascade (tn) can look similar to an exponential, but may proceed from a multistep single-path reaction. Thus the exponential needs to be ascertained with some accuracy. (b) It is possible that some or all of the lag results from a stochastic process, i.e., formation of a single nucleus being observed. This, however, is likely to be accompanied by a secondary process, as few techniques are sensitive enough to detect a single polymer at a time, and having one nucleus form many polymers is a hallmark of a secondary process. Thus, the reproducibility of the kinetics must be established to rule out stochastics. If data show wide variation, stochastic methods as described earlier may be employed. (c) Given a secondary process, one must separate the primary nucleation process from the secondary process (by stochastic means or by use of the product B2A, as

  10. Neurofilament protein aggregation in a cell line model system.

    PubMed

    Hull, Elizabeth; Spoja, Christoffer; Cordova, Matt; Cohlberg, Jeffrey A

    2008-02-01

    Protein aggregates are associated with many diseases and even aggregates of proteins that have no role in disease are inherently toxic to both neuronal and non-neuronal cells. We have developed a model system to explore the mechanism of protein aggregation using a mouse muscle cell line expressing chimeric neurofilament (NF) proteins, a constituent of the protein aggregates in ALS, Lewy body dementia, and Charcot-Marie-Tooth disease. Formation of protein aggregates in these cells leads to reduced cell viability and activated caspases. Aggregates contained both chimeric NF proteins and ubiquitin by immunolocalization and were predominately cytosolic when proteins were expressed at low levels or for shorter periods of time but were present in the nucleus when expression levels increased. This system represents a flexible, new tool to decipher the molecular mechanism of protein aggregation and the contributions of aggregation to cell toxicity.

  11. Tau protein and tau aggregation inhibitors.

    PubMed

    Bulic, Bruno; Pickhardt, Marcus; Mandelkow, Eva-Maria; Mandelkow, Eckhard

    2010-01-01

    Alzheimer disease is characterized by pathological aggregation of two proteins, tau and Abeta-amyloid, both of which are considered to be toxic to neurons. In this review we summarize recent advances on small molecule inhibitors of protein aggregation with emphasis on tau, with activities mediated by the direct interference of self-assembly. The inhibitors can be clustered in several compound classes according to their chemical structure, with subsequent description of the structure-activity relationships, showing that hydrophobic interactions are prevailing. The description is extended to the pharmacological profile of the compounds in order to evaluate their drug-likeness, with special attention to toxicity and bioavailability. The collected data indicate that following the improvements of the in vitro inhibitory potencies, the consideration of the in vivo pharmacokinetics is an absolute prerequisite for the development of compounds suitable for a transfer from bench to bedside.

  12. Role of prion protein aggregation in neurotoxicity.

    PubMed

    Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Florio, Tullio

    2012-01-01

    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.

  13. Role of Prion Protein Aggregation in Neurotoxicity

    PubMed Central

    Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Florio, Tullio

    2012-01-01

    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. PMID:22942726

  14. Protein oleogels from heat-set whey protein aggregates.

    PubMed

    de Vries, Auke; Wesseling, Anne; van der Linden, Erik; Scholten, Elke

    2017-01-15

    In this research we use heat-set whey protein aggregates (diameter∼200nm) as novel building blocks for structure formation in liquid oil to form oleogels. To transfer the aggregates to the oil phase, a solvent exchange procedure to sunflower oil was applied using acetone as an intermediate solvent. We found that agglomeration of the aggregates was prevented and the particle size in oil did not change from that in the initial aqueous phase. The small protein aggregates assemble into a space-spanning network, thereby providing solid-like properties to liquid oil. From oscillatory rheology we conclude that the aggregates are highly effective in forming a network. Already at ∼3% we found that G'>G″ and G' scales with protein concentration as G'∼cp(5.3). Applying a fractal gel network theory to the rheological data we deduce that the gels are in the strong link regime with a fractal dimension of 2.2. The results show that protein aggregates, besides their well-known functionality in aqueous solvents, are capable of forming a network in liquid oil. This provides a novel and promising way to design oleogels with tuneable rheological properties, applicable to e.g. foods, pharmaceuticals and/or cosmetics.

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

    SciTech Connect

    Giese, Armin . E-mail: Armin.Giese@med.uni-muenchen.de; Bader, Benedikt; Bieschke, Jan; Schaffar, Gregor; Odoy, Sabine; Kahle, Philipp J.; Haass, Christian; Kretzschmar, Hans

    2005-08-12

    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 {alpha}-synuclein forms co-aggregates with a mutant variant found in familial Parkinson's disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects.

  16. Subassembly aggregates of papaya mosaic virus protein.

    PubMed

    Erickson, J W; Hallett, F R; Bancroft, J B

    1983-08-01

    An examination of the number of subunits in small aggregates of papaya mosaic virus (PMV) coat protein is presented based on a model system which gives results consistent with the experimental observation that the 14 S subassembly species is a double disc, composed of two rows of nine subunits each. The estimated hydration of the disc, about 0.85 g 1H20/9 protein, is unusually large and indicates a cavitated structure for the disc. Comparison with other rod-shaped viruses suggests that the flexuous nature of PMV is a consequence of sparse axial inter-subunit contacts at high radius.

  17. Effect of antimicrobial preservatives on partial protein unfolding and aggregation.

    PubMed

    Hutchings, Regina L; Singh, Surinder M; Cabello-Villegas, Javier; Mallela, Krishna M G

    2013-02-01

    One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation 'hot-spot' reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol. All these enhanced the partial protein unfolding that includes a local region which was predicted to be the aggregation 'hot-spot'. The extent of destabilization correlated with the extent of aggregation. Further, we show that stabilizing the 'hot-spot' reduces aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas chlorobutanol causes the least protein aggregation. The same protein region acts as the 'hot-spot' for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others. Copyright © 2012 Wiley Periodicals, Inc.

  18. Emerging methods for structural analysis of protein aggregation.

    PubMed

    Khan, Eshan; Kumar, Amit

    2017-02-06

    Protein misfolding and aggregation is a key attribute of different neurodegenerative diseases. Misfolded and aggregated proteins are intrinsically disordered and rule out structure based drug design. The comprehensive characterization of misfolded proteins and associated aggregation pathway is prerequisite to develop therapeutics for neurodegenerative diseases caused due to the protein aggregation. Visible protein aggregates used to be the final stage during aggregation mechanism. The structural analysis of intermediate steps in such protein aggregates will help us to discern the conformational role and subsequently involved pathways. The structural analysis of protein aggregation using various biophysical methods may aid the improved therapeutics for protein misfolding and aggregation related neurodegenerative diseases. In this mini review, we have summarized different spectroscopic methods such as fluorescence spectroscopy, circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy for structural analysis of protein aggregation. We believe that the understanding of invisible intermediate of misfolded proteins and the key steps involved during protein aggregation mechanisms may advance the therapeutic approaches for targeting neurological diseases that are caused due to misfolded proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. In vitro aggregation of the regulated secretory protein chromogranin A.

    PubMed Central

    Jain, Renu K; Chang, Wen Tzu; Geetha, Chitta; Joyce, Paul B M; Gorr, Sven-Ulrik

    2002-01-01

    Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation. PMID:12175332

  20. ProADD: A database on Protein Aggregation Diseases

    PubMed Central

    Shobana, Ramesh; Pandaranayaka, Eswari PJ

    2014-01-01

    ProADD, a database for protein aggregation diseases, is developed to organize the data under a single platform to facilitate easy access for researchers. Diseases caused due to protein aggregation and the proteins involved in each of these diseases are integrated. The database helps in classification of proteins involved in the protein aggregation diseases based on sequence and structural analysis. Analysis of proteins can be done to mine patterns prevailing among the aggregating proteins. Availability http://bicmku.in/ProADD PMID:25097386

  1. Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations.

    PubMed

    Barnett, Gregory V; Qi, Wei; Amin, Samiul; Neil Lewis, E; Roberts, Christopher J

    2015-12-01

    Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).

  2. Investigation of protein aggregation dynamics with a Bloch surface wave sensor

    NASA Astrophysics Data System (ADS)

    Paeder, Vincent; Santi, Sara; Musi, Valeria; Herzig, Hans Peter

    2011-07-01

    We present a study of the dynamics of protein aggregation using a common path heterodyne Bloch surface wave sensing scheme. We demonstrate the ability to detect, during thermal incubation, the early events linked to the aggregation of proteins related to conformational diseases. Alzheimer's amyloid-β 1-42 is used to demonstrate the efficiency of the method. A model based on elementary interactions is shown to describe accurately the aggregation process. The described sensing scheme is sensitive to the early events of the aggregation process. is hence proposed as a method for the detection of early stages of the evolution of conformational diseases.

  3. Dynamics of proteins aggregation. I. Universal scaling in unbounded media

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

    It is well understood that in some cases proteins do not fold correctly and, depending on their environment, even properly-folded proteins change their conformation spontaneously, taking on a misfolded state that leads to protein aggregation and formation of large aggregates. An important factor that contributes to the aggregation is the interactions between the misfolded proteins. Depending on the aggregation environment, the aggregates may take on various shapes forming larger structures, such as protein plaques that are often toxic. Their deposition in tissues is a major contributing factor to many neuro-degenerative diseases, such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, and prion. This paper represents the first part in a series devoted to molecular simulation of protein aggregation. We use the PRIME, a meso-scale model of proteins, together with extensive discontinuous molecular dynamics simulation to study the aggregation process in an unbounded fluid system, as the first step toward MD simulation of the same phenomenon in crowded cellular environments. Various properties of the aggregates have been computed, including dynamic evolution of aggregate-size distribution, mean aggregate size, number of peptides that contribute to the formation of β sheets, number of various types of hydrogen bonds formed in the system, radius of gyration of the aggregates, and the aggregates' diffusivity. We show that many of such quantities follow dynamic scaling, similar to those for aggregation of colloidal clusters. In particular, at long times the mean aggregate size S(t) grows with time as, S(t) ˜ tz, where z is the dynamic exponent. To our knowledge, this is the first time that the qualitative similarity between aggregation of proteins and colloidal aggregates has been pointed out.

  4. Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

    PubMed

    Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

    2015-06-02

    Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac.

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

    PubMed Central

    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

  6. Amorphous protein aggregation monitored using fluorescence self-quenching.

    PubMed

    Jozawa, Hiroki; Kabir, Md Golam; Zako, Tamotsu; Maeda, Mizuo; Chiba, Kazuhiro; Kuroda, Yutaka

    2016-10-01

    Biophysical understanding of amorphous protein aggregation can significantly impact diverse area of biotechnology. Here, we report the time dependent salt-induced formation of amorphous aggregation as monitored by fluorescence self-quenching and compare the results with conventional methods for detecting protein aggregation [static light scattering (LS) and dynamic light scattering (DLS)]. As a model protein, we used a bovine pancreatic trypsin inhibitor (BPTI) variant extended by two glycines (C2G) at its C terminus, and three variants where three types of Solubility Controlling Peptide tags (SCP tags) made of five serines (C5S), alanines (C5A) or aspartic acids (C5D) were added to the C terminus of C2G. All variants have a native-like BPTI structure and trypsin inhibitory activity, but different solubilities controlled by the SCP tags. The BPTIs were labeled using NHS-Fluorescein (FAM) conjugated to BPTI's lysines, and we measured the changes in fluorescence intensity occurring upon the addition of NaCl. The fluorescence of all FAM-BPTIs decreased almost immediately, albeit to a different extent, upon addition of salt and became constant after 10 min for 24 h or more. On the other hand, LS and DLS signal changes were dependent on the type of tags. Namely, C2G's LS and DLS signals changed immediately, the signals of C5S and C5A tagged FAM-BPTIs increased slowly from 10 min to 24 h, and those of C5D remained constant. These observations indicated the presence of at least one intermediate step, with increased protein-protein interaction yielding a 'molecular condensation' phase. According to this model, C2G would rapidly turn from 'condensates' to aggregates, whereas C5S and C5A tagged FAM-BPTIs would do so slowly, and the soluble C5D tagged variant would remain in the molecular condensation state. © 2016 Federation of European Biochemical Societies.

  7. Protein aggregates in Huntington’s disease

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain.

    PubMed

    Currais, Antonio; Fischer, Wolfgang; Maher, Pamela; Schubert, David

    2017-01-01

    Age is, by far, the greatest risk factor for Alzheimer's disease (AD), yet few AD drug candidates have been generated that target pathways specifically associated with the aging process itself. Two ubiquitous features of the aging brain are the intracellular accumulation of aggregated proteins and inflammation. As intraneuronal amyloid protein is detected before markers of inflammation, we argue that old, age-associated, aggregated proteins in neurons can induce inflammation, resulting in multiple forms of brain toxicities. The consequence is the increased risk of old, age-associated, neurodegenerative diseases. As most of these diseases are associated with the accumulation of aggregated proteins, it is possible that any therapeutic that reduces intracellular protein aggregation will benefit all.-Currais, A., Fischer, W., Maher, P., Schubert, D. Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain.

  10. Protein misfolding, aggregation, and autophagy after brain ischemia.

    PubMed

    Luo, Tianfei; Park, Yujung; Sun, Xin; Liu, Chunli; Hu, Bingren

    2013-12-01

    Ischemic brain injury is a common disorder linked to a variety of diseases. Significant progress has been made in our understanding of the underlying mechanisms. Previous studies show that protein misfolding, aggregation, and multiple organelle damage are major pathological events in postischemic neurons. The autophagy pathway is the chief route for bulk degradation of protein aggregates and damaged organelles. The latest studies suggest that impairment of autophagy contributes to abnormal protein aggregation and organelle damages after brain ischemia. This article reviews recent studies of protein misfolding, aggregation, and impairment of autophagy after brain ischemia.

  11. Cellular Strategies for Regulating Functional and Nonfunctional Protein Aggregation

    PubMed Central

    Gsponer, Jörg; Babu, M. Madan

    2012-01-01

    Summary Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier’s principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. PMID:23168257

  12. Contrasting effects of nanoparticle-protein attraction on amyloid aggregation.

    PubMed

    Radic, Slaven; Davis, Thomas P; Ke, Pu Chun; Ding, Feng

    2015-01-01

    Nanoparticles (NPs) have been experimentally found to either promote or inhibit amyloid aggregation of proteins, but the molecular mechanisms for such complex behaviors remain unknown. Using coarse-grained molecular dynamics simulations, we investigated the effects of varying the strength of nonspecific NP-protein attraction on amyloid aggregation of a model protein, the amyloid-beta peptide implicated in Alzheimer's disease. Specifically, with increasing NP-peptide attraction, amyloid aggregation on the NP surface was initially promoted due to increased local protein concentration on the surface and destabilization of the folded state. However, further increase of NP-peptide attraction decreased the stability of amyloid fibrils and reduced their lateral diffusion on the NP surface necessary for peptide conformational changes and self-association, thus prohibiting amyloid aggregation. Moreover, we found that the relative concentration between protein and NPs also played an important role in amyloid aggregation. With a high NP/protein ratio, NPs that intrinsically promote protein aggregation may display an inhibitive effect by depleting the proteins in solution while having a low concentration of the proteins on each NP's surface. Our coarse-grained molecular dynamics simulation study offers a molecular mechanism for delineating the contrasting and seemingly conflicting effects of NP-protein attraction on amyloid aggregation and highlights the potential of tailoring anti-aggregation nanomedicine against amyloid diseases.

  13. Heme and iron induce protein aggregation.

    PubMed

    Travassos, Leonardo H; Vasconcellos, Luiz R C; Bozza, Marcelo T; Carneiro, Leticia A M

    2017-03-04

    Heme is an essential molecule expressed in many tissues where it plays key roles as the prosthetic group of several proteins involved in vital physiological and metabolic processes such as gas and electron transport. Structurally, heme is a tetrapyrrole ring containing an atom of iron (Fe) in its center. When released into the extracellular milieu, heme exerts several deleterious effects, which make it an important player in infectious and noninfectious hemolytic diseases where large amounts of free heme are observed such as malaria, dengue fever, β-thalassemia, sickle cell disease and ischemia-reperfusion. Our recent work has uncovered an unappreciated cellular response triggered by heme or Fe, one of its degradation products, on macrophages, which is the formation of protein aggregates known as aggresome-like induced structres (ALIS). This response was shown to be fully dependent on ROS production and the activation of the transcription factor NFE2L2/NRF2. In addition, we have demonstrated that heme degradation by HMOX1/HO-1 (heme oxygenase 1) is required and that Fe is essential for the formation of ALIS, as heme analogs lacking the central atom of Fe are not able to induce these structures. ALIS formation is also observed in vivo, in a model of phenylhydrazine (PHZ)-induced hemolysis, indicating that it is an integral part of the host response to excessive free heme and that it may play a role in cellular homeostasis.

  14. Targeting Protein Aggregation for the Treatment of Degenerative Diseases

    PubMed Central

    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

  15. A mechanistic insight into protein-ligand interaction, folding, misfolding, aggregation and inhibition of protein aggregates: An overview.

    PubMed

    Chandel, Tajalli Ilm; Zaman, Masihuz; Khan, Mohsin Vahid; Ali, Maroof; Rabbani, Gulam; Ishtikhar, Mohd; Khan, Rizwan Hasan

    2017-09-08

    This review article summarises the possible mechanisms of the protein-ligand interaction, folding, misfolding, aggregation and inhibition of protein aggregates. Under certain stressed condition the folding process deviates from its path and results into misfolding and aggregation of proteins. So aggregates have to be inhibited in order to cure the diseases. In some cases of protein-ligand interaction studies we have seen that the interaction of a protein with more than one ligand may show both type of quenching mechanisms i.e. dynamic as well as static quenching rather than single type of quenching mechanism, that result can be entirely different by the result of binding study utilising single ligand. So, likewise it is hypothesized that if the aggregates are inhibited by using more than one inhibitor may give more fruitful results rather than application of single inhibitor in inhibition and disaggregation of the preformed aggregates. Therefore, we have hypothesized mechanisms for the inhibition of protein aggregates that may assist in curing the neurodegenerative diseases. Thus, besides the mechanism of protein-ligand interaction, folding, misfolding and aggregation; the hypothesized mechanisms for the inhibition of protein aggregates may show new route to researchers either directly or indirectly in treating the diseases. Copyright © 2017. Published by Elsevier B.V.

  16. Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity.

    PubMed

    Galm, Lara; Amrhein, Sven; Hubbuch, Jürgen

    2016-02-08

    The aggregation of proteins became one of the major challenges in the development of biopharmaceu-ticals since the formation of aggregates can affect drug quality and immunogenicity. However, aggregation mechanisms are highly complex and the investigation requires cost, time, and material intensive experi-mental effort. In the present work, the predictive power of protein characteristics for the phase behavior of three different proteins which are very similar in size and structure was studied. In particular, the surface hydrophobicity, zeta potential, and conformational flexibility of human lysozyme, lysozyme from chicken egg white, and α-lactalbumin at pH 3, 5, 7, and 9 were assessed and examined for correlation with experimental stability studies focusing on protein phase behavior induced by sodium chloride and ammonium sulfate. The molecular dynamics (MD) simulation based study of the conformational flexibility without precipitants was able to identify highly flexible protein regions which could be associated to the less regular secondary structure elements and random coiled and terminal regions in particular. Conformational flex-ibility of the entire protein structure and protein surface hydrophobicity could be correlated to differing aggregation propensities among the studied proteins and could be identified to be applicable for predic-tion of protein phase behavior in aqueous solution without precipitants. For prediction of protein phase behavior and aggregation propensity in aqueous solution with precipitants, protein flexibility was further studied in dependency of salt concentration and species by means of human lysozyme. Even though the results of the salt dependent MD simulations could not be shown to be sufficient for prediction of salt depending phase behavior, this study revealed a more pronounced destabilizing effect of ammonium sulfate in comparison to sodium chloride and thus, was found to be in good agreement with theoretical considerations

  17. Computational approaches to understanding protein aggregation in neurodegeneration.

    PubMed

    Redler, Rachel L; Shirvanyants, David; Dagliyan, Onur; Ding, Feng; Kim, Doo Nam; Kota, Pradeep; Proctor, Elizabeth A; Ramachandran, Srinivas; Tandon, Arpit; Dokholyan, Nikolay V

    2014-04-01

    The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggregation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute significantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both all-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.

  18. Colloidal Aggregation Causes Inhibition of G Protein-Coupled Receptors

    PubMed Central

    2013-01-01

    Colloidal aggregation is the dominant mechanism for artifactual inhibition of soluble proteins, and controls against it are now widely deployed. Conversely, investigating this mechanism for membrane-bound receptors has proven difficult. Here we investigate the activity of four well-characterized aggregators against three G protein-coupled receptors (GPCRs) recognizing peptide and protein ligands. Each of the aggregators was active at micromolar concentrations against the three GPCRs in cell-based assays. This activity could be attenuated by either centrifugation of the inhibitor stock solution or by addition of Tween-80 detergent. In the absence of agonist, the aggregators acted as inverse agonists, consistent with a direct receptor interaction. Meanwhile, several literature GPCR ligands that resemble aggregators themselves formed colloids, by both physical and enzymological tests. These observations suggest that some GPCRs may be artifactually antagonized by colloidal aggregates, an effect that merits the attention of investigators in this field. PMID:23437772

  19. Ion exchange chromatography of proteins and clearance of aggregates.

    PubMed

    Yigzaw, Y; Hinckley, P; Hewig, A; Vedantham, G

    2009-06-01

    Clearance of product related aggregates in therapeutic proteins is a major focus of purification process development. A typical purification process will have one or two chromatographic steps that remove these product related aggregates to an acceptable level. Both cation exchange and anion exchange chromatography can provide robust clearance of aggregates. The primary factors that are critical for aggregate clearance are: resin chemistry, binding and elution condition, peak collection and column load factor. This review covers how these factors can be optimized to increase the effectiveness of ion exchange chromatography in removing aggregates.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Conformational stability as a design target to control protein aggregation.

    PubMed

    Costanzo, Joseph A; O'Brien, Christopher J; Tiller, Kathryn; Tamargo, Erin; Robinson, Anne Skaja; Roberts, Christopher J; Fernandez, Erik J

    2014-05-01

    Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

  2. An Evolutionary Trade-Off between Protein Turnover Rate and Protein Aggregation Favors a Higher Aggregation Propensity in Fast Degrading Proteins

    PubMed Central

    De Baets, Greet; Reumers, Joke; Delgado Blanco, Javier; Dopazo, Joaquin; Schymkowitz, Joost; Rousseau, Frederic

    2011-01-01

    We previously showed the existence of selective pressure against protein aggregation by the enrichment of aggregation-opposing ‘gatekeeper’ residues at strategic places along the sequence of proteins. Here we analyzed the relationship between protein lifetime and protein aggregation by combining experimentally determined turnover rates, expression data, structural data and chaperone interaction data on a set of more than 500 proteins. We find that selective pressure on protein sequences against aggregation is not homogeneous but that short-living proteins on average have a higher aggregation propensity and fewer chaperone interactions than long-living proteins. We also find that short-living proteins are more often associated to deposition diseases. These findings suggest that the efficient degradation of high-turnover proteins is sufficient to preclude aggregation, but also that factors that inhibit proteasomal activity, such as physiological ageing, will primarily affect the aggregation of short-living proteins. PMID:21731483

  3. The influence of protein aggregation on adsorption kinetics

    NASA Astrophysics Data System (ADS)

    Rovner, Joel; Roberts, Christopher; Furst, Eric; Hudson, Steven

    2015-03-01

    When proteins adsorb to an air-water interface they lower the surface tension and may form an age-dependent viscoelastic film. Protein adsorption to surfaces is relevant to both commercial uses and biological function. The rate at which the surface tension decreases depends strongly on temperature, solution pH, and protein structure. These kinetics also depend on the degree to which the protein is aggregated in solution. Here we explore these differences using Chymotrypsinogen as a model protein whose degree of aggregation is adjusted through controlled heat treatment and measured by chromatography. To study these effects we have used a micropipette tensiometer to produce a spherical-cap bubble whose interfacial pressure was controlled - either steady or oscillating. Short heat treatment produced small soluble aggregates, and these adsorbed faster than the original protein monomer. Longer heat treatment produced somewhat larger soluble aggregates which adsorbed more slowly. These results point to complex interactions during protein adsorption.

  4. Protein Aggregation and Its Impact on Product Quality

    PubMed Central

    Roberts, Christopher J.

    2014-01-01

    Protein pharmaceutical products are typically active as folded monomers that are composed of one or more protein chains, such as the heavy and light chains in monoclonal antibodies that are a mainstay of current drug pipelines. There are numerous possible aggregated states for a given protein, some of which are potentially useful, while most of which are considered deleterious from the perspective of pharmaceutical product quality and performance. This review provides an overview of how and why different aggregated states of proteins occur, how this potentially impacts product quality and performance, fundamental approaches to control aggregate formation, and the practical approaches that are currently used in the pharmaceutical industry. PMID:25173826

  5. Comparison of heat-induced aggregation of globular proteins.

    PubMed

    Delahaije, Roy J B M; Wierenga, Peter A; Giuseppin, Marco L F; Gruppen, Harry

    2015-06-03

    Typically, heat-induced aggregation of proteins is studied using a single protein under various conditions (e.g., temperature). Because different studies use different conditions and methods, a mechanistic relationship between molecular properties and the aggregation behavior of proteins has not been identified. Therefore, this study investigates the kinetics of heat-induced aggregation and the size/density of formed aggregates for three different proteins (ovalbumin, β-lactoglobulin, and patatin) under various conditions (pH, ionic strength, concentration, and temperature). The aggregation rate of β-lactoglobulin was slower (>10 times) than that of ovalbumin and patatin. Moreover, the conditions (pH, ionic strength, and concentration) affected the aggregation kinetics of β-lactoglobulin more strongly than for ovalbumin and patatin. In contrast to the kinetics, for all proteins the aggregate size/density increased with decreasing electrostatic repulsion. By comparing these proteins under these conditions, it became clear that the aggregation behavior cannot easily be correlated to the molecular properties (e.g., charge and exposed hydrophobicity).

  6. Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids.

    PubMed

    Horrocks, Mathew H; Lee, Steven F; Gandhi, Sonia; Magdalinou, Nadia K; Chen, Serene W; Devine, Michael J; Tosatto, Laura; Kjaergaard, Magnus; Beckwith, Joseph S; Zetterberg, Henrik; Iljina, Marija; Cremades, Nunilo; Dobson, Christopher M; Wood, Nicholas W; Klenerman, David

    2016-03-16

    The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.

  7. Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids

    PubMed Central

    2016-01-01

    The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson’s disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders. PMID:26800462

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:27043825

  10. Solubis: a webserver to reduce protein aggregation through mutation.

    PubMed

    Van Durme, Joost; De Baets, Greet; Van Der Kant, Rob; Ramakers, Meine; Ganesan, Ashok; Wilkinson, Hannah; Gallardo, Rodrigo; Rousseau, Frederic; Schymkowitz, Joost

    2016-08-01

    Protein aggregation is a major factor limiting the biotechnological and therapeutic application of many proteins, including enzymes and monoclonal antibodies. The molecular principles underlying aggregation are by now sufficiently understood to allow rational redesign of natural polypeptide sequences for decreased aggregation tendency, and hence potentially increased expression and solubility. Given that aggregation-prone regions (APRs) tend to contribute to the stability of the hydrophobic core or to functional sites of the protein, mutations in these regions have to be carefully selected in order not to disrupt protein structure or function. Therefore, we here provide access to an automated pipeline to identify mutations that reduce protein aggregation by reducing the intrinsic aggregation propensity of the sequence (using the TANGO algorithm), while taking care not to disrupt the thermodynamic stability of the native structure (using the empirical force-field FoldX). Moreover, by providing a plot of the intrinsic aggregation propensity score of APRs corrected by the local stability of that region in the folded structure, we allow users to prioritize those regions in the protein that are most in need of improvement through protein engineering. The method can be accessed at http://solubis.switchlab.org/. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. Effect of heparin on protein aggregation: inhibition versus promotion.

    PubMed

    Xu, Yisheng; Seeman, Daniel; Yan, Yunfeng; Sun, Lianhong; Post, Jared; Dubin, Paul L

    2012-05-14

    The effect of heparin on both native and denatured protein aggregation was investigated by turbidimetry and dynamic light scattering (DLS). Turbidimetric data show that heparin is capable of inhibiting and reversing the native aggregation of bovine serum albumin (BSA), β-lactoglobulin (BLG), and Zn-insulin at a pH near pI and at low ionic strength I; however, the results vary with regard to the range of pH, I, and protein-heparin stoichiometry required to achieve these effects. The kinetics of this process were studied to determine the mechanism by which interaction with heparin could result in inhibition or reversal of native protein aggregates. For each protein, the binding of heparin to distinctive intermediate aggregates formed at different times in the aggregation process dictates the outcome of complexation. This differential binding was explained by changes in the affinity of a given protein for heparin, partly due to the effects of protein charge anisotropy as visualized by electrostatic modeling. The heparin effect can be further extended to include inhibition of denaturing protein aggregation, as seen from the kinetics of BLG aggregation under conditions of thermally induced unfolding with and without heparin.

  12. Prion-like spread of protein aggregates in neurodegeneration.

    PubMed

    Polymenidou, Magdalini; Cleveland, Don W

    2012-05-07

    Protein misfolding is common to most neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Recent work using animal models with intracellular α-synuclein and tau inclusions adds decisively to a growing body of evidence that misfolded protein aggregates can induce a self-perpetuating process that leads to amplification and spreading of pathological protein assemblies. When coupled with the progressive nature of neurodegeneration, recognition of such cell-to-cell aggregate spread suggests a unifying mechanism underlying the pathogenesis of these disorders.

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

    PubMed

    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.

  14. Delaying aging in Caenorhabditis elegans with protein aggregation inhibitors.

    PubMed

    Cuanalo-Contreras, Karina; Park, Kyung-Won; Mukherjee, Abhisek; Millán-Pérez Peña, Lourdes; Soto, Claudio

    2017-01-01

    Recent evidence suggests that during aging there is widespread accumulation of aggregated insoluble proteins, even in the absence of pathological conditions. Pharmacological manipulation of protein aggregation might be helpful to unveil the involvement of protein aggregates during aging, as well as to develop novel strategies to delay aging. Here we investigated the effect of known protein aggregation inhibitors on the lifespan and health-span of Caenorhabditis elegans. For this purpose, we selected various structurally diverse anti-aggregation compounds and screened them in liquid and solid medium for their ability to alter the rate of aging in vivo. Our results show that treatment of C. elegans with diverse aggregation inhibitors significantly increases the animal lifespan and health-span. These findings indicate that protein misfolding and aggregation may play an important role in cellular dysfunction during aging, opening a novel approach to increase longevity and enhance the quality of life during aging. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Mechanisms of m-cresol induced protein aggregation studied using a model protein cytochrome c†

    PubMed Central

    Singh, Surinder M.; Hutchings, Regina L.; Mallela, Krishna M.G.

    2014-01-01

    Multi-dose protein formulations require an effective antimicrobial preservative (AP) to inhibit microbial growth during long-term storage of unused formulations. m-cresol is one such AP, but has been shown to cause protein aggregation. However, the fundamental physical mechanisms underlying such AP-induced protein aggregation are not understood. In this study, we used a model protein cytochrome c to identify the protein unfolding that triggers protein aggregation. m-cresol induced cytochrome c aggregation at preservative concentrations that are commonly used to inhibit microbial growth. Addition of m-cresol decreased the temperature at which the protein aggregated and increased the aggregation rate. However, m-cresol did not perturb the tertiary or secondary structure of cytochrome c. Instead, it populated an “invisible” partially unfolded intermediate where a local protein region around the methionine residue at position 80 was unfolded. Stabilizing the Met80 region drastically decreased the protein aggregation, which conclusively shows that this local protein region acts as an aggregation “hot-spot”. Based on these results, we propose that APs induce protein aggregation by partial rather than global unfolding. Because of the availability of site-specific probes to monitor different levels of protein unfolding, cytochrome c provided a unique advantage in characterizing the partial protein unfolding that triggers protein aggregation. PMID:21229618

  16. Aggregating tags for column-free protein purification.

    PubMed

    Lin, Zhanglin; Zhao, Qing; Xing, Lei; Zhou, Bihong; Wang, Xu

    2015-12-01

    Protein purification remains a central need for biotechnology. In recent years, a class of aggregating tags has emerged, which offers a quick, cost-effective and column-free alternative for producing recombinant proteins (and also peptides) with yield and purity comparable to that of the popular His-tag. These column-free tags induce the formation of aggregates (during or after expression) when fused to a target protein or peptide, and upon separation from soluble impurities, the target protein or peptide is subsequently released via a cleavage site. In this review, we categorize these tags as follows: (i) tags that induce inactive protein aggregates in vivo; (ii) tags that induce active protein aggregates in vivo; and (iii) tags that induce soluble expression in vivo, but aggregates in vitro. The respective advantages and disadvantages of these tags are discussed, and compared to the three conventional tags (His-tag, maltose-binding protein [MBP] tag, and intein-mediated purification with a chitin-binding tag [IMPACT-CN]). While this new class of aggregating tags is promising, more systematic tests are required to further the use. It is conceivable, however, that the combination of these tags and the more traditional columns may significantly reduce the costs for resins and columns, particularly for the industrial scale.

  17. Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls.

    PubMed

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Parcon, Paul A; Barger, Steven W; Griffin, W Sue T; Alla, Ramani; Tackett, Alan J; Mackintosh, Samuel G; Petricoin, Emanuel; Zhou, Weidong; Shmookler Reis, Robert J

    2016-10-01

    Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease-specific 'seed' proteins; however, roles of other co-aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control-subject pools using magnetic-bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high-resolution proteomics. Although total detergent-insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or Aβ1-42 pulldown, the protein constituents of Alzheimer-derived aggregates were more abundant, diverse, and post-translationally modified than those from controls. Tau- and Aβ-containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer-specific protein enrichment in tau-containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co-aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co-localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer-specific enrichment in tau-containing aggregates, were assessed for aggregation-promoting roles in C. elegans by RNA-interference 'knockdown'. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ1-42 . These observations imply shared mechanisms driving both types of aggregation, and/or aggregate-mediated cross-talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common

  18. Structural Transitions and Aggregation in Amyloidogenic Proteins

    NASA Astrophysics Data System (ADS)

    Steckmann, Timothy; Chapagain, Prem; Gerstman, Bernard; Computational and Theoretical Biophysics Group at Florida International University Team

    2014-03-01

    Amyloid fibrils are a common component in many debilitating human neurological diseases such as Alzheimer's and Parkinson's. A detailed molecular-level understanding of the formation process of amyloid fibrils is crucial for developing methods to slow down or prevent these horrific diseases. Alpha-helix to beta-sheet structural transformation is commonly observed in the process of fibril formation. We performed replica-exchange molecular dynamics simulations of structural transformations in an engineered model peptide cc-beta. Several sets of simulations with different number of cc-beta monomers were considered. Conversion of alpha-helix monomers to beta strands and the aggregation of beta strand monomers into sheets were analyzed as a function of the system size. Hydrogen bond analysis was performed and the beta-aggregate structures were characterized by a nematic order parameter.

  19. Applying microfluidic techniques in quantitative studies of protein aggregation

    NASA Astrophysics Data System (ADS)

    Herling, Therese

    2012-02-01

    Protein aggregation and fibrillation is involved in a number of devastating diseases, of which we have a limited understanding at present. Microfluidic techniques can be used in developing quantitative assays to study individual aspects of protein aggregation. Under certain conditions bovine insulin aggregates to give spherulites; spherical structures with fibrils growing and branching out from a central core. Drawing a parallel to actin polymerisation of the cell's cytoskeleton, fibril growth generates force. The force generated by polymerisation at fibril ends during spherulite growth can be measured in a microfluidic environment (TPJ Knowles et al, PNAS, 2009). By measuring the bending of four polydimethylsiloxane walls by a growing spherulite positioned in the centre, the force generated by polymerisation at fibril termini can be calculated. By growing the spherulites with a constant flow of monomer, the maximum force able to be generated by fibril growth, the stall force, can be calculated. This gives insight into the energy landscape of protein aggregation.

  20. When proteostasis goes bad: Protein aggregation in the cell.

    PubMed

    Radwan, Mona; Wood, Rebecca J; Sui, Xiaojing; Hatters, Danny M

    2017-02-01

    Protein aggregation is a hallmark of the major neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's and motor neuron and is a symptom of a breakdown in the management of proteome foldedness. Indeed, it is remarkable that under normal conditions cells can keep their proteome in a highly crowded and confined space without uncontrollable aggregation. Proteins pose a particular challenge relative to other classes of biomolecules because upon synthesis they must typically follow a complex folding pathway to reach their functional conformation (native state). Non-native conformations, including the unfolded nascent chain, are highly prone to aberrant interactions, leading to aggregation. Here we review recent advances in knowledge of proteostasis, approaches to monitor proteostasis and the impact that protein aggregation has on biology. We also include discussion of the outstanding challenges. © 2017 IUBMB Life, 69(2):49-54, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  1. Strain-dependent profile of misfolded prion protein aggregates

    PubMed Central

    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

    2016-01-01

    Prions are composed of the misfolded prion protein (PrPSc) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrPSc aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrPSc aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrPSc 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 PrPSc aggregates and the incubation periods for the strains studied. The relative presence of PrPSc 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 PrPSc aggregates in prion-induced neurodegeneration. PMID:26877167

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

    PubMed

    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.

  3. Tannin-assisted aggregation of natively unfolded proteins

    NASA Astrophysics Data System (ADS)

    Zanchi, D.; Narayanan, T.; Hagenmuller, D.; Baron, A.; Guyot, S.; Cabane, B.; Bouhallab, S.

    2008-06-01

    Tannin-protein interactions are essentially physical: hydrophobic and hydrogen-bond-mediated. We explored the tannin-assisted protein aggregation on the case of β-casein, which is a natively unfolded protein known for its ability to form micellar aggregates. We used several tannins with specified length. Our SAXS results show that small tannins increase the number of proteins per micelle, but keeping their size constant. It leads to a tannin-assisted compactization of micelles. Larger tannins, with linear dimensions greater than the crown width of micelles, lead to the aggregation of micelles by a bridging effect. Experimental results can be understood within a model where tannins are treated as effective enhancers of hydrophobic attraction between specific sites in proteins.

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

    PubMed Central

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

    2016-01-01

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

  5. Multi-block poloxamer surfactants suppress aggregation of denatured proteins.

    PubMed

    Mustafi, Devkumar; Smith, Catherine M; Makinen, Marvin W; Lee, Raphael C

    2008-01-01

    On the basis of elastic light scattering, we have compared the capacity of the multi-block, surfactant copolymers Poloxamer 108 (P108), Poloxamer 188 (P188), and Tetronic 1107 (T1107), of average molecular weight 4700, 8400, and 15,000, respectively, with that of polyethylene glycol (PEG, molecular weight 8000) to suppress aggregation of heat-denatured hen egg white lysozyme (HEWL) and bovine serum albumin (BSA). We also compared the capacity of P188 to that of PEG to suppress aggregation of carboxypeptidase A denatured in the presence of trifluoroethanol and to facilitate recovery of catalytic activity. In contrast to the multi-block copolymers, PEG had no effect in inhibiting aggregation of HEWL or of carboxypeptidase A with the recovery of catalytic activity. At very high polymer:protein ratios (>or=10:1), PEG increased aggregation of heat-denatured HEWL and BSA, consistent with its known properties to promote macromolecular crowding and crystallization of proteins. At a polymer:protein ratio of 2:1, the tetra-block copolymer T1107 was the most effective of the three surfactant copolymers, completely suppressing aggregation of heat-denatured HEWL. At a T1107:BSA ratio of 10:1, the poloxamer suppressed aggregation of heat-denatured BSA by 50% compared to that observed in the absence of the polymer. We showed that the extent of suppression of aggregation of heat-denatured proteins by multi-block surfactant copolymers is dependent on the size of the protein and the copolymer:protein molar ratio. We also concluded that at least one of the tertiary nitrogens in the ethylene-1,2-diamine structural core of the T1107 copolymer is protonated, and that this electrostatic factor underlies its capacity to suppress aggregation of denatured proteins more effectively than nonionic, multi-block poloxamers. These results indicate that amphiphilic, surfactant, multi-block copolymers are efficient as additives to suppress aggregation and to facilitate refolding of denatured

  6. The importance of sequence diversity in the aggregation and evolution of proteins.

    PubMed

    Wright, Caroline F; Teichmann, Sarah A; Clarke, Jane; Dobson, Christopher M

    2005-12-08

    Incorrect folding of proteins, leading to aggregation and amyloid formation, is associated with a group of highly debilitating medical conditions including Alzheimer's disease and late-onset diabetes. The issue of how unwanted protein association is normally avoided in a living system is particularly significant in the context of the evolution of multidomain proteins, which account for over 70% of all eukaryotic proteins, where the effective local protein concentration in the vicinity of each domain is very high. Here we describe the aggregation kinetics of multidomain protein constructs of immunoglobulin domains and the ability of different homologous domains to aggregate together. We show that aggregation of these proteins is a specific process and that the efficiency of coaggregation between different domains decreases markedly with decreasing sequence identity. Thus, whereas immunoglobulin domains with more than about 70% identity are highly prone to coaggregation, those with less than 30-40% sequence identity do not detectably interact. A bioinformatics analysis of consecutive homologous domains in large multidomain proteins shows that such domains almost exclusively have sequence identities of less than 40%, in other words below the level at which coaggregation is likely to be efficient. We propose that such low sequence identities could have a crucial and general role in safeguarding proteins against misfolding and aggregation.

  7. Artificially controlled aggregation of proteins and targeting in hematopoietic cells.

    PubMed

    Rosen, Hanna; Gao, Ying; Johnsson, Ellinor; Olsson, Inge

    2003-11-01

    The targeting mechanisms for granule proteins in hematopoietic cells are largely unknown. Aggregation is believed to be important for protein sorting-for-entry and sorting-by-retention in endocrine and neuroendocrine cells. We asked whether artificially induced multimerization/aggregation of chimeric proteins could affect their sorting in hematopoietic cells. A system was used that permits ligand-controlled intracellular oligomerization of hybrid proteins containing the FK506-binding protein (FKBP). The hybrid proteins ELA-(FKBP)3 with neutrophil elastase (ELA) and (FKBP*)4-FCS-hGH with a furin cleavage site (FCS) and human growth hormone (hGH) were expressed in the myeloblastic 32D and the rat basophilic leukemia (RBL-1) hematopoietic cell lines. ELA alone is normally targeted to secretory lysosomes. However, the hybrid proteins and ligand-induced aggregates of them were constitutively secreted and not targeted. The hGH that was released at the FCS in (FKBP*)4-FCS-hGH was also constitutively secreted. We conclude that protein multimerization/aggregation per se is not enough to facilitate sorting-for-entry to secretory lysosomes in hematopoietic cells and that improperly folded proteins may be eliminated from sorting by constitutive secretion.

  8. Protein aggregate spreading in neurodegenerative diseases: Problems and perspectives

    PubMed Central

    Lee, Seung-Jae; Lim, Hee-Sun; Masliah, Eliezer; Lee, He-Jin

    2014-01-01

    Progressive accumulation of specific protein aggregates is a defining feature of many major neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, fronto-temporal dementia, Huntington’s disease, and Creutzfeldt–Jakob disease (CJD). Findings from several recent studies have suggested that aggregation-prone proteins, such as tau, α-synuclein, polyglutamine-containing proteins, and amyloid-β, can spread to other cells and brain regions, a phenomenon considered unique to prion disorders, such as CJD and bovine spongiform encephalopathy. Cell-to-cell propagation of protein aggregates may be the general underlying principle for progressive deterioration of neurodegenerative diseases. This may also have significant implications in cell replacement therapies, as evidenced by the propagation of α-synuclein aggregates from host to grafted cells in long-term transplants in Parkinson’s patients. Here, we review recent progress in protein aggregate propagation in experimental model systems and discuss outstanding questions and future perspectives. Understanding the mechanisms of this pathological spreading may open the way to unique opportunities for development of diagnostic techniques and novel therapies for protein misfolding-associated neurodegenerative diseases. PMID:21624403

  9. Steroidal Surfactants: Detection of Premicellar Aggregation, Secondary Aggregation Changes in Micelles, and Hosting of a Highly Charged Negative Substance.

    PubMed

    Barnadas-Rodríguez, Ramon; Cladera, Josep

    2015-08-25

    CHAPSO and CHAPS are zwitterionic surfactants derived from bile salts which are usually employed in protein purification and for the preparation of liposomes and bicelles. Despite their spread use, there are significant discrepancies on the critical concentrations that determine their aggregation behavior. In this work, we study the interaction between these surfactants with the negative fluorescent dye pyranine (HPTS) by absorbance, fluorescence, and infrared spectrometry to establish their concentration-dependent aggregation. For the studied surfactants, we detect three critical concentrations showing their concentration-dependent presence as a monomeric form, premicellar aggregates, micelles, and a second type of micelle in aqueous medium. The nature of the interaction of HPTS with the surfactants was studied using analogues of their tails and the negative bile salt taurocholate (TC) as reference for the sterol ring. The results indicate that the chemical groups involved are the hydroxyl groups of the polar face of the sterol ring and the sulfonate groups of the dye. This interaction causes not only the incorporation of the negative dye in CHAPSO and CHAPS micelles but also its association with their premicellar aggregates. Surprisingly, this hosting behavior for a negative charged molecule was also detected for the negative bile salt TC, bypassing, in this way, the electrostatic repulsion between the guest and the host.

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

    PubMed

    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.

  11. Effects of Soy Protein Nanoparticle Aggregate Size on the Viscoelastic Properties of Styrene-Butadiene Composites

    USDA-ARS?s Scientific Manuscript database

    Soy protein nanoparticle aggregates were prepared by alkaline hydrolysis of soy protein isolate (SPI). Light scattering measurements indicated a narrow size distribution of SPI aggregates. Nanocomposites were formed by mixing hydrolyzed SPI (HSPI) nanoparticle aggregates with styrene-butadiene (SB...

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

    PubMed

    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.

  13. Protein aggregation and its impact on product quality.

    PubMed

    Roberts, Christopher J

    2014-12-01

    Protein pharmaceutical products are typically active as folded monomers that are composed of one or more protein chains, such as the heavy and light chains in monoclonal antibodies that are a mainstay of current drug pipelines. There are numerous possible aggregated states for a given protein, some of which are potentially useful, while most of which are considered deleterious from the perspective of pharmaceutical product quality and performance. This review provides an overview of how and why different aggregated states of proteins occur, how this potentially impacts product quality and performance, fundamental approaches to control aggregate formation, and the practical approaches that are currently used in the pharmaceutical industry. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Cellular factors modulating the mechanism of tau protein aggregation

    PubMed Central

    Fontaine, Sarah N.; Sabbagh, Jonathan J.; Baker, Jeremy; Martinez-Licha, Carlos R.; Darling, April

    2015-01-01

    Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer’s disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer’s disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones. PMID:25666877

  15. The structure and functionality of contractile forisome protein aggregates.

    PubMed

    Jaeger, Magnus S; Uhlig, Katja; Clausen-Schaumann, Hauke; Duschl, Claus

    2008-01-01

    Although they have been discovered decades ago, only in the last years forisome protein aggregates received broader attention due to their ability to convert chemical into mechanical energy. In contrast to most other motor proteins, these proteins from Fabaceae plants are independent of high-energy chemical compounds, like e.g. ATP, but undergo an anisotropic shape transition (longitudinally expanded to contracted) in response to ion concentration changes (Ca(2+), H(+), etc.), instead. We present morphological and functional data on forisomes obtained using atomic force microscopy (AFM). High-aspect ratio AFM tips allow the detailed elucidation of structural characteristics that are inaccessible with standard AFM tips. Microindentation measurements were employed to calculate the elasticity of the forisome material. Young's moduli were found to be approximately 32.7 kPa in the expanded state and approximately 2.748 kPa in the contracted state of the polymer. These results are compared to investigations where a tipless AFM cantilever was utilized to exert a load against the shape transition. In the latter experiments, an energy conversion of approximately 2.29 pJ per stroke was detected. Energetical considerations support the hypothesis that the switching process is accompanied by a change in cross-linking of the constituent subunits and allow estimating the extent of cooperativity during the pH-induced transition. Finally, useful parameters were identified and characterized that are crucial for the application of forisomes as functional elements in microfluidic chips.

  16. Interface interactions of natural rubber and protein/fiber aggregates

    USDA-ARS?s Scientific Manuscript database

    Mechanical properties of natural rubber are improved with a renewable filler for rubber applications. Aggregates of protein and fiber that constitute soy protein concentrate were shear-reduced and used to enhance the tensile modulus of the natural rubber. The aqueous dispersion of the shear-reduced ...

  17. Conformation-based assay of tau protein aggregation.

    PubMed

    Fichou, Yann; Eschmann, Neil A; Keller, Timothy J; Han, Songi

    2017-01-01

    Amyloid fiber-forming proteins are predominantly intrinsically disordered proteins (IDPs). The protein tau, present mostly in neurons, is no exception. There is a significant interest in the study of tau protein aggregation mechanisms, given the direct correlation between the deposit of β-sheet structured neurofibrillary tangles made of tau and pathology in several neurodegenerative diseases, including Alzheimer's disease. Among the core unresolved questions is the nature of the initial step triggering aggregation, with increasing attention placed on the question whether a conformational change of the IDPs plays a key role in the early stages of aggregation. Specifically, there is growing evidence that a shift in the conformation ensemble of tau is involved in its aggregation pathway, and might even dictate structural and pathological properties of mature fibers. Yet, because IDPs lack a well-defined 3D structure and continuously exchange between different conformers, it has been technically challenging to characterize their structural changes on-pathway to aggregation. Here, we make a case that double spin labeling of the β-sheet stacking region of tau combined with pulsed double electron-electron resonance spectroscopy is a powerful method to assay conformational changes occurring during the course of tau aggregation, by probing intramolecular distances around aggregation-prone domains. We specifically demonstrate the potential of this approach by presenting recent results on conformation rearrangement of the β-sheet stacking segment VQIINK (known as PHF6*) of tau. We highlight a canonical shift of the conformation ensemble, on-pathway and occurring at the earliest stage of aggregation, toward an opening of PHF6*. We expect this method to be applicable to other critical segments of tau and other IDPs. © 2017 Elsevier Inc. All rights reserved.

  18. An in vivo platform for identifying inhibitors of protein aggregation

    PubMed Central

    Mahood, Rachel A.; Jackson, Matthew P.; Revill, Charlotte H.; Foster, Richard J.; Smith, D. Alastair; Ashcroft, Alison E.; Brockwell, David J.; Radford, Sheena E.

    2015-01-01

    Protein aggregation underlies an array of human diseases, yet only one small molecule therapeutic has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of IAPP aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation. PMID:26656088

  19. Protein/Peptide Aggregation and Amyloidosis on Biointerfaces

    PubMed Central

    Lu, Qi; Tang, Qiuhan; Xiong, Yuting; Qing, Guangyan; Sun, Taolei

    2016-01-01

    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. PMID:28773858

  20. Autoimmune Responses to Soluble Aggregates of Amyloidogenic Proteins Involved in Neurodegenerative Diseases: Overlapping Aggregation Prone and Autoimmunogenic regions.

    PubMed

    Kumar, Sandeep; Thangakani, A Mary; Nagarajan, R; Singh, Satish K; Velmurugan, D; Gromiha, M Michael

    2016-02-29

    Why do patients suffering from neurodegenerative diseases generate autoantibodies that selectively bind soluble aggregates of amyloidogenic proteins? Presently, molecular basis of interactions between the soluble aggregates and human immune system is unknown. By analyzing sequences of experimentally validated T-cell autoimmune epitopes, aggregating peptides, amyloidogenic proteins and randomly generated peptides, here we report overlapping regions that likely drive aggregation as well as generate autoantibodies against the aggregates. Sequence features, that make short peptides susceptible to aggregation, increase their incidence in human T-cell autoimmune epitopes by 4-6 times. Many epitopes are predicted to be significantly aggregation prone (aggregation propensities ≥10%) and the ones containing experimentally validated aggregating regions are enriched in hydrophobicity by 10-20%. Aggregate morphologies also influence Human Leukocyte Antigen (HLA)--types recognized by the aggregating regions containing epitopes. Most (88%) epitopes that contain amyloid fibril forming regions bind HLA-DR, while majority (63%) of those containing amorphous β-aggregating regions bind HLA-DQ. More than two-thirds (70%) of human amyloidogenic proteins contain overlapping regions that are simultaneously aggregation prone and auto-immunogenic. Such regions help clear soluble aggregates by generating selective autoantibodies against them. This can be harnessed for early diagnosis of proteinopathies and for drug/vaccine design against them.

  1. Autoimmune Responses to Soluble Aggregates of Amyloidogenic Proteins Involved in Neurodegenerative Diseases: Overlapping Aggregation Prone and Autoimmunogenic regions

    PubMed Central

    Kumar, Sandeep; Thangakani, A. Mary; Nagarajan, R.; Singh, Satish K.; Velmurugan, D.; Gromiha, M. Michael

    2016-01-01

    Why do patients suffering from neurodegenerative diseases generate autoantibodies that selectively bind soluble aggregates of amyloidogenic proteins? Presently, molecular basis of interactions between the soluble aggregates and human immune system is unknown. By analyzing sequences of experimentally validated T-cell autoimmune epitopes, aggregating peptides, amyloidogenic proteins and randomly generated peptides, here we report overlapping regions that likely drive aggregation as well as generate autoantibodies against the aggregates. Sequence features, that make short peptides susceptible to aggregation, increase their incidence in human T-cell autoimmune epitopes by 4–6 times. Many epitopes are predicted to be significantly aggregation prone (aggregation propensities ≥10%) and the ones containing experimentally validated aggregating regions are enriched in hydrophobicity by 10–20%. Aggregate morphologies also influence Human Leukocyte Antigen (HLA) - types recognized by the aggregating regions containing epitopes. Most (88%) epitopes that contain amyloid fibril forming regions bind HLA-DR, while majority (63%) of those containing amorphous β-aggregating regions bind HLA-DQ. More than two-thirds (70%) of human amyloidogenic proteins contain overlapping regions that are simultaneously aggregation prone and auto-immunogenic. Such regions help clear soluble aggregates by generating selective autoantibodies against them. This can be harnessed for early diagnosis of proteinopathies and for drug/vaccine design against them. PMID:26924748

  2. SANS simulation of aggregated protein in aqueous solution

    NASA Astrophysics Data System (ADS)

    Sugiyama, Masaaki; Hamada, Kei; Kato, Koichi; Kurimoto, Eiji; Okamoto, Kenta; Morimoto, Yukio; Ikeda, Susumu; Naito, Sachio; Furusaka, Michihiko; Itoh, Keiji; Mori, Kazuhiro; Fukunaga, Toshiharu

    2009-02-01

    Small-angle neutron scattering (SANS) of aggregated protein in an aqueous solution is simulated based on the crystallographic data of the protein. After obtaining the crystallographic data of the target protein, hydrogen atoms are added to the data and then some hydrogen atoms are replaced with deuterium atoms. The structure models are made with this data and then their gyration radii and SANS intensities are calculated. Compared the calculated SANS data with the experimental one, the most probable structure is determined. With this analysis method, the aggregate structure of proteasome α7-subunit (PRSα) in an aqueous solution was investigated. Three structural models, a simple monomer and two types of dimers, were supposed as the aggregated structure of PRSα. The analysis showed that the best compromised structure was the dimer, which was consistent with electron microscopy observation.

  3. Protein aggregation induced during glass bead lysis of yeast

    PubMed Central

    Papanayotou, Irene; Sun, Beimeng; Roth, Amy F.; Davis, Nicholas G.

    2013-01-01

    Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein–protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation. PMID:20641011

  4. Effects of osmolytes on protein folding and aggregation in cells.

    PubMed

    Ignatova, Zoya; Gierasch, Lila M

    2007-01-01

    Nature has developed many strategies to ensure that the complex and challenging protein folding reaction occurs in vivo with adequate efficiency and fidelity for the success of the organism. Among the strategies widely employed in a huge range of species and cell types is the elaboration of small organic molecules called osmolytes that offset the potentially damaging effects of osmotic stress. While considerable knowledge has been gained in vitro regarding the influence of osmolytes on protein structure and folding, it is of great interest to probe the effects of osmolytes in cells. We have developed an in-cell fluorescent-labeling method that enables the study of protein stability and also protein aggregation in vivo. We utilize a genetically encoded tag called a tetra-Cys motif that binds specifically to a bis-arsenical fluorescein-based dye "FlAsH"; we inserted the tetra-Cys motif into a protein of interest in such a way that the FlAsH signal reported on the state of folding or aggregation of the protein. Then, we designed protocols to assess how various osmolytes influence the stability and propensity to aggregate of our protein of interest. These are described here. Not only are there potential biotechnological applications of osmolytes in the quest to produce greater quantities of well-folded proteins, but also osmolytes may serve as tools and points of departure for therapeutic intervention in protein folding and aggregation diseases. Having in vivo methods to analyze how osmolytes affect folding and aggregation enhances our ability to further these goals greatly.

  5. Protein aggregates and dementia: is there a common toxicity?

    PubMed Central

    Lovestone, S; McLoughlin, D

    2002-01-01

    This review considers some of the recent advances made in the understanding of the pathogenic proteins known to aggregate and be implicated in neurodegenerative dementing disorders. It concentrates on the two most obvious candidates for the role of toxic protein in Alzheimer's disease (AD)—ß-amyloid peptide and tau—but also considers other proteins in this disorder and in less common but equally devastating diseases. PMID:11796764

  6. Applied Electric Fields and the Aggregation of Highly Charged Proteins

    NASA Astrophysics Data System (ADS)

    Nemzer, Louis; Flanders, Bret; Sorensen, Christopher

    2011-03-01

    The abnormal aggregation of misfolded proteins is associated with the onset of Alzheimer's disease, along with other neurodegenerative disorders, and there is increasing evidence that prefibrillar clusters, rather than fully-formed amyloid plaques, are primarily responsible. Therefore, weakly invasive methods, such as dynamic light scattering, which can probe the size distribution and structure factor of early nuclei and proto-aggregate clusters, can serve an important role in understanding this process, and may lead to insights regarding future therapeutic interventions. Here we study a highly charged model protein, lysozyme, under the influence of applied AC and DC fields in an effort to evaluate general models of protein aggregation, including the coarse-grained ``patchy protein'' method of visualizing charge heterogeneity. This anisotropy in the interprotein interaction can lead to frustrated crystalline order, resulting in low density phases. Dynamic measurements of the size distribution and structure factor can reveal local ordering, hierarchical clustering, and fractal properties of the aggregates. Early results show that applied fields affect early cluster growth by modulating local protein and counterion concentrations, in addition to their influence on protein alignment.

  7. Prediction of "aggregation-prone" and "aggregation-susceptible" regions in proteins associated with neurodegenerative diseases.

    PubMed

    Pawar, Amol P; Dubay, Kateri F; Zurdo, Jesús; Chiti, Fabrizio; Vendruscolo, Michele; Dobson, Christopher M

    2005-07-08

    Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.

  8. Prion-like transmission of protein aggregates in neurodegenerative diseases

    PubMed Central

    Brundin, Patrik; Melki, Ronald; Kopito, Ron

    2010-01-01

    Neurodegenerative diseases are commonly associated with the accumulation of intracellular or extracellular protein aggregates. Recent studies suggest that these aggregates are capable of crossing cellular membranes and can directly contribute to the propagation of neurodegenerative disease pathogenesis. We propose that, once initiated, neuropathological changes might spread in a ‘prion-like’ manner and that disease progression is associated with the intercellular transfer of pathogenic proteins. The transfer of naked infectious particles between cells could therefore be a target for new disease-modifying therapies. PMID:20308987

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

    PubMed

    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.

  10. The Effects of Excipients on Protein Aggregation During Agitation: An Interfacial Shear Rheology Study

    PubMed Central

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

    2014-01-01

    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 spectroscopy and microflow imaging (MFI). 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, PS 80 substantially inhibited agitation-induced KGF-2 aggregation but facilitated protein particulate formation in quiescent solutions. The combination of PS 80 and heparin or sucrose completely prevented protein aggregation during both non-agitated 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 minutes 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. PMID:23712900

  11. Lessons learned from protein aggregation: toward technological and biomedical applications.

    PubMed

    Avila, César L; Chaves, Silvina; Socias, Sergio B; Vera-Pingitore, Esteban; González-Lizárraga, Florencia; Vera, Cecilia; Ploper, Diego; Chehín, Rosana

    2017-09-13

    The close relationship between protein aggregation and neurodegenerative diseases has been the driving force behind the renewed interest in a field where biophysics, neurobiology and nanotechnology converge in the study of the aggregate state. On one hand, knowledge of the molecular principles that govern the processes of protein aggregation has a direct impact on the design of new drugs for high-incidence pathologies that currently can only be treated palliatively. On the other hand, exploiting the benefits of protein aggregation in the design of new nanomaterials could have a strong impact on biotechnology. Here we review the contributions of our research group on novel neuroprotective strategies developed using a purely biophysical approach. First, we examine how doxycycline, a well-known and innocuous antibiotic, can reshape α-synuclein oligomers into non-toxic high-molecular-weight species with decreased ability to destabilize biological membranes, affect cell viability and form additional toxic species. This mechanism can be exploited to diminish the toxicity of α-synuclein oligomers in Parkinson's disease. Second, we discuss a novel function in proteostasis for extracellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in combination with a specific glycosaminoglycan (GAG) present in the extracellular matrix. GAPDH, by changing its quaternary structure from a tetramer to protofibrillar assembly, can kidnap toxic species of α-synuclein, and thereby interfere with the spreading of the disease. Finally, we review a brighter side of protein aggregation, that of exploiting the physicochemical advantages of amyloid aggregates as nanomaterials. For this, we designed a new generation of insoluble biocatalysts based on the binding of photo-immobilized enzymes onto hybrid protein:GAG amyloid nanofibrils. These new nanomaterials can be easily functionalized by attaching different enzymes through dityrosine covalent bonds.

  12. Proteostasis impairment in protein-misfolding and -aggregation diseases.

    PubMed

    Hipp, Mark S; Park, Sae-Hun; Hartl, F Ulrich

    2014-09-01

    Cells possess an extensive network of components to safeguard proteome integrity and maintain protein homeostasis (proteostasis). When this proteostasis network (PN) declines in performance, as may be the case during aging, newly synthesized proteins are no longer able to fold efficiently and metastable proteins lose their functionally active conformations, particularly under conditions of cell stress. Apart from loss-of-function effects, a critical consequence of PN deficiency is the accumulation of cytotoxic protein aggregates, which are also associated with many age-dependent neurodegenerative diseases and other medical disorders. Here we discuss recent evidence that the chronic production of aberrantly folded and aggregated proteins in these diseases is harmful by overtaxing PN capacity, setting in motion a vicious cycle of increasing proteome imbalance that eventually leads to PN collapse and cell death. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. CCT complex restricts neuropathogenic protein aggregation via autophagy

    PubMed Central

    Pavel, Mariana; Imarisio, Sara; Menzies, Fiona M.; Jimenez-Sanchez, Maria; Siddiqi, Farah H.; Wu, Xiaoting; Renna, Maurizio; O'Kane, Cahir J.; Crowther, Damian C.; Rubinsztein, David C.

    2016-01-01

    Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity. PMID:27929117

  14. Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans.

    PubMed

    Kim, Surin A; D'Acunto, Victoria F; Kokona, Bashkim; Hofmann, Jennifer; Cunningham, Nicole R; Bistline, Emily M; Garcia, F Jay; Akhtar, Nabeel M; Hoffman, Susanna H; Doshi, Seema H; Ulrich, Kathleen M; Jones, Nicholas M; Bonini, Nancy M; Roberts, Christine M; Link, Christopher D; Laue, Thomas M; Fairman, Robert

    2017-09-05

    At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington's disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.

  15. PASTA 2.0: an improved server for protein aggregation prediction.

    PubMed

    Walsh, Ian; Seno, Flavio; Tosatto, Silvio C E; Trovato, Antonio

    2014-07-01

    The formation of amyloid aggregates upon protein misfolding is related to several devastating degenerative diseases. The propensities of different protein sequences to aggregate into amyloids, how they are enhanced by pathogenic mutations, the presence of aggregation hot spots stabilizing pathological interactions, the establishing of cross-amyloid interactions between co-aggregating proteins, all rely at the molecular level on the stability of the amyloid cross-beta structure. Our redesigned server, PASTA 2.0, provides a versatile platform where all of these different features can be easily predicted on a genomic scale given input sequences. The server provides other pieces of information, such as intrinsic disorder and secondary structure predictions, that complement the aggregation data. The PASTA 2.0 energy function evaluates the stability of putative cross-beta pairings between different sequence stretches. It was re-derived on a larger dataset of globular protein domains. The resulting algorithm was benchmarked on comprehensive peptide and protein test sets, leading to improved, state-of-the-art results with more amyloid forming regions correctly detected at high specificity. The PASTA 2.0 server can be accessed at http://protein.bio.unipd.it/pasta2/. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    PubMed

    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.

  17. Aggregation propensity of critical regions of the protein Tau

    NASA Astrophysics Data System (ADS)

    Muthee, Micaiah; Ahmed, Azka; Larini, Luca

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

  18. Monte Carlo Studies of Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Jónsson, Sigurður Ægir; Staneva, Iskra; Mohanty, Sandipan; Irbäck, Anders

    The disease-linked amyloid β (Aβ) and α-synuclein (αS) proteins are both fibril-forming and natively unfolded in free monomeric form. Here, we discuss two recent studies, where we used extensive implicit solvent all-atom Monte Carlo (MC) simulations to elucidate the conformational ensembles sampled by these proteins. For αS, we somewhat unexpectedly observed two distinct phases, separated by a clear free-energy barrier. The presence of the barrier makes αS, with 140 residues, a challenge to simulate. By using a two-step simulation procedure based on flat-histogram techniques, it was possible to alleviate this problem. The barrier may in part explain why fibril formation is much slower for αS than it is for Aβ

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Evaluation of Nanoparticle Tracking for Characterization of Fibrillar Protein Aggregates

    PubMed Central

    Yang, Dennis T.; Lu, Xiaomeng; Fan, Yamin; Murphy, Regina M.

    2015-01-01

    Amyloidogenesis is the process of formation of protein aggregates with fibrillar morphology. Because amyloidogenesis is linked to neurodegenerative disease, there is interest in understanding the mechanism of fibril growth. Kinetic models of amyloidogenesis require data on the number concentration and size distribution of aggregates, but this information is difficult to obtain using conventional methods. Nanoparticle tracking analysis (NTA) is a relatively new technique that may be uniquely suited for obtaining these data. In NTA, the two-dimensional (2-D) trajectory of individual particles is tracked, from which the diffusion coefficient, and, hence, hydrodynamic radius is obtained. Here we examine the validity of NTA in tracking number concentration and size of DNA, as a model of a fibrillar macromolecule. We use NTA to examine three amyloidogenic materials: beta-amyloid, transthyretin, and polyglutamine-containing peptides. Our results are instructive in demonstrating the advantages and some limitations of single-particle diffusion measurements for investigating aggregation in protein systems. PMID:25843955

  1. Multimolecule test-tube simulations of protein unfolding and aggregation

    PubMed Central

    McCully, Michelle E.; Beck, David A. C.; Daggett, Valerie

    2012-01-01

    Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein–water hydrogen bonds were also replaced with protein–protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein–protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level. PMID:23091038

  2. Theory for the aggregation of proteins and copolymers

    SciTech Connect

    Fields, G.B.; Alonso, D.O.V.; Stigter, D.; Dill, K.A.

    1992-05-14

    We develop mean-field lattice statistical mechanics theory for the equilibrium between denatured and aggregated states of proteins and other random copolymers of hydrophobic and polar monomers in aqueous solution. We suppose that the aggregated state is a mixture of amorphous polymer plus solvent and that the driving forces are the hydrophobic interaction, which favors aggregation, and conformational and translational entropies, which favor disaggregation. The theory predicts that the phase diagram for thermal aggregation is an asymmetric closed loop, and for denaturants (guanidinium hydrochloride of urea) it is asymmetric with an upper consolute point. The theory predicts that a copolymer in a poor solvent will expand with increasing polymer concentration because of {open_quotes}screening{close_quotes} of the solvent interactions by the other chains; the chain ultimately reaches a theta-like state in the absence of solvent. The screening concentration depends strongly on the copolymer composition. We find two striking features of these copolymer phase diagrams. First, they are extraordinarily sensitive to the copolymer composition; a change of one amino acid can substantially change the aggregation behavior. Second, relative to homopolymers, copolymers should be stable against aggregation at concentrations that are higher by many orders of magnitude. 43 refs., 13 figs.

  3. Quantitative thermophoretic study of disease-related protein aggregates.

    PubMed

    Wolff, Manuel; Mittag, Judith J; Herling, Therese W; Genst, Erwin De; Dobson, Christopher M; Knowles, Tuomas P J; Braun, Dieter; Buell, Alexander K

    2016-03-17

    Amyloid fibrils are a hallmark of a range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. A detailed understanding of the physico-chemical properties of the different aggregated forms of proteins, and of their interactions with other compounds of diagnostic or therapeutic interest, is crucial for devising effective strategies against such diseases. Protein aggregates are situated at the boundary between soluble and insoluble structures, and are challenging to study because classical biophysical techniques, such as scattering, spectroscopic and calorimetric methods, are not well adapted for their study. Here we present a detailed characterization of the thermophoretic behavior of different forms of the protein α-synuclein, whose aggregation is associated with Parkinson's disease. Thermophoresis is the directed net diffusional flux of molecules and colloidal particles in a temperature gradient. Because of their low volume requirements and rapidity, analytical methods based on this effect have considerable potential for high throughput screening for drug discovery. In this paper we rationalize and describe in quantitative terms the thermophoretic behavior of monomeric, oligomeric and fibrillar forms of α-synuclein. Furthermore, we demonstrate that microscale thermophoresis (MST) is a valuable method for screening for ligands and binding partners of even such highly challenging samples as supramolecular protein aggregates.

  4. Quantitative thermophoretic study of disease-related protein aggregates

    PubMed Central

    Wolff , Manuel; Mittag, Judith J.; Herling, Therese W.; Genst, Erwin De; Dobson, Christopher M.; Knowles, Tuomas P. J.; Braun, Dieter; Buell, Alexander K.

    2016-01-01

    Amyloid fibrils are a hallmark of a range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. A detailed understanding of the physico-chemical properties of the different aggregated forms of proteins, and of their interactions with other compounds of diagnostic or therapeutic interest, is crucial for devising effective strategies against such diseases. Protein aggregates are situated at the boundary between soluble and insoluble structures, and are challenging to study because classical biophysical techniques, such as scattering, spectroscopic and calorimetric methods, are not well adapted for their study. Here we present a detailed characterization of the thermophoretic behavior of different forms of the protein α-synuclein, whose aggregation is associated with Parkinson’s disease. Thermophoresis is the directed net diffusional flux of molecules and colloidal particles in a temperature gradient. Because of their low volume requirements and rapidity, analytical methods based on this effect have considerable potential for high throughput screening for drug discovery. In this paper we rationalize and describe in quantitative terms the thermophoretic behavior of monomeric, oligomeric and fibrillar forms of α-synuclein. Furthermore, we demonstrate that microscale thermophoresis (MST) is a valuable method for screening for ligands and binding partners of even such highly challenging samples as supramolecular protein aggregates. PMID:26984748

  5. Role of arginine in the stabilization of proteins against aggregation.

    PubMed

    Baynes, Brian M; Wang, Daniel I C; Trout, Bernhardt L

    2005-03-29

    The amino acid arginine is frequently used as a solution additive to stabilize proteins against aggregation, especially in the process of protein refolding. Despite arginine's prevalence, the mechanism by which it stabilizes proteins is not presently understood. We propose that arginine deters aggregation by slowing protein-protein association reactions, with only a small concomitant effect on protein folding. The associated rate effect was observed experimentally in association of globular proteins (insulin and a monoclonal anti-insulin) and in refolding of carbonic anhydrase. We suggest that this effect arises because arginine is preferentially excluded from protein-protein encounter complexes but not from dissociated protein molecules. Such an effect is predicted by our gap effect theory [Baynes and Trout (2004) Biophys. J. 87, 1631] for "neutral crowder" additives such as arginine which are significantly larger than water but have only a small effect on the free energies of isolated protein molecules. The effect of arginine on refolding of carbonic anhydrase was also shown to be consistent with this hypothesis.

  6. Folding of Aggregated Proteins to Functionally Active Form

    DTIC Science & Technology

    2006-06-01

    detergent [31]. The detergent is then removed by washes with cyclodextrin . Because the pro- cedure can be carried out on gravity-flow columns, it can be...capitalizes on the ability of chemicals such as cyclodextrin to prevent the aggregation of renatured protein when denaturants and/or detergents are removed... cyclodextrin . The cyclodextrin is then removed by another solvent exchange and the properly folded protein released from the column. In a variation of this

  7. Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils

    PubMed Central

    Ramshini, Hassan; Parrini, Claudia; Relini, Annalisa; Zampagni, Mariagioia; Mannini, Benedetta; Pesce, Alessandra; Saboury, Ali Akbar; Nemat-Gorgani, Mohsen; Chiti, Fabrizio

    2011-01-01

    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. PMID:21249193

  8. Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection.

    PubMed

    Kokona, Bashkim; May, Carrie A; Cunningham, Nicole R; Richmond, Lynn; Jay Garcia, F; Durante, Julia C; Ulrich, Kathleen M; Roberts, Christine M; Link, Christopher D; Stafford, Walter F; Laue, Thomas M; Fairman, Robert

    2016-03-01

    This work explores the heterogeneity of aggregation of polyglutamine fusion constructs in crude extracts of transgenic Caenorhabditis elegans animals. The work takes advantage of the recent technical advances in fluorescence detection for the analytical ultracentrifuge. Further, new sedimentation velocity methods, such as the multi-speed method for data capture and wide distribution analysis for data analysis, are applied to improve the resolution of the measures of heterogeneity over a wide range of sizes. The focus here is to test the ability to measure sedimentation of polyglutamine aggregates in complex mixtures as a prelude to future studies that will explore the effects of genetic manipulation and environment on aggregation and toxicity. Using sedimentation velocity methods, we can detect a wide range of aggregates, ranging from robust analysis of the monomer species through an intermediate and quite heterogeneous population of oligomeric species, and all the way up to detecting species that likely represent intact inclusion bodies based on comparison to an analysis of fluorescent puncta in living worms by confocal microscopy. Our results support the hypothesis that misfolding of expanded polyglutamine tracts into insoluble aggregates involves transitions through a number of stable intermediate structures, a model that accounts for how an aggregation pathway can lead to intermediates that can have varying toxic or protective attributes. An understanding of the details of intermediate and large-scale aggregation for polyglutamine sequences, as found in neurodegenerative diseases such as Huntington's Disease, will help to more precisely identify which aggregated species may be involved in toxicity and disease. © 2015 The Protein Society.

  9. Multirobot autonomous landmine detection using distributed multisensor information aggregation

    NASA Astrophysics Data System (ADS)

    Jumadinova, Janyl; Dasgupta, Prithviraj

    2012-06-01

    We consider the problem of distributed sensor information fusion by multiple autonomous robots within the context of landmine detection. We assume that different landmines can be composed of different types of material and robots are equipped with different types of sensors, while each robot has only one type of landmine detection sensor on it. We introduce a novel technique that uses a market-based information aggregation mechanism called a prediction market. Each robot is provided with a software agent that uses sensory input of the robot and performs calculations of the prediction market technique. The result of the agent's calculations is a 'belief' representing the confidence of the agent in identifying the object as a landmine. The beliefs from different robots are aggregated by the market mechanism and passed on to a decision maker agent. The decision maker agent uses this aggregate belief information about a potential landmine and makes decisions about which other robots should be deployed to its location, so that the landmine can be confirmed rapidly and accurately. Our experimental results show that, for identical data distributions and settings, using our prediction market-based information aggregation technique increases the accuracy of object classification favorably as compared to two other commonly used techniques.

  10. Stability and structure of protein-polysaccharide coacervates in the presence of protein aggregates.

    PubMed

    Sanchez, Christian; Renard, Denis

    2002-08-21

    We have studied at pH 4.2 and three protein (Pr):polysaccharide (Pol) weight ratios (8:1, 2:1 and 1:1) the structure and stability of beta-lactoglobulin/acacia gum/water dispersions containing protein aggregates (BLG/AG/W) or free from aggregates (AF-BLG/AG/W). Phase diagrams were characteristic of complex coacervation. BLG/AG/W dispersions displayed a larger biphasic area than AF-BLG/AG/W dispersions, that moved towards the protein axis. It was concluded that protein aggregates affected complex coacervation both by entropic (size and molecular masses of aggregates) and enthalpic (surface properties of aggregates) effects. Laser light scattering measurements revealed that the particles diameter (d(43)) induced by demixing was controlled by protein aggregates in AF-BLG/AG/W dispersions. At 1 wt.% biopolymer concentration, particles were 15-20 times larger in AF-BLG/AG/W dispersions than in BLG/AG/W dispersions at (Pr:Pol) ratios of 2:1 or 1:1. Confocal scanning laser microscopy showed that AF-BLG/AG/W dispersions only contained spherical coacervates. BLG/AG/W dispersions contained both coacervates and aggregates coated with AG or/and BLG/AG coacervates. At a (Pr:Pol) ratio of 2:1 and 1:1, coacervates were vesicular or multivesicular. Coacervates were smaller in BLG/AG/W dispersions than in AF-BLG/AG/W dispersions. It was concluded that protein aggregates have the intrinsic ability to stabilize complex coacervates and could be used to design multifunctional delivery systems. This study showed that composite dispersions containing both protein aggregates embedded in protein-polysaccharide coacervates and free coacervates may be performed. In this respect, the design of protein aggregates with controlled size distribution and surface properties could open new possibilities both in the non-chemical control of coacervates stability and in the development of multifunctional delivery systems.

  11. Exploring energy landscapes of protein folding and aggregation.

    PubMed

    Mousseau, Normand; Derreumaux, Philippe

    2008-05-01

    Human diseases, such as Alzheimer's and Creutzfeldt-Jakob's are associated with misfolding and aggregation of specific proteins into amyloid fibrils sharing a generic cross-beta structure. The self-assembly process is complex, but once a nucleus is formed, rapid fibril formation occurs. Insight into the structures of the oligomers during the lag phase, varying between hours and days, is very difficult experimentally because these species are transient, and numerically using all-atom molecular dynamics because the time scale explored is on the order of 10-100 ns. It is therefore important to develop simplified protein models and alternative methods to sample more efficiently the conformational space. In the past few years, we have developed the activation-relaxation technique (ART nouveau) coupled to the OPEP coarse-grained force field. This review reports the application of ART-OPEP on protein folding and aggregation.

  12. Modeling of chemical inhibition from amyloid protein aggregation kinetics

    PubMed Central

    2014-01-01

    Backgrounds The process of amyloid proteins aggregation causes several human neuropathologies. In some cases, e.g. fibrillar deposits of insulin, the problems are generated in the processes of production and purification of protein and in the pump devices or injectable preparations for diabetics. Experimental kinetics and adequate modelling of chemical inhibition from amyloid aggregation are of practical importance in order to study the viable processing, formulation and storage as well as to predict and optimize the best conditions to reduce the effect of protein nucleation. Results In this manuscript, experimental data of insulin, Aβ42 amyloid protein and apomyoglobin fibrillation from recent bibliography were selected to evaluate the capability of a bivariate sigmoid equation to model them. The mathematical functions (logistic combined with Weibull equation) were used in reparameterized form and the effect of inhibitor concentrations on kinetic parameters from logistic equation were perfectly defined and explained. The surfaces of data were accurately described by proposed model and the presented analysis characterized the inhibitory influence on the protein aggregation by several chemicals. Discrimination between true and apparent inhibitors was also confirmed by the bivariate equation. EGCG for insulin (working at pH = 7.4/T = 37°C) and taiwaniaflavone for Aβ42 were the compounds studied that shown the greatest inhibition capacity. Conclusions An accurate, simple and effective model to investigate the inhibition of chemicals on amyloid protein aggregation has been developed. The equation could be useful for the clear quantification of inhibitor potential of chemicals and rigorous comparison among them. PMID:24572069

  13. Pathophysiology of protein aggregation and extended phenotyping in filaminopathy

    PubMed Central

    Serdaroglu-Oflazer, Piraye; Leber, Yvonne; Odgerel, Zagaa; van der Ven, Peter F. M.; Olivé, Montse; Ferrer, Isidro; Onipe, Adekunle; Mihaylov, Mariya; Bilbao, Juan M.; Lee, Hee S.; Höhfeld, Jörg; Djinović-Carugo, Kristina; Kong, Kester; Tegenthoff, Martin; Peters, Sören A.; Stenzel, Werner; Vorgerd, Matthias; Goldfarb, Lev G.; Fürst, Dieter O.

    2012-01-01

    Mutations in FLNC cause two distinct types of myopathy. Disease associated with mutations in filamin C rod domain leading to expression of a toxic protein presents with progressive proximal muscle weakness and shows focal destructive lesions of polymorphous aggregates containing desmin, myotilin and other proteins in the affected myofibres; these features correspond to the profile of myofibrillar myopathy. The second variant associated with mutations in the actin-binding domain of filamin C is characterized by weakness of distal muscles and morphologically by non-specific myopathic features. A frameshift mutation in the filamin C rod domain causing haploinsufficiency was also found responsible for distal myopathy with some myofibrillar changes but no protein aggregation typical of myofibrillar myopathies. Controversial data accumulating in the literature require re-evaluation and comparative analysis of phenotypes associated with the position of the FLNC mutation and investigation of the underlying disease mechanisms. This is relevant and necessary for the refinement of diagnostic criteria and developing therapeutic approaches. We identified a p.W2710X mutation in families originating from ethnically diverse populations and re-evaluated a family with a p.V930_T933del mutation. Analysis of the expanded database allows us to refine clinical and myopathological characteristics of myofibrillar myopathy caused by mutations in the rod domain of filamin C. Biophysical and biochemical studies indicate that certain pathogenic mutations in FLNC cause protein misfolding, which triggers aggregation of the mutant filamin C protein and subsequently involves several other proteins. Immunofluorescence analyses using markers for the ubiquitin–proteasome system and autophagy reveal that the affected muscle fibres react to protein aggregate formation with a highly increased expression of chaperones and proteins involved in proteasomal protein degradation and autophagy. However

  14. 4D imaging of protein aggregation in live cells.

    PubMed

    Spokoini, Rachel; Shamir, Maya; Keness, Alma; Kaganovich, Daniel

    2013-04-05

    One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances. The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state, target them for degradation by the ubiquitin-proteasome system, or direct them to protective aggregation inclusions. In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not

  15. 4D Imaging of Protein Aggregation in Live Cells

    PubMed Central

    Kaganovich, Daniel

    2013-01-01

    One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances. The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9. In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded

  16. Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae

    PubMed Central

    Plateau, Pierre; Saveanu, Cosmin; Lestini, Roxane; Dauplais, Marc; Decourty, Laurence; Jacquier, Alain; Blanquet, Sylvain; Lazard, Myriam

    2017-01-01

    Selenomethionine, a dietary supplement with beneficial health effects, becomes toxic if taken in excess. To gain insight into the mechanisms of action of selenomethionine, we screened a collection of ≈5900 Saccharomyces cerevisiae mutants for sensitivity or resistance to growth-limiting amounts of the compound. Genes involved in protein degradation and synthesis were enriched in the obtained datasets, suggesting that selenomethionine causes a proteotoxic stress. We demonstrate that selenomethionine induces an accumulation of protein aggregates by a mechanism that requires de novo protein synthesis. Reduction of translation rates was accompanied by a decrease of protein aggregation and of selenomethionine toxicity. Protein aggregation was supressed in a ∆cys3 mutant unable to synthetize selenocysteine, suggesting that aggregation results from the metabolization of selenomethionine to selenocysteine followed by translational incorporation in the place of cysteine. In support of this mechanism, we were able to detect random substitutions of cysteinyl residues by selenocysteine in a reporter protein. Our results reveal a novel mechanism of toxicity that may have implications in higher eukaryotes. PMID:28303947

  17. Failure of RQC machinery causes protein aggregation and proteotoxic stress.

    PubMed

    Choe, Young-Jun; Park, Sae-Hun; Hassemer, Timm; Körner, Roman; Vincenz-Donnelly, Lisa; Hayer-Hartl, Manajit; Hartl, F Ulrich

    2016-03-10

    Translation of messenger RNAs lacking a stop codon results in the addition of a carboxy-terminal poly-lysine tract to the nascent polypeptide, causing ribosome stalling. Non-stop proteins and other stalled nascent chains are recognized by the ribosome quality control (RQC) machinery and targeted for proteasomal degradation. Failure of this process leads to neurodegeneration by unknown mechanisms. Here we show that deletion of the E3 ubiquitin ligase Ltn1p in yeast, a key RQC component, causes stalled proteins to form detergent-resistant aggregates and inclusions. Aggregation is dependent on a C-terminal alanine/threonine tail that is added to stalled polypeptides by the RQC component, Rqc2p. Formation of inclusions additionally requires the poly-lysine tract present in non-stop proteins. The aggregates sequester multiple cytosolic chaperones and thereby interfere with general protein quality control pathways. These findings can explain the proteotoxicity of ribosome-stalled polypeptides and demonstrate the essential role of the RQC in maintaining proteostasis.

  18. Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates

    PubMed Central

    Filipe, Vasco; Hawe, Andrea

    2010-01-01

    Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates. Methods Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA. Results NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates. Conclusion NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates. PMID:20204471

  19. Benchtop Detection of Proteins

    NASA Technical Reports Server (NTRS)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2007-01-01

    A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein

  20. Computational Simulations of the Early Steps of Protein Aggregation

    PubMed Central

    Wei, Guanghong; Mousseau, Normand

    2007-01-01

    There is strong evidence that the oligomers of key proteins, formed during the early steps of aggregation, could be the primary toxic species associated with human neurodegenerative diseases, such as Alzheimer's and prion diseases. Here, we review recent progress in the development of computational approaches in order to understand the structures, dynamics and free energy surfaces of oligomers. We also discuss possible research directions for the coming years. PMID:19164927

  1. A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

    PubMed Central

    Eronina, Tatyana; Borzova, Vera; Maloletkina, Olga; Kleymenov, Sergey; Asryants, Regina; Markossian, Kira; Kurganov, Boris

    2011-01-01

    To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = Kagg(T−T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets. PMID:21760963

  2. Mechanisms of protein stabilization and prevention of protein aggregation by glycerol.

    PubMed

    Vagenende, Vincent; Yap, Miranda G S; Trout, Bernhardt L

    2009-11-24

    The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

  3. The ER Stress Surveillance (ERSU) pathway regulates daughter cell ER protein aggregate inheritance.

    PubMed

    Piña, Francisco J; Niwa, Maho

    2015-09-01

    Stress induced by cytoplasmic protein aggregates can have deleterious consequences for the cell, contributing to neurodegeneration and other diseases. Protein aggregates are also formed within the endoplasmic reticulum (ER), although the fate of ER protein aggregates, specifically during cell division, is not well understood. By simultaneous visualization of both the ER itself and ER protein aggregates, we found that ER protein aggregates that induce ER stress are retained in the mother cell by activation of the ER Stress Surveillance (ERSU) pathway, which prevents inheritance of stressed ER. In contrast, under conditions of normal ER inheritance, ER protein aggregates can enter the daughter cell. Thus, whereas cytoplasmic protein aggregates are retained in the mother cell to protect the functional capacity of daughter cells, the fate of ER protein aggregates is determined by whether or not they activate the ERSU pathway to impede transmission of the cortical ER during the cell cycle.

  4. Oligomeric and polymeric aggregates formed by proteins containing expanded polyglutamine

    PubMed Central

    Iuchi, S.; Hoffner, G.; Verbeke, P.; Djian, P.; Green, H.

    2003-01-01

    Neurological diseases resulting from proteins containing expanded polyglutamine (polyQ) are characteristically associated with insoluble neuronal inclusions, usually intranuclear, and neuronal death. We describe here oligomeric and polymeric aggregates formed in cells by expanded polyQ. These aggregates are not dissociated by concentrated formic acid, an extremely effective solvent for otherwise insoluble proteins. Perinuclear inclusions formed in cultured cells by expanded polyQ can be completely dissolved in concentrated formic acid, but a soluble protein oligomer containing the expanded polyQ and released by the formic acid is not dissociated to monomer. In Huntington's disease, a formic acid-resistant oligomer is present in cerebral cortex, but not in cerebellum. Cortical nuclei contain a polymeric aggregate of expanded polyQ that is insoluble in formic acid, does not enter polyacrylamide gels, but is retained on filters. This finding shows that the process of polymerization is more advanced in the cerebral cortex than in cultured cells. The resistance of oligomer and polymer to formic acid suggests the participation of covalent bonds in their stabilization. PMID:12591956

  5. Natural biomolecules and protein aggregation: emerging strategies against amyloidogenesis.

    PubMed

    Sgarbossa, Antonella

    2012-12-14

    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.

  6. Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide.

    PubMed

    Gambin, Yann; Polinkovsky, Mark; Francois, Bill; Giles, Nichole; Bhumkar, Akshay; Sierecki, Emma

    2016-04-30

    Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule "counting" methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.

  7. Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide

    PubMed Central

    Gambin, Yann; Polinkovsky, Mark; Francois, Bill; Giles, Nichole; Bhumkar, Akshay; Sierecki, Emma

    2016-01-01

    Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule “counting” methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays. PMID:27144560

  8. A polymer physics perspective on driving forces and mechanisms for protein aggregation.

    PubMed

    Pappu, Rohit V; Wang, Xiaoling; Vitalis, Andreas; Crick, Scott L

    2008-01-01

    Protein aggregation is a commonly occurring problem in biology. Cells have evolved stress-response mechanisms to cope with problems posed by protein aggregation. Yet, these quality control mechanisms are overwhelmed by chronic aggregation-related stress and the resultant consequences of aggregation become toxic to cells. As a result, a variety of systemic and neurodegenerative diseases are associated with various aspects of protein aggregation and rational approaches to either inhibit aggregation or manipulate the pathways to aggregation might lead to an alleviation of disease phenotypes. To develop such approaches, one needs a rigorous and quantitative understanding of protein aggregation. Much work has been done in this area. However, several unanswered questions linger, and these pertain primarily to the actual mechanism of aggregation as well as to the types of inter-molecular associations and intramolecular fluctuations realized at low protein concentrations. It has been suggested that the concepts underlying protein aggregation are similar to those used to describe the aggregation of synthetic polymers. Following this suggestion, the relevant concepts of polymer aggregation are introduced. The focus is on explaining the driving forces for polymer aggregation and how these driving forces vary with chain length and solution conditions. It is widely accepted that protein aggregation is a nucleation-dependent process. This view is based mainly on the presence of long times for the accumulation of aggregates and the elimination of these lag times with "seeds". In this sense, protein aggregation is viewed as being analogous to the aggregation of colloidal particles. The theories for polymer aggregation reviewed in this work suggest an alternative mechanism for the origin of long lag times in protein aggregation. The proposed mechanism derives from the recognition that polymers have unique dynamics that distinguish them from other aggregation-prone systems such as

  9. A polymer physics perspective on driving forces and mechanisms for protein aggregation

    PubMed Central

    Pappu, Rohit V.; Wang, Xiaoling; Vitalis, Andreas; Crick, Scott L.

    2008-01-01

    Protein aggregation is a commonly occurring problem in biology. Cells have evolved stress-response mechanisms to cope with problems posed by protein aggregation. Yet, these quality control mechanisms are overwhelmed by chronic aggregation-related stress and the resultant consequences of aggregation become toxic to cells. As a result, a variety of systemic and neurodegenerative diseases are associated with various aspects of protein aggregation and rational approaches to either inhibit aggregation or manipulate the pathways to aggregation might lead to an alleviation of disease phenotypes. To develop such approaches, one needs a rigorous and quantitative understanding of protein aggregation. Much work has been done in this area. However, several unanswered questions linger, and these pertain primarily to the actual mechanism of aggregation as well as to the types of intermolecular associations and intramolecular fluctuations realized at low protein concentrations. It has been suggested that the concepts underlying protein aggregation are similar to those used to describe the aggregation of synthetic polymers. Following this suggestion, the relevant concepts of polymer aggregation are introduced. The focus is on explaining the driving forces for polymer aggregation and how these driving forces vary with chain length and solution conditions. It is widely accepted that protein aggregation is a nucleation-dependent process. This view is based mainly on the presence of long times for the accumulation of aggregates and the elimination of these lag times with “seeds”. In this sense, protein aggregation is viewed as being analogous to the aggregation of colloidal particles. The theories for polymer aggregation reviewed in this work suggest an alternative mechanism for the origin of long lag times in protein aggregation. The proposed mechanism derives from the recognition that polymers have unique dynamics that distinguish them from other aggregation-prone systems such

  10. Protein aggregation, misfolding and consequential human neurodegenerative diseases.

    PubMed

    Sami, Neha; Rahman, Safikur; Kumar, Vijay; Zaidi, Sobia; Islam, Asimul; Ali, Sher; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2017-11-01

    Proteins are major components of the biological functions in a cell. Biology demands that a protein must fold into its stable three-dimensional structure to become functional. In an unfavorable cellular environment, protein may get misfolded resulting in its aggregation. These conformational disorders are directly related to the tissue damage resulting in cellular dysfunction giving rise to different diseases. This way, several neurodegenerative diseases such as Alzheimer, Parkinson Huntington diseases and amyotrophic lateral sclerosis are caused. Misfolding of the protein is prevented by innate molecular chaperones of different classes. It is envisaged that work on this line is likely to translate the knowledge into the development of possible strategies for early diagnosis and efficient management of such related human diseases. The present review deals with the human neurodegenerative diseases caused due to the protein misfolding highlighting pathomechanisms and therapeutic intervention.

  11. Detection and quantification of tau aggregation using a membrane filter assay.

    PubMed

    Chang, Edward; Kuret, Jeff

    2008-02-15

    Aggregation of the microtubule-associated protein tau contributes to the formation of neurofibrillary lesions in Alzheimer's disease and is a useful marker of disease progression. Although filter trap assays have been employed to assess the extent of tau aggregation in cells and tissues as well as in vitro, their performance relative to other assay modalities has not been reported. To clarify this issue, the ability of the filter trap approach to quantify aggregation of purified recombinant full-length tau protein in vitro was examined as a function of membrane chemistry in a 96-well format. Results showed that nitrocellulose yielded the greatest assay sensitivity relative to polyvinylidene fluoride or cellulose acetate at equal membrane porosity. However, all combinations of filter chemistries, porosities, and monoclonal detection antibodies yielded nonlinear correlations between signal intensity and analyte concentration. When corrected for nonlinearity, the filter trap assay determined a value for the critical monomer concentration for tau aggregation that was statistically identical to determinations made by electron microscopy assay. The data suggest conditions under which filter trap assays can be used to estimate tau aggregation kinetics.

  12. Distinct stress conditions result in aggregation of proteins with similar properties

    PubMed Central

    Weids, Alan J.; Ibstedt, Sebastian; Tamás, Markus J.; Grant, Chris M.

    2016-01-01

    Protein aggregation is the abnormal association of proteins into larger aggregate structures which tend to be insoluble. This occurs during normal physiological conditions and in response to age or stress-induced protein misfolding and denaturation. In this present study we have defined the range of proteins that aggregate in yeast cells during normal growth and after exposure to stress conditions including an oxidative stress (hydrogen peroxide), a heavy metal stress (arsenite) and an amino acid analogue (azetidine-2-carboxylic acid). Our data indicate that these three stress conditions, which work by distinct mechanisms, promote the aggregation of similar types of proteins probably by lowering the threshold of protein aggregation. The proteins that aggregate during physiological conditions and stress share several features; however, stress conditions shift the criteria for protein aggregation propensity. This suggests that the proteins in aggregates are intrinsically aggregation-prone, rather than being proteins which are affected in a stress-specific manner. We additionally identified significant overlaps between stress aggregating yeast proteins and proteins that aggregate during ageing in yeast and C. elegans. We suggest that similar mechanisms may apply in disease- and non-disease settings and that the factors and components that control protein aggregation may be evolutionary conserved. PMID:27086931

  13. Distinct stress conditions result in aggregation of proteins with similar properties.

    PubMed

    Weids, Alan J; Ibstedt, Sebastian; Tamás, Markus J; Grant, Chris M

    2016-04-18

    Protein aggregation is the abnormal association of proteins into larger aggregate structures which tend to be insoluble. This occurs during normal physiological conditions and in response to age or stress-induced protein misfolding and denaturation. In this present study we have defined the range of proteins that aggregate in yeast cells during normal growth and after exposure to stress conditions including an oxidative stress (hydrogen peroxide), a heavy metal stress (arsenite) and an amino acid analogue (azetidine-2-carboxylic acid). Our data indicate that these three stress conditions, which work by distinct mechanisms, promote the aggregation of similar types of proteins probably by lowering the threshold of protein aggregation. The proteins that aggregate during physiological conditions and stress share several features; however, stress conditions shift the criteria for protein aggregation propensity. This suggests that the proteins in aggregates are intrinsically aggregation-prone, rather than being proteins which are affected in a stress-specific manner. We additionally identified significant overlaps between stress aggregating yeast proteins and proteins that aggregate during ageing in yeast and C. elegans. We suggest that similar mechanisms may apply in disease- and non-disease settings and that the factors and components that control protein aggregation may be evolutionary conserved.

  14. Multiple benefits of gregariousness cover detectability costs in aposematic aggregations.

    PubMed

    Riipi, M; Alatalo, R V; Lindström, L; Mappes, J

    2001-10-04

    Understanding the early evolution of aposematic (warning) coloration has been a challenge for scientists, as a new conspicuous morph in a population of cryptic insects would have a high predation risk and would probably die out before local predators learnt to avoid it. Fisher presented the idea of aggregation benefit through the survival of related individuals; however, his theory has been strongly debated as the mechanisms that favour grouping have never been explored experimentally with the incorporation of detectability costs. Here we create a comprehensive 'novel world' experiment with the great tit (Parus major) as a predator to explore simultaneously the predation-related benefits and costs for aposematic aggregated prey, manipulating both group size and signal strength. Our results show that grouping would have been highly beneficial for the first aposematic prey individuals surrounded by naive predators, because (1) detectability risk increased only asymptotically with group size; (2) additional detectability costs due to conspicuous signals were marginal in groups; (3) even naive predators deserted the group after detecting unpalatability (dilution effect); and (4) avoidance learning of signal was faster in groups. None of these mechanisms require kin selection.

  15. Tom40 protein import channel binds to non-native proteins and prevents their aggregation.

    PubMed

    Esaki, Masatoshi; Kanamori, Takashi; Nishikawa, Shuh-ichi; Shin, Injae; Schultz, Peter G; Endo, Toshiya

    2003-12-01

    Mitochondria contain the translocator of the outer mitochondrial membrane (TOM) for protein entry into the organelle, and its subunit Tom40 forms a protein-conducting channel. Here we report the role of Tom40 in protein translocation across the membrane. The site-specific photocrosslinking experiment revealed that translocating unfolded or loosely folded precursor segments of up to 90 residues can be associated with Tom40. Purified Tom40 bound to non-native proteins and suppressed their aggregation when they are prone to aggregate. A denatured protein bound to the Tom40 channel blocked the protein import into mitochondria. These results indicate that, in contrast to the nonstick tunnel of the ribosome for polypeptide exit, the Tom40 channel offers an optimized environment to translocating non-native precursor proteins by preventing their aggregation.

  16. Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

    PubMed Central

    Shin, I.; Wachtel, E.; Roth, E.; Bon, C.; Silman, I.; Weiner, L.

    2002-01-01

    A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45°C, of single molecules entrapped within the reverse micelles (0.031 min−1), was higher than in aqueous solution (0.007 min−1) or in the presence of normal micelles (0.020 min−1). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53°C, were 0.317 and 0.342 min−1, respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles. PMID:12142456

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

    PubMed

    Povarova, Natalia V; Petri, Natalia D; Blokhina, Anna E; Bogdanov, Alexey M; Gurskaya, Nadya G; Lukyanov, Konstantin A

    2017-07-12

    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.

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

    PubMed Central

    Povarova, Natalia V.; Petri, Natalia D.; Blokhina, Anna E.; Bogdanov, Alexey M.; Lukyanov, Konstantin A.

    2017-01-01

    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. PMID:28704934

  19. A multi-dimensional Structure-Activity Relationship of a protein in its aggregated states

    PubMed Central

    Wang, Lei; Schubert, David; Sawaya, Michael R.; Eisenberg, David; Riek, Roland

    2010-01-01

    Protein aggregates are both associated with disease and function. Because a variety of factors induce protein aggregation, a given protein can aggregate into different states. Here, we compare the structures and activities of five distinct protein aggregates of a single protein. Despite the diverse chemical, physical and biological treatments used to induce aggregation, all aggregate types contain the cross-β-sheet motif. However, they are structurally distinct, having different segments of the protein sequence involved in secondary structure formation. Because of these structural differences each aggregate has a unique set of properties. These include affinity to ATP, Thioflavin T, DNA, and membrane mimics, and interference with cell viability. The key to their multiple properties may be that the repetitive nature of the cross-β-sheet motif guarantees for many potent activities through cooperativity. The observed multidimensional structure-activity relationship of protein aggregates may be important for amyloid diseases but may also be advantageous in nanotechnology. PMID:20397175

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

    PubMed Central

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

    2016-01-01

    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. PMID:27612497

  1. Probing Proteins and Differentiating Their Native and Denatured States with Aggregation-Induced Emission Fluorogen

    NASA Astrophysics Data System (ADS)

    Leung, Chris Wai Tung; Hong, Yuning; Tang, Ben Zhong

    2013-06-01

    The tertiary 3D structures of proteins determine their unique functions. Perturbation of their native state including denaturation may cause loss of the protein functions. In this work, water-soluble tetraphenylethylene (TPE) fluorophore, sodium 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene (BSPOTPE), with aggregation-induced emission (AIE) characteristics is utilized as a fluorescent probe for protein detection and for differentiating their folding modes. Owing to hydrophobic interaction between the proteins and BSPOTPE, it provides a fast and simple method to differentiate the native and denatured states of the proteins through monitoring fluorescence change in solution and PAGE gels. Six proteins are chosen as model proteins in the study. Among them, cytochrome c shows distinctive behavior to other proteins due to the presence of heme group. A comprehensive study of cytochrome c and human serum albumin is carried out in this work.

  2. Formaldehyde at Low Concentration Induces Protein Tau into Globular Amyloid-Like Aggregates In Vitro and In Vivo

    PubMed Central

    Nie, Chun Lai; Wei, Yan; Chen, Xinyong; Liu, Yan Ying; Dui, Wen; Liu, Ying; Davies, Martyn C.; Tendler, Saul J.B.; He, Rong Giao

    2007-01-01

    Recent studies have shown that neurodegeneration is closely related to misfolding and aggregation of neuronal tau. Our previous results show that neuronal tau aggregates in formaldehyde solution and that aggregated tau induces apoptosis of SH-SY5Y and hippocampal cells. In the present study, based on atomic force microscopy (AFM) observation, we have found that formaldehyde at low concentrations induces tau polymerization whilst acetaldehyde does not. Neuronal tau misfolds and aggregates into globular-like polymers in 0.01–0.1% formaldehyde solutions. Apart from globular-like aggregation, no fibril-like polymerization was observed when the protein was incubated with formaldehyde for 15 days. SDS-PAGE results also exhibit tau polymerizing in the presence of formaldehyde. Under the same experimental conditions, polymerization of bovine serum albumin (BSA) or α-synuclein was not markedly detected. Kinetic study shows that tau significantly misfolds and polymerizes in 60 minutes in 0.1% formaldehyde solution. However, presence of 10% methanol prevents protein tau from polymerization. This suggests that formaldehyde polymerization is involved in tau aggregation. Such aggregation process is probably linked to the tau's special “worm-like” structure, which leaves the ε-amino groups of Lys and thiol groups of Cys exposed to the exterior. Such a structure can easily bond to formaldehyde molecules in vitro and in vivo. Polymerizing of formaldehyde itself results in aggregation of protein tau. Immunocytochemistry and thioflavin S staining of both endogenous and exogenous tau in the presence of formaldehyde at low concentrations in the cell culture have shown that formaldehyde can induce tau into amyloid-like aggregates in vivo during apoptosis. The significant protein tau aggregation induced by formaldehyde and the severe toxicity of the aggregated tau to neural cells may suggest that toxicity of methanol and formaldehyde ingestion is related to tau misfolding and

  3. Amyloid Aggregation and Membrane Disruption by Amyloid Proteins

    NASA Astrophysics Data System (ADS)

    Ramamoorthy, Ayyalusamy

    2013-03-01

    Amyloidogenesis has been the focus of intense basic and clinical research, as an increasing number of amyloidogenic proteins have been linked to common and incurable degenerative diseases including Alzheimer's, type II diabetes, and Parkinson's. Recent studies suggest that the cell toxicity is mainly due to intermediates generated during the assembly process of amyloid fibers, which have been proposed to attack cells in a variety of ways. Disruption of cell membranes is believed to be one of the key components of amyloid toxicity. However, the mechanism by which this occurs is not fully understood. Our research in this area is focused on the investigation of the early events in the aggregation and membrane disruption of amyloid proteins, Islet amyloid polypeptide protein (IAPP, also known as amylin) and amyloid-beta peptide, on the molecular level. Structural insights into the mechanisms of membrane disruption by these amyloid proteins and the role of membrane components on the membrane disruption will be presented.

  4. Control the aggregation of model amyloid insulin protein under ac-electric fields

    NASA Astrophysics Data System (ADS)

    Zheng, Zhongli; Jing, Benxin; Zhu, Y. Elaine

    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 an alternative engineering approach to manipulate and control protein aggregation, we have investigated the aggregation kinetics of insulin, a well-established amyloid model protein, under applied ac-electric fields of varied ac-frequency and voltage at room temperature. Using fluorescence correlation spectroscopy and fluorescence imaging, we have observed that the insulin aggregation can occur at much shortened lag time under applied ac-electric fields, when 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 difference of 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.

  5. Protein engineering to stabilize soluble amyloid β-protein aggregates for structural and functional studies.

    PubMed

    Härd, Torleif

    2011-10-01

    The molecular biology underlying protein aggregation and neuronal death in Alzheimer's disease is not yet completely understood, but small soluble nonamyloid aggregates of the amyloid β-protein (Aβ) have been shown to play a fundamental neurotoxic role. The composition and biological action of such aggregates, known as oligomers and protofibrils, are therefore areas of intense study. However, research is complicated by the multitude of different interconverting aggregates that Aβ can form in vitro and in vivo, and by the inhomogeneity and instability of in vitro preparations. Here we review recent studies in which protein engineering, and in particular disulfide engineering, has been applied to stabilize different Aβ aggregates. For example, several techniques now exist to obtain stable and neurotoxic protofibrillar forms of Aβ, and engineered Aβ dimers, or larger aggregates formed by these, have been shown to specifically induce neuronal damage in a way that mimics Alzheimer's disease pathology. Disulfide engineering has also revealed structural properties of neurotoxic aggregates, for instance that Aβ in protofibrils and globular oligomers adopts a β-hairpin conformation that is similar to, but topologically distinct from, the conformation of Aβ in mature amyloid fibrils. Protein engineering is therefore a workable strategy to address many of the outstanding questions relating to the structure, interconversion and biological effects of oligomers and protofibrils of Aβ.

  6. F-box protein 7 mutations promote protein aggregation in mitochondria and inhibit mitophagy.

    PubMed

    Zhou, Zhi Dong; Xie, Shao Ping; Sathiyamoorthy, Sushmitha; Saw, Wuan Ting; Sing, Tan Ye; Ng, Shin Hui; Chua, Heidi Pek Hup; Tang, Alyssa Mei Yan; Shaffra, Fathima; Li, Zeng; Wang, Hongyan; Ho, Patrick Ghim Hoe; Lai, Mitchell Kim Peng; Angeles, Dario C; Lim, Tit Meng; Tan, Eng-King

    2015-11-15

    The mutations of F-box protein 7 (FBXO7) gene (T22M, R378G and R498X) are associated with a severe form of autosomal recessive juvenile-onset Parkinson's disease (PD) (PARK 15). Here we demonstrated that wild-type (WT) FBXO7 is a stress response protein and it can play both cytoprotective and neurotoxic roles. The WT FBXO7 protein is vital to cell mitophagy and can facilitate mitophagy to protect cells, whereas mutant FBXO7 inhibits mitophagy. Upon stress, the endogenous WT FBXO7 gets up-regulated, concentrates into mitochondria and forms FBXO7 aggregates in mitochondria. However, FBXO7 mutations aggravate deleterious FBXO7 aggregation in mitochondria. The FBXO7 aggregation and toxicity can be alleviated by Proline, glutathione (GSH) and coenzyme Q10, whereas deleterious FBXO7 aggregation in mitochondria can be aggravated by prohibitin 1 (PHB1), a mitochondrial protease inhibitor. The overexpression of WT FBXO7 could lead to FBXO7 protein aggregation and dopamine neuron degeneration in transgenic Drosophila heads. The elevated FBXO7 expression and aggregation were identified in human fibroblast cells from PD patients. FBXO7 can also form aggregates in brains of PD and Alzheimer's disease. Our study provides novel pathophysiologic insights and suggests that FBXO7 may be a potential therapeutic target in FBXO7-linked neuron degeneration in PD.

  7. Self-Templated Nucleation in Peptide and Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Auer, Stefan; Dobson, Christopher M.; Vendruscolo, Michele; Maritan, Amos

    2008-12-01

    Peptides and proteins exhibit a common tendency to assemble into highly ordered fibrillar aggregates, whose formation proceeds in a nucleation-dependent manner that is often preceded by the formation of oligomeric assemblies. This process has received much attention because disordered oligomeric aggregates have been associated with neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Here we describe a self-templated nucleation mechanism that determines the transition between the initial condensation of polypeptide chains into disordered assemblies and their reordering into fibrillar structures. The results that we present show that at the molecular level this transition is due to the ability of polypeptide chains to reorder within oligomers into fibrillar assemblies whose surfaces act as templates that stabilize the disordered assemblies.

  8. Inhibiting toxic aggregation of amyloidogenic proteins: a therapeutic strategy for protein misfolding diseases.

    PubMed

    Cheng, Biao; Gong, Hao; Xiao, Hongwen; Petersen, Robert B; Zheng, Ling; Huang, Kun

    2013-10-01

    The deposition of self-assembled amyloidogenic proteins is associated with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases. In this perspective review, we provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clinical applications thereof. Compounds such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity. Some inhibitors have been tested in clinical trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases. © 2013.

  9. Anomalous Formation of Multilayer Protein Aggregates on the Surface of Nanotubular TiO2

    NASA Astrophysics Data System (ADS)

    Forstater, Jacob; Kleinhammes, Alfred; Wu, Yue

    2012-02-01

    Significant evidence links protein aggregation to the pathology and progression of most protein misfolding diseases. Protein aggregation also poses a significant problem for the safe and cost-effective production of therapeutic proteins. A comprehensive understanding of these problems requires both a detailed understanding native protein-protein interactions as well as an understanding of how protein-material interactions may alter protein aggregation phenomenon. Here we report on the anomalous formation of multilayered protein aggregates of globular proteins on the surface of TiO2 nanotubes. Our findings suggest that minor alterations of the surface hydration properties of the nanotubes may drastically alter protein aggregation phenomenon. We further highlight the role of electrostatic and Van der Waals forces in this aggregation process.

  10. Designed protein aggregates entrapping carbon nanotubes for bioelectrochemical oxygen reduction.

    PubMed

    Garcia, Kristen E; Babanova, Sofia; Scheffler, William; Hans, Mansij; Baker, David; Atanassov, Plamen; Banta, Scott

    2016-11-01

    The engineering of robust protein/nanomaterial interfaces is critical in the development of bioelectrocatalytic systems. We have used computational protein design to identify two amino acid mutations in the small laccase protein (SLAC) from Streptomyces coelicolor to introduce new inter-protein disulfide bonds. The new dimeric interface introduced by these disulfide bonds in combination with the natural trimeric structure drive the self-assembly of SLAC into functional aggregates. The mutations had a minimal effect on kinetic parameters, and the enzymatic assemblies exhibited an increased resistance to irreversible thermal denaturation. The SLAC assemblies were combined with single-walled carbon nanotubes (SWNTs), and explored for use in oxygen reduction electrodes. The incorporation of SWNTs into the SLAC aggregates enabled operation at an elevated temperature and reduced the reaction overpotential. A current density of 1.1 mA/cm(2) at 0 V versus Ag/AgCl was achieved in an air-breathing cathode system. Biotechnol. Bioeng. 2016;113: 2321-2327. © 2016 Wiley Periodicals, Inc.

  11. Orthogonal cross-seeding: an approach to explore protein aggregates in living cells.

    PubMed

    Hinz, Justyna; Gierasch, Lila M; Ignatova, Zoya

    2008-04-08

    Protein aggregation is associated with the pathology of many diseases, especially neurodegenerative diseases. A variety of structurally polymorphic aggregates or preaggregates including amyloid fibrils is accessible to any aggregating protein. Preaggregates are now believed to be the toxic culprits in pathologies rather than mature aggregates. Although clearly valuable, understanding the mechanism of formation and the structural characteristics of these prefibrillar species is currently lacking. We report here a simple new approach to map the nature of the aggregate core of transient aggregated species directly in the cell. The method is conceptually based on the highly discriminating ability of aggregates to recruit new monomeric species with equivalent molecular structure. Different soluble segments comprising parts of an amyloidogenic protein were transiently pulse-expressed in a tightly controlled, time-dependent manner along with the parent aggregating full-length protein, and their recruitment into the insoluble aggregate was monitored immunochemically. We used this approach to determine the nature of the aggregate core of the metastable aggregate species formed during the course of aggregation of a chimera containing a long polyglutamine repeat tract in a bacterial host. Strikingly, we found that different segments of the full-length protein dominated the aggregate core at different times during the course of aggregation. In its simplicity, the approach is also potentially amenable to screen also for compounds that can reshape the aggregate core and induce the formation of alternative nonamyloidogenic species.

  12. Simulating protein folding and aggregation on the 10 second timescale

    NASA Astrophysics Data System (ADS)

    Pande, Vijay

    2007-03-01

    Understanding how proteins self-assemble or ``fold'' is a fundamental problem in biophysics. Moreover, the ability to understand and quantitatively predict folding kinetics would have many implications, especially in the area of diseases related to protein misfolding, such as Alzheimer's Disease. However, there are many challenges to simulating folding, most notably the great computational challenges of simulating protein folding with models with sufficient accuracy to make quantitative predictions of experiments. In my talk, I will discuss our recent work to combine distributed computing with a new theoretical technique (Markov State Models) in order to simulate folding on long timescales as well as the direct and quantitative experimental tests of these methods. I will conclude with the application of these methods to the study of the Abeta peptide, whose aggregation has been directly implicated as the toxic element in Alzheimer's Disease.

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

    NASA Astrophysics Data System (ADS)

    Dasgupta, Anushka

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

  14. Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA

    PubMed Central

    Pathak, Bani K.; Mondal, Surojit; Banerjee, Senjuti; Ghosh, Amar Nath; Barat, Chandana

    2017-01-01

    An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity. PMID:28169307

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

    PubMed Central

    Ibstedt, Sebastian; Sideri, Theodora C.; Grant, Chris M.; Tamás, Markus J.

    2014-01-01

    ABSTRACT 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. PMID:25217615

  16. Rapid detection of Aβ aggregation and inhibition by dual functions of gold nanoplasmic particles: catalytic activator and optical reporter.

    PubMed

    Choi, Inhee; Lee, Luke P

    2013-07-23

    One of the primary pathological hallmarks of Alzheimer's diseases (AD) is amyloid-β (Aβ) aggregation and its extracellular accumulation. However, current in vitro Aβ aggregation assays require time-consuming and labor-intensive steps, which delay the process of drug discovery and understanding the mechanism of Aβ induced neurotoxicity. Here, we propose a rapid detection method for studying Aβ aggregation and inhibition under an optimized acidic perturbation condition by dual functions of gold nanoplasmonic particles (GNPs): (1) catalytic activator and (2) optical reporter. Because of roles of GNPs as effective nucleation sites for fast-catalyzing Aβ aggregation and colorimetric optical reporters for tracking Aβ aggregation, we accomplished the fast aggregation assay in less than 1 min by the naked eyes. Our detection method is based on spontaneous clustering of unconjugated (unmodified) GNPs along with the aggregated Aβ network under an aggregation-promoting condition. As a proof-of-concept demonstration, we employed the acidic perturbation permitting rapid cooperative assemblies of GNPs and Aβ peptides via their surface charge modulation. Under the optimized acidic perturbation condition around pH 2 to 3, we characterized the concentration-dependent colorimetric responses for aggregation at physiologically relevant Aβ concentration levels (from 100 μM to 10 nM). We also demonstrated the GNP/acidic condition-based rapid inhibition assay of Aβ aggregation by using well-known binding reagents such as antibody and serum albumin. The proposed methodology can be a powerful alternative method for screening drugs for AD as well as studying molecular biophysics of protein aggregations, and further extended to explore other protein conformational diseases such as neurodegenerative disease.

  17. Competitive inhibition reaction mechanisms for the two-step model of protein aggregation.

    PubMed

    Whidden, Mark; Ho, Allison; Ivanova, Magdalena I; Schnell, Santiago

    2014-01-01

    We propose three new reaction mechanisms for competitive inhibition of protein aggregation for the two-step model of protein aggregation. The first mechanism is characterized by the inhibition of native protein, the second is characterized by the inhibition of aggregation-prone protein and the third mechanism is characterized by the mixed inhibition of native and aggregation-prone proteins. Rate equations are derived for these mechanisms, and a method is described for plotting kinetic results to distinguish these three types of inhibitors. The derived rate equations provide a simple way of estimating the inhibition constant of native or aggregation-prone protein inhibitors in protein aggregation. The new approach is used to estimate the inhibition constants of different peptide inhibitors of insulin aggregation. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Inactivation of enveloped virus by laser-driven protein aggregation

    NASA Astrophysics Data System (ADS)

    Tsen, Shaw-Wei D.; Chapa, Travis; Beatty, Wandy; Tsen, Kong-Thon; Yu, Dong; Achilefu, Samuel

    2012-12-01

    Ultrafast lasers in the visible and near-infrared range have emerged as a potential new method for pathogen reduction of blood products and pharmaceuticals. However, the mechanism of enveloped virus inactivation by this method is unknown. We report the inactivation as well as the molecular and structural effects caused by visible (425 nm) femtosecond laser irradiation on murine cytomegalovirus (MCMV), an enveloped, double-stranded DNA virus. Our results show that laser irradiation (1) caused a 5-log reduction in MCMV titer, (2) did not cause significant changes to the global structure of MCMV virions including membrane and capsid, as assessed by electron microscopy, (3) produced no evidence of double-strand breaks or crosslinking in MCMV genomic DNA, and (4) caused selective aggregation of viral capsid and tegument proteins. We propose a model in which ultrafast laser irradiation induces partial unfolding of viral proteins by disrupting hydrogen bonds and/or hydrophobic interactions, leading to aggregation of closely associated viral proteins and inactivation of the virus. These results provide new insight into the inactivation of enveloped viruses by visible femtosecond lasers at the molecular level, and help pave the way for the development of a new ultrafast laser technology for pathogen reduction.

  19. Interactions of Lipid Membranes with Fibrillar Protein Aggregates.

    PubMed

    Gorbenko, Galyna; Trusova, Valeriya; Girych, Mykhailo; Adachi, Emi; Mizuguchi, Chiharu; Saito, Hiroyuki

    2015-01-01

    Amyloid fibrils are an intriguing class of protein aggregates with distinct physicochemical, structural and morphological properties. They display peculiar membrane-binding behavior, thus adding complexity to the problem of protein-lipid interactions. The consensus that emerged during the past decade is that amyloid cytotoxicity arises from a continuum of cross-β-sheet assemblies including mature fibrils. Based on literature survey and our own data, in this chapter we address several aspects of fibril-lipid interactions, including (i) the effects of amyloid assemblies on molecular organization of lipid bilayer; (ii) competition between fibrillar and monomeric membrane-associating proteins for binding to the lipid surface; and (iii) the effects of lipids on the structural morphology of fibrillar aggregates. To illustrate some of the processes occurring in fibril-lipid systems, we present and analyze fluorescence data reporting on lipid bilayer interactions with fibrillar lysozyme and with the N-terminal 83-residue fragment of amyloidogenic mutant apolipoprotein A-I, 1-83/G26R/W@8. The results help understand possible mechanisms of interaction and mutual remodeling of amyloid fibers and lipid membranes, which may contribute to amyloid cytotoxicity.

  20. SODA: prediction of protein solubility from disorder and aggregation propensity.

    PubMed

    Paladin, Lisanna; Piovesan, Damiano; Tosatto, Silvio C E

    2017-05-13

    Solubility is an important, albeit not well understood, feature determining protein behavior. It is of paramount importance in protein engineering, where similar folded proteins may behave in very different ways in solution. Here we present SODA, a novel method to predict the changes of protein solubility based on several physico-chemical properties of the protein. SODA uses the propensity of the protein sequence to aggregate as well as intrinsic disorder, plus hydrophobicity and secondary structure preferences to estimate changes in solubility. It has been trained and benchmarked on two different datasets. The comparison to other recently published methods shows that SODA has state-of-the-art performance and is particularly well suited to predict mutations decreasing solubility. The method is fast, returning results for single mutations in seconds. A usage example estimating the full repertoire of mutations for a human germline antibody highlights several solubility hotspots on the surface. The web server, complete with RESTful interface and extensive help, can be accessed from URL: http://protein.bio.unipd.it/soda. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    PubMed

    David, Della C; Ollikainen, Noah; Trinidad, Jonathan C; Cary, Michael P; Burlingame, Alma L; Kenyon, Cynthia

    2010-08-10

    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.

  2. Widespread Protein Aggregation as an Inherent Part of Aging in C. elegans

    PubMed Central

    David, Della C.; Ollikainen, Noah; Trinidad, Jonathan C.; Cary, Michael P.; Burlingame, Alma L.; Kenyon, Cynthia

    2010-01-01

    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 β-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. PMID:20711477

  3. Sequence-Specific Protein Aggregation Generates Defined Protein Knockdowns in Plants1[OPEN

    PubMed Central

    Vuylsteke, Marnik; Aesaert, Stijn; Rombaut, Debbie; De Smet, Frederik; Xu, Jie; Van Lijsebettens, Mieke; Rousseau, Frederic

    2016-01-01

    Protein aggregation is determined by short (5–15 amino acids) aggregation-prone regions (APRs) of the polypeptide sequence that self-associate in a specific manner to form β-structured inclusions. Here, we demonstrate that the sequence specificity of APRs can be exploited to selectively knock down proteins with different localization and function in plants. Synthetic aggregation-prone peptides derived from the APRs of either the negative regulators of the brassinosteroid (BR) signaling, the glycogen synthase kinase 3/Arabidopsis SHAGGY-like kinases (GSK3/ASKs), or the starch-degrading enzyme α-glucan water dikinase were designed. Stable expression of the APRs in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) induced aggregation of the target proteins, giving rise to plants displaying constitutive BR responses and increased starch content, respectively. Overall, we show that the sequence specificity of APRs can be harnessed to generate aggregation-associated phenotypes in a targeted manner in different subcellular compartments. This study points toward the potential application of induced targeted aggregation as a useful tool to knock down protein functions in plants and, especially, to generate beneficial traits in crops. PMID:27208282

  4. Opioid peptides derived from food proteins suppress aggregation and promote reactivation of partly unfolded stressed proteins.

    PubMed

    Artemova, N V; Bumagina, Z M; Kasakov, A S; Shubin, V V; Gurvits, B Ya

    2010-02-01

    A new view of the opioid peptides is presented. The potential of small peptides derived from precursor food proteins, to bind to partly unfolded stressed proteins to prevent their irreversible aggregation and inactivation has been demonstrated in various in vitro test systems: dithiothreitol-induced aggregation of alpha-lactalbumin (LA), heat-induced aggregation of alcohol dehydrogenase (ADH), and aggregation and inactivation of bovine erythrocyte carbonic anhydrase (CA) in the process of its refolding after removal of stress conditions. Using dynamic light scattering (DLS), turbidimetry, fluorescence, and circular dichroism measurements protective effects of the synthetic opioid peptides: exorphin C from wheat gluten (Tyr-Pro-Ile-Ser-Leu), rubiscolin-5 from spinach ribulose-bisphosphate-carboxylase/oxygenase (Rubisco) (Tyr-Pro-Leu-Asp-Leu), and hemorphin-6 from bovine hemoglobin (Tyr-Pro-Trp-Thr-Gln-Arg) have been revealed. We have demonstrated the concentration-dependent suppression of light scattering intensity of aggregates of LA and ADH in the presence of the peptides, the population of nanoparticles with higher hydrodynamic radii being shifted to the lower ones, accompanied by an increase in the lag period of aggregation. The presence of the peptides in the refolding solution was shown to assist reactivation of CA and enhance the yield of the CA soluble protein. The results suggest that bioactive food protein fragments may be regarded as exogenous supplements to the endogenous defense mechanisms of the human organism under stress conditions.

  5. Fractal dimensions of soy protein nanoparticle aggregates determined by dynamic mechanical method

    USDA-ARS?s Scientific Manuscript database

    The fractal dimension of the protein aggregates can be estimated by dynamic mechanical methods when the particle aggregates are imbedded in a polymer matrix. Nanocomposites were formed by mixing hydrolyzed soy protein isolate (HSPI) nanoparticle aggregates with styrene-butadiene (SB) latex, followe...

  6. Protein detection system

    DOEpatents

    Fruetel, Julie A [Livermore, CA; Fiechtner, Gregory J [Bethesda, MD; Kliner, Dahv A. V. [San Ramon, CA; McIlroy, Andrew [Livermore, CA

    2009-05-05

    The present embodiment describes a miniature, microfluidic, absorption-based sensor to detect proteins at sensitivities comparable to LIF but without the need for tagging. This instrument utilizes fiber-based evanescent-field cavity-ringdown spectroscopy, in combination with faceted prism microchannels. The combination of these techniques will increase the effective absorption path length by a factor of 10.sup.3 to 10.sup.4 (to .about.1-m), thereby providing unprecedented sensitivity using direct absorption. The coupling of high-sensitivity absorption with high-performance microfluidic separation will enable real-time sensing of biological agents in aqueous samples (including aerosol collector fluids) and will provide a general method with spectral fingerprint capability for detecting specific bio-agents.

  7. Denatured state aggregation parameters derived from concentration dependence of protein stability.

    PubMed

    Schön, Arne; Clarkson, Benjamin R; Siles, Rogelio; Ross, Patrick; Brown, Richard K; Freire, Ernesto

    2015-11-01

    Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated.

  8. PIP3-binding proteins promote age-dependent protein aggregation and limit survival in C. elegans.

    PubMed

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Johnson, Jay; Alla, Ramani; Mackintosh, Samuel G; Shmookler Reis, Robert J

    2016-08-02

    Class-I phosphatidylinositol 3-kinase (PI3KI) converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 comprises two fatty-acid chains that embed in lipid-bilayer membranes, joined by glycerol to inositol triphosphate. Proteins with domains that specifically bind that head-group (e.g. pleckstrin-homology [PH] domains) are thus tethered to the inner plasma-membrane surface where they have an enhanced likelihood of interaction with other PIP3-bound proteins, in particular other components of their signaling pathways. Null alleles of the C. elegans age-1 gene, encoding the catalytic subunit of PI3KI, lack any detectable class-I PI3K activity and so cannot form PIP3. These mutant worms survive almost 10-fold longer than the longest-lived normal control, and are highly resistant to a variety of stresses including oxidative and electrophilic challenges. Traits associated with age-1 mutation are widely believed to be mediated through AKT-1, which requires PIP3 for both tethering and activation. Active AKT complex phosphorylates and thereby inactivates the DAF-16/FOXO transcription factor. However, extensive evidence indicates that pleiotropic effects of age-1-null mutations, including extreme longevity, cannot be explained by insulin like-receptor/AKT/FOXO signaling alone, suggesting involvement of other PIP3-binding proteins. We used ligand-affinity capture to identify membrane-bound proteins downstream of PI3KI that preferentially bind PIP3. Computer modeling supports a subset of candidate proteins predicted to directly bind PIP3 in preference to PIP2, and functional testing by RNAi knockdown confirmed candidates that partially mediate the stress-survival, aggregation-reducing and longevity benefits of PI3KI disruption. PIP3-specific candidate sets are highly enriched for proteins previously reported to affect translation, stress responses, lifespan, proteostasis, and lipid transport.

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

    PubMed

    Holmberg, Mats; Nollen, Ellen A A

    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 expression of fluorescently tagged aggregation-prone proteins have been used to screen for genetic modifiers of aggregation. To establish the role of modifying factors in the generation of aggregation intermediates, a method has been developed using native agarose gel electrophoresis (NAGE) that enables parallel screening of aggregation patterns of fluorescently labeled aggregation-prone proteins. Together with microscopy-based genetic screens this method can be used to identify modifiers of protein aggregation and characterize their molecular function. Although described here for analyzing aggregates in C. elegans, NAGE can be adjusted for use in other model organisms as well as for cultured cells.

  10. A universal method for detection of amyloidogenic misfolded proteins.

    PubMed

    Yam, Alice Y; Wang, Xuemei; Gao, Carol Man; Connolly, Michael D; Zuckermann, Ronald N; Bleu, Thieu; Hall, John; Fedynyshyn, Joseph P; Allauzen, Sophie; Peretz, David; Salisbury, Cleo M

    2011-05-24

    Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.

  11. A Dynamic Study of Protein Secretion and Aggregation in the Secretory Pathway

    PubMed Central

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

    2014-01-01

    Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC. PMID:25279560

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

    PubMed

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

    2014-01-01

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

  13. Raman studies of gluten proteins aggregation induced by dietary fibres.

    PubMed

    Nawrocka, Agnieszka; Szymańska-Chargot, Monika; Miś, Antoni; Kowalski, Radosław; Gruszecki, Wiesław I

    2016-03-01

    Interactions between gluten proteins and dietary fibre preparations are crucial in the baking industry. The addition of dietary fibre to bread causes significant reduction in its quality which is influenced by changes in the structure of gluten proteins. Fourier transform Raman spectroscopy was applied to determine changes in the structure of gluten proteins modified by seven dietary fibres. The commercially available gluten proteins without starch were mixed with the fibres in three concentrations: 3%, 6% and 9%. The obtained results showed that all fibres, regardless of their origin, caused the same kind of changes i.e. decrease in the α-helix content with a simultaneous increase in the content of antiparallel-β-sheet. The results indicated that presence of cellulose was the probable cause of these changes, and lead to aggregation or abnormal folding of the gluten proteins. Other changes observed in the gluten structure concerning β-structures, conformation of disulphide bridges, and aromatic amino acid environment, depended on the fibres chemical composition.

  14. Nanoparticles in relation to peptide and protein aggregation

    PubMed Central

    Zaman, Masihuz; Ahmad, Ejaz; Qadeer, Atiyatul; Rabbani, Gulam; Khan, Rizwan Hasan

    2014-01-01

    Over the past two decades, there has been considerable research interest in the use of nanoparticles in the study of protein and peptide aggregation, and of amyloid-related diseases. The influence of nanoparticles on amyloid formation yields great interest due to its small size and high surface area-to-volume ratio. Targeting nucleation kinetics by nanoparticles is one of the most searched for ways to control or induce this phenomenon. The observed effect of nanoparticles on the nucleation phase is determined by particle composition, as well as the amount and nature of the particle’s surface. Various thermodynamic parameters influence the interaction of proteins and nanoparticles in the solution, and regulate the protein assembly into fibrils, as well as the disaggregation of preformed fibrils. Metals, organic particles, inorganic particles, amino acids, peptides, proteins, and so on are more suitable candidates for nanoparticle formulation. In the present review, we attempt to explore the effects of nanoparticles on protein and peptide fibrillation processes from both perspectives (ie, as inducers and inhibitors on nucleation kinetics and in the disaggregation of preformed fibrils). Their formulation and characterization by different techniques have been also addressed, along with their toxicological effects, both in vivo and in vitro. PMID:24611007

  15. Distinct β-sheet structure in protein aggregates determined by ATR-FTIR spectroscopy.

    PubMed

    Shivu, Bhavana; Seshadri, Sangita; Li, Jie; Oberg, Keith A; Uversky, Vladimir N; Fink, Anthony L

    2013-08-06

    Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the conformation of aggregated proteins in vivo and in vitro. Several different protein aggregates, including amyloid fibrils from several peptides and polypeptides, inclusion bodies, folding aggregates, soluble oligomers, and protein extracts from stressed cells, were examined in this study. All protein aggregates demonstrate a characteristic new β structure with lower-frequency band positions. All protein aggregates acquire this new β band following the aggregation process involving intermolecular interactions. The β sheets in some proteins arise from regions of the polypeptide that are helical or non β in the native conformation. For a given protein, all types of the aggregates (e.g., inclusion bodies, folding aggregates, and thermal aggregates) showed similar spectra, indicating that they arose from a common partially folded species. All of the aggregates have some nativelike secondary structure and nonperiodic structure as well as the specific new β structure. The new β could be most likely attributed to stronger hydrogen bonds in the intermolecular β-sheet structure present in the protein aggregates.

  16. Delicate balance between functionally required flexibility and aggregation risk in a β-rich protein.

    PubMed

    Ferrolino, Mylene C; Zhuravleva, Anastasia; Budyak, Ivan L; Krishnan, Beena; Gierasch, Lila M

    2013-12-10

    Susceptibility to aggregation is general to proteins because of the potential for intermolecular interactions between hydrophobic stretches in their amino acid sequences. Protein aggregation has been implicated in several catastrophic diseases, yet we still lack in-depth understanding about how proteins are channeled to this state. Using a predominantly β-sheet protein whose folding has been explored in detail, cellular retinoic acid-binding protein 1 (CRABP1), as a model, we have tackled the challenge of understanding the links between a protein's natural tendency to fold, 'breathe', and function with its propensity to misfold and aggregate. We identified near-native dynamic species that lead to aggregation and found that inherent structural fluctuations in the native protein, resulting in opening of the ligand-entry portal, expose hydrophobic residues on the most vulnerable aggregation-prone sequences in CRABP1. CRABP1 and related intracellullar lipid-binding proteins have not been reported to aggregate inside cells, and we speculate that the cellular concentration of their open, aggregation-prone conformations is sufficient for ligand binding but below the critical concentration for aggregation. Our finding provides an example of how nature fine-tunes a delicate balance between protein function, conformational variability, and aggregation vulnerability and implies that with the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable but controllable risk.

  17. Use of whey protein soluble aggregates for thermal stability-a hypothesis paper.

    PubMed

    Ryan, Kelsey N; Zhong, Qixin; Foegeding, Edward A

    2013-08-01

    Forming whey proteins into soluble aggregates is a modification shown to improve or expand the applications in foaming, emulsification, gelation, film-formation, and encapsulation. Whey protein soluble aggregates are defined as aggregates that are intermediates between monomer proteins and an insoluble gel network or precipitate. The conditions under which whey proteins denature and aggregate have been extensively studied and can be used as guiding principles of producing soluble aggregates. These conditions are reviewed for pH, ion type and concentration, cosolutes, and protein concentration, along with heating temperature and duration. Combinations of these conditions can be used to design soluble aggregates with desired physicochemical properties including surface charge, surface hydrophobicity, size, and shape. These properties in turn can be used to obtain target macroscopic properties, such as viscosity, clarity, and stability, of the final product. A proposed approach to designing soluble aggregates with improved thermal stability for beverage applications is presented.

  18. Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size

    PubMed Central

    Pryor, N. Elizabeth; Moss, Melissa A.; Hestekin, Christa N.

    2012-01-01

    The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-β protein (Aβ) associated with Alzheimer’s disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aβ species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting. PMID:22489141

  19. Altering protein surface charge with chemical modification modulates protein-gold nanoparticle aggregation

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

    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.

  20. A delicate balance between functionally required flexibility and aggregation risk in a β-rich protein

    PubMed Central

    Ferrolino, Mylene C.; Zhuravleva, Anastasia; Budyak, Ivan L.; Krishnan, Beena; Gierasch, Lila M.

    2014-01-01

    Susceptibility to aggregation is general to proteins because of the potential for intermolecular interactions between hydrophobic stretches in their amino acid sequences. Protein aggregation has been implicated in several catastrophic diseases, yet we still lack in-depth understanding about how proteins are channeled to this state. Using a predominantly β-sheet protein whose folding has been explored in detail: cellular retinoic acid-binding protein 1 (CRABP1), as a model, we have tackled the challenge of understanding the links between a protein’s natural tendency to fold, ‘breathe’, and function with its propensity to misfold and aggregate. We identified near-native dynamic species that lead to aggregation and found that inherent structural fluctuations in the native protein, resulting in opening of the ligand entry portal, expose hydrophobic residues on the most vulnerable aggregation-prone sequences in CRABP1. CRABP1 and related intracellullar lipid-binding proteins have not been reported to aggregate inside cells, and we speculate that the cellular concentration of their open, aggregation-prone conformations is sufficient for ligand binding but below the critical concentration for aggregation. Our finding provides an example of how nature fine-tunes a delicate balance between protein function, conformational variability, and aggregation vulnerability, and implies that with the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable but controllable risk. PMID:24236614

  1. Antimicrobial preservatives induce aggregation of interferon alpha-2a: The order in which preservatives induce protein aggregation is independent of the protein

    PubMed Central

    Bis, Regina L.; Mallela, Krishna M.G.

    2014-01-01

    Antimicrobial preservatives (APs) are included in liquid multi-dose protein formulations to combat the growth of microbes and bacteria. These compounds have been shown to cause protein aggregation, which leads to serious immunogenic and toxic side-effects in patients. Our earlier work on a model protein cytochrome c (Cyt c) demonstrated that APs cause protein aggregation in a specific manner. The aim of this study is to validate the conclusions obtained from our model protein studies on a pharmaceutical protein. Interferon α-2a (IFNA2) is available as a therapeutic treatment for numerous immune-compromised disorders including leukemia and hepatitis c, and APs have been used in its multi-dose formulation. Similar to Cyt c, APs induced IFNA2 aggregation, demonstrated by the loss of soluble monomer and increase in solution turbidity. The extent of IFNA2 aggregation increased with the increase in AP concentration. IFNA2 aggregation also depended on the nature of AP, and followed the order m-cresol > phenol > benzyl alcohol > phenoxyethanol. This specific order exactly matched with that observed for the model protein Cyt c. These and previously published results on antibodies and other recombinant proteins suggest that the general mechanism by which APs induce protein aggregation may be independent of the protein. PMID:24974985

  2. Antimicrobial preservatives induce aggregation of interferon alpha-2a: the order in which preservatives induce protein aggregation is independent of the protein.

    PubMed

    Bis, Regina L; Mallela, Krishna M G

    2014-09-10

    Antimicrobial preservatives (APs) are included in liquid multi-dose protein formulations to combat the growth of microbes and bacteria. These compounds have been shown to cause protein aggregation, which leads to serious immunogenic and toxic side-effects in patients. Our earlier work on a model protein cytochrome c (Cyt c) demonstrated that APs cause protein aggregation in a specific manner. The aim of this study is to validate the conclusions obtained from our model protein studies on a pharmaceutical protein. Interferon α-2a (IFNA2) is available as a therapeutic treatment for numerous immune-compromised disorders including leukemia and hepatitis C, and APs have been used in its multi-dose formulation. Similar to Cyt c, APs induced IFNA2 aggregation, demonstrated by the loss of soluble monomer and increase in solution turbidity. The extent of IFNA2 aggregation increased with the increase in AP concentration. IFNA2 aggregation also depended on the nature of AP, and followed the order m-cresol>phenol>benzyl alcohol>phenoxyethanol. This specific order exactly matched with that observed for the model protein Cyt c. These and previously published results on antibodies and other recombinant proteins suggest that the general mechanism by which APs induce protein aggregation may be independent of the protein. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Techniques for Monitoring Protein Misfolding and Aggregation in Vitro and in Living Cells

    PubMed Central

    Gregoire, Simpson; Irwin, Jacob; Kwon, Inchan

    2012-01-01

    Protein misfolding and aggregation have been considered important in understanding many neurodegenerative diseases and recombinant biopharmaceutical production. Therefore, various traditional and modern techniques have been utilized to monitor protein aggregation in vitro and in living cells. Fibril formation, morphology and secondary structure content of amyloidogenic proteins in vitro have been monitored by molecular probes, TEM/AFM, and CD/FTIR analyses, respectively. Protein aggregation in living cells has been qualitatively or quantitatively monitored by numerous molecular folding reporters based on either fluorescent protein or enzyme. Aggregation of a target protein is directly correlated to the changes in fluorescence or enzyme activity of the folding reporter fused to the target protein, which allows non-invasive monitoring aggregation of the target protein in living cells. Advances in the techniques used to monitor protein aggregation in vitro and in living cells have greatly facilitated the understanding of the molecular mechanism of amyloidogenic protein aggregation associated with neurodegenerative diseases, optimizing culture conditions to reduce aggregation of biopharmaceuticals expressed in living cells, and screening of small molecule libraries in the search for protein aggregation inhibitors. PMID:23565019

  4. A permeation method for detection of self-aggregation of doxorubicin in aqueous environment.

    PubMed

    Fülöp, Zoltán; Gref, Ruxandra; Loftsson, Thorsteinn

    2013-09-15

    For pharmaceutical scientists, it is important to know if dissolved drug molecules are present only as monomers or in the form of aggregates in a test solution or formulation. Amphiphilic or hydrophobic drugs frequently self-associate to form dimers, trimers or higher order aggregates. Doxorubicin aggregation was examined by a previously developed permeation technique to detect oligosaccharide aggregation in aqueous solutions. At very low doxorubicin concentrations dimers and trimers have been observed, but in aqueous 0.5mg/ml doxorubicin solutions aggregates containing about 40 molecules were observed. The permeation studies were supported by TEM studies. The results indicate that neutral doxorubicin molecules aggregate more readily than the protonated ones. Doxorubicin aggregation is a stepwise process resulting in formation of aggregates of variable sizes are enhanced aggregation with increasing doxorubicin concentration. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Flow cytometric quantification and characterization of intracellular protein aggregates in yeast.

    PubMed

    Shiber, Ayala; Breuer, William; Ravid, Tommer

    2014-01-01

    The sequestration of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. Methods for systematic analysis of cellular aggregate content are still largely limited to fluorescence microscopy and to separation by biochemical techniques. Here, we describe an alternative approach, using flow cytometric analysis, applied to protein aggregates released from their intracellular milieu by mild lysis of yeast cells. Protein aggregates were induced in yeast by heat shock or by chaperone deprivation and labeled using GFP- or mCherry-tagged quality control substrate proteins and chaperones. The fluorescence-labeled aggregate particles were distinguishable from cell debris by flow cytometry. The assay was used to quantify the number of fluorescent aggregates per μg of cell lysate protein and for monitoring changes in the cellular content and properties of aggregates, induced by stress. The results were normalized to the frequencies of fluorescent reporter expression in the cell population, allowing quantitative comparison. The assay also provided a quantitative measure of co-localization of aggregate components, such as chaperones and quality control substrates, within the same aggregate particle. This approach may be extended by fluorescence-activated sorting and isolation of various protein aggregates, including those harboring proteins associated with conformation disorders.

  6. Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine

    NASA Astrophysics Data System (ADS)

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

    2010-01-01

    Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by ~300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.

  7. Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine.

    PubMed

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

    2010-01-01

    Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by approximately 300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.

  8. Optical protein detection based on magnetic clusters rotation.

    PubMed

    Ramiandrisoa, Donatien; Brient-Litzler, Elodie; Daynes, Aurélien; Compain, Eric; Bibette, Jérôme; Baudry, Jean

    2015-09-25

    In this paper we present a simple method to quantify aggregates of 200nm magnetic particles. This method relies on the optical and magnetic anisotropy of particle aggregates, whereas dispersed particles are optically isotropic. We orientate aggregates by applying short pulses of a magnetic field, and we measure optical density variation directly linked to this reorientation. By computing the scattering efficiency of doublets and singlets, we demonstrate the absolute quantification of a few % of doublets in a well dispersed suspension. More generally, these optical variations are related to the aggregation state of the sample. This method can be easily applied to an agglutination assay, where target proteins induce aggregation of colloidal particles. By observing only aligned clusters, we increase sensitivity and we reduce the background noise as compared to a classical agglutination assay: we obtain a detection limit on the C-reactive protein of less than 3pM for a total assay time of 10min.

  9. Reactivation of Aggregated Proteins by the ClpB/DnaK Bi-chaperone System

    PubMed Central

    Zolkiewski, Michal; Chesnokova, Liudmila S.; Witt, Stephan N.

    2016-01-01

    Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme glucose-6-phosphate dehydrogenase mediated by ClpB from Escherichia coli in cooperation with another molecular chaperone DnaK. The procedures for purification of these chaperones are also described. PMID:26836408

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

    PubMed

    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 two major proteolytic machineries, the ubiquitin proteasome system (UPS) and the autophagy/lysosomal system, in the pathogenesis of neurodegenerative diseases. These proteolytic systems cooperate to maintain the proteostasis, as is indicated by intricate cross talk. In addition, the UPS and autophagy are regulated by stress pathways that are activated by disturbed proteostasis, like the unfolded protein response (UPR). We will specifically discuss how these proteolytic pathways are affected in neurodegenerative diseases. We will show that there is a differential involvement of the UPS and autophagy in different neurodegenerative disorders. In addition, the proteolytic impairment may be primary or secondary to the pathology. These differences have important implications for the design of therapeutic strategies. The opportunities and caveats of targeting the UPS and autophagy/lysosomal system as a therapeutic strategy in neurodegeneration will be discussed.

  11. A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells.

    PubMed

    Gregoire, Simpson; Kwon, Inchan

    2012-10-01

    Protein misfolding and aggregation play important roles in many physiological processes. These include pathological protein aggregation in neurodegenerative diseases and biopharmaceutical protein aggregation during production in mammalian cells. To develop a simple non-invasive assay for protein misfolding and aggregation in mammalian cells, the folding reporter green fluorescent protein (GFP) system, originally developed for bacterial cells, was evaluated. As a folding reporter, GFP was fused to the C-terminus of a panel of human copper/zinc superoxide dismutase (SOD1) mutants with varying misfolding/aggregation propensities. Flow cytometric analysis of transfected HEK293T and NSC-34 cells revealed that the mean fluorescence intensities of the cells expressing GFP fusion of SOD1 variants exhibited an inverse correlation with the misfolding/aggregation propensities of the four SOD1 variants. Our results support the hypothesis that the extent of misfolding/aggregation of a target protein in mammalian cells can be quantitatively estimated by measuring the mean fluorescence intensity of the cells expressing GFP fusion. The assay method developed herein will facilitate the understanding of aggregation process of SOD1 variants and the identification of aggregation inhibitors. The method also has great promise for misfolding/aggregation studies of other proteins in mammalian cells. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions.

    PubMed

    Creusot, Nathalie; Gruppen, Harry

    2008-11-12

    In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications.

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Robust quantitation of basic-protein higher-order aggregates using size-exclusion chromatography.

    PubMed

    Gervais, David; Downer, Andrew; King, Darryl; Kanda, Patrick; Foote, Nicholas; Smith, Stuart

    2017-05-30

    Detection of higher-order aggregates (HOA) using size-exclusion chromatography (SEC) was found to be variable for a basic protein, using exposed-silanol or diol-silica-based SEC columns. Preparations of the tetrameric biopharmaceutical enzyme Erwinia chrysanthemil-asparaginase (ErA), which has an isoelectric point of 8.6, were analysed using a diol-silica SEC column. Although the proportions of ErA main peak and octamer species were unaffected, HOA recovery and detection were extremely variable and had poor agreement with an orthogonal measurement technique, analytical ultracentrifugation (AUC). The observation that only HOA was selectively affected by non-specific silanol interactions was unexpected, so alternatives were sought. Coated-silica SEC columns improved the resolution and reproducibility of HOA detection for this alkaline-pI protein, and improved the agreement of HOA with the AUC method. Basic proteins, such as ErA, should be thoroughly evaluated in SEC method development, to ensure that resolution of larger aggregate species is not compromised.

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

    PubMed

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

    2011-05-01

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

  16. Legal but lethal: functional protein aggregation at the verge of toxicity

    PubMed Central

    Falsone, Angelika; Falsone, S. Fabio

    2015-01-01

    Many neurodegenerative disorders are linked to irreversible protein aggregation, a process that usually comes along with toxicity and serious cellular damage. However, it is emerging that protein aggregation can also serve for physiological purposes, as impressively shown for prions. While the aggregation of this protein family was initially considered exclusively toxic in mammalians organisms, it is now almost clear that many other proteins adopt prion-like attributes to rationally polymerize into higher order complexes with organized physiologic roles. This implies that cells can tolerate at least in some measure the accumulation of inherently dangerous protein aggregates for functional profit. This review summarizes currently known strategies that living organisms adopt to preserve beneficial aggregation, and to prevent the catastrophic accumulation of toxic aggregates that frequently accompany neurodegeneration. PMID:25741240

  17. Proteomic characterization of aggregating proteins after the inhibition of the ubiquitin proteasome system.

    PubMed

    Wilde, Inga B; Brack, Maria; Winget, Jason M; Mayor, Thibault

    2011-03-04

    Protein aggregation, which is associated with the impairment of the ubiquitin proteasome system, is a hallmark of many neurodegenerative diseases. To better understand the contribution of proteasome inhibition in aggregation, we analyzed which proteins may potentially localize in chemically induced aggregates in human neuroblastoma tissue culture cells. We enriched for proteins in high-density structures by using a sucrose gradient in combination with stable isotope labeling with amino acids in cell culture (SILAC). The quantitative analysis allowed us to distinguish which proteins were specifically affected by the proteasome inhibition. We identified 642 potentially aggregating proteins, including the p62/sequestosome 1 and NBR1 ubiquitin-binding proteins involved in aggregation. We also identified the ubiquitin-associated protein 2 like (UBAP2L). We verified that it cofractionated with ubiquitin in the high-density fraction and that it was colocalized in the ubiquitin-containing aggregates after proteasome inhibition. In addition, we identified several chaperone proteins and used data from protein interaction networks to show that they potentially interact with distinct subgroups of proteins within the aggregating structures. Several other proteins associated with neurodegenerative diseases, like UCHL1, were identified, further underlining the potential of our analysis to better understand the aggregation process and proteotoxic stress caused by proteasome inhibition.

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

    PubMed

    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.

  19. Protein folding, misfolding, and aggregation. Formation of inclusion bodies and aggresomes.

    PubMed

    Markossian, K A; Kurganov, B I

    2004-09-01

    In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.

  20. Protein Misfolding and Aggregation in Cataract Disease and Prospects for Prevention

    PubMed Central

    Moreau, Kate L.; King, Jonathan A.

    2012-01-01

    The transparency of the eye lens depends on maintaining the native tertiary structures and solubility of the lens crystallin proteins over a lifetime. Cataract, the leading cause of blindness worldwide, is caused by protein aggregation within the protected lens environment. With age, covalent protein damage accumulates through pathways thought to include UV radiation, oxidation, deamidation, and truncations. Experiments suggest that the resulting protein destabilization leads to partially unfolded, aggregation-prone intermediates and the formation of insoluble, light-scattering protein aggregates. These aggregates either include or overwhelm the protein chaperone content of the lens. Here we review the causes of cataracts and non-surgical methods being investigated to inhibit or delay cataract development, including natural product-based therapies, modulators of oxidation, and protein aggregation inhibitors. PMID:22520268

  1. Identification of an RNA Polymerase III Regulator Linked to Disease-Associated Protein Aggregation.

    PubMed

    Sin, Olga; de Jong, Tristan; Mata-Cabana, Alejandro; Kudron, Michelle; Zaini, Mohamad Amr; Aprile, Francesco A; Seinstra, Renée I; Stroo, Esther; Prins, Roméo Willinge; Martineau, Céline N; Wang, Hai Hui; Hogewerf, Wytse; Steinhof, Anne; Wanker, Erich E; Vendruscolo, Michele; Calkhoven, Cornelis F; Reinke, Valerie; Guryev, Victor; Nollen, Ellen A A

    2017-03-16

    Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer's and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases.

  2. A multiscale view of therapeutic protein aggregation: a colloid science perspective.

    PubMed

    Nicoud, Lucrèce; Owczarz, Marta; Arosio, Paolo; Morbidelli, Massimo

    2015-03-01

    The formation of aggregates in protein-based pharmaceuticals is a major issue that can compromise drug safety and drug efficacy. With a view to improving protein stability, considerable effort is put forth to unravel the fundamental mechanisms underlying the aggregation process. However, therapeutic protein aggregation is a complex multistep phenomenon that involves time and length scales spanning several orders of magnitude, and strategies addressing protein aggregation inhibition are currently still largely empirical in practice. Here, we review how key concepts developed in the frame of colloid science can be applied to gain knowledge on the kinetics and thermodynamics of therapeutic protein aggregation across different length scales. In particular, we discuss the use of coarse-grained molecular interaction potentials to quantify protein colloidal stability. We then show how population balance equations simulations can provide insights into the mechanisms of aggregate formation at the mesoscale, and we highlight the strength of the concept of fractal scaling to quantify irregular aggregate morphologies. Finally, we correlate the macroscopic rheological properties of protein solutions with the occupied volume fraction and the aggregate structure. Overall, this work illustrates the power and limitations of colloidal approaches in the multiscale description of the aggregation of therapeutic proteins.

  3. Localization of Protein Aggregation in Escherichia coli Is Governed by Diffusion and Nucleoid Macromolecular Crowding Effect

    PubMed Central

    Coquel, Anne-Sophie; Jacob, Jean-Pascal; Primet, Mael; Demarez, Alice; Dimiccoli, Mariella; Julou, Thomas; Moisan, Lionel

    2013-01-01

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

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

    PubMed

    Coquel, Anne-Sophie; Jacob, Jean-Pascal; Primet, Mael; Demarez, Alice; Dimiccoli, Mariella; Julou, Thomas; Moisan, Lionel; Lindner, Ariel B; Berry, Hugues

    2013-04-01

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

  5. Self-Assembling NanoLuc Luciferase Fragments as Probes for Protein Aggregation in Living Cells.

    PubMed

    Zhao, Jia; Nelson, Travis J; Vu, Quyen; Truong, Tiffany; Stains, Cliff I

    2016-01-15

    Given the clear role of protein aggregation in human disease, there is a critical need for assays capable of quantifying protein aggregation in living systems. We hypothesized that the inherently low background and biocompatibility of luminescence signal readouts could provide a potential solution to this problem. Herein, we describe a set of self-assembling NanoLuc luciferase (Nluc) fragments that produce a tunable luminescence readout that is dependent upon the solubility of a target protein fused to the N-terminal Nluc fragment. To demonstrate this approach, we employed this assay in bacteria to assess mutations known to disrupt amyloid-beta (Aβ) aggregation as well as disease-relevant mutations associated with familial Alzheimer's diseases. The luminescence signal from these experiments correlates with the reported aggregation potential of these Aβ mutants and reinforces the increased aggregation potential of disease-relevant mutations in Aβ1-42. To further demonstrate the utility of this approach, we show that the effect of small molecule inhibitors on Aβ aggregation can be monitored using this system. In addition, we demonstrate that aggregation assays can be ported into mammalian cells. Taken together, these results indicate that this platform could be used to rapidly screen for mutations that influence protein aggregation as well as inhibitors of protein aggregation. This method offers a novel, genetically encodable luminescence readout of protein aggregation in living cells.

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

    PubMed

    Urbonaite, V; de Jongh, H H J; van der Linden, E; Pouvreau, L

    2015-10-14

    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 (1%, v/v) prepared in the presence of 3.5 mM divalent salts increased in size and decreased in apparent density following the salt type order MgSO4, MgCl2, CaSO4, and CaCl2. In the absence of applied (centrifugal) forces, larger and less dense aggregates entrap more water. When force is applied from larger and more deformable aggregates, more water can be displaced. Entrapped water of ∼8-13 g of water/g of protein is associated with (pelleted) aggregates, of which approximately 4.5-8.5 g of water/g of protein is not constrained in exchangeability with the solvent. The amount of confined water within aggregates was found to be independent of the aggregate density and accounted for ∼3.5 g of water/g of protein. Confined water in aggregates is hindered in its diffusion because of physical structure constraints and, therefore, not directly exchangeable with the solvent. These insights in the protein aggregate size and deformability in relation to water entrapment and confinement could be used to tune water holding on larger length scales when force is applied.

  7. Effects of arginine on heat-induced aggregation of concentrated protein solutions.

    PubMed

    Shah, Dhawal; Shaikh, Abdul Rajjak; Peng, Xinxia; Rajagopalan, Raj

    2011-01-01

    Arginine is one of the commonly used additives to enhance refolding yield of proteins, to suppress aggregation of proteins, and to increase solubility of proteins, and yet the molecular interactions that contribute to the role of arginine are unclear. Here, we present experiments, using bovine serum albumin (BSA), lysozyme (LYZ), and β-lactoglobulin (BLG) as model proteins, to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. Results show that the enhancement in aggregation is caused only for BSA and BLG, but not for LYZ, indicating that arginine's preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine's role as an enhancer or suppressor of aggregation of proteins. The experimental and computational results suggest that the guanidinium group of arginine promotes aggregation through the hydrogen-bond-based bridging interactions with the acidic residues of a protein, whereas the binding of the guanidinium group to aromatic residues (aggregation-prone) contributes to the stability and solubilization of the proteins. The approach, we describe here, can be used to select suitable additives to stabilize a protein solution at high concentrations based on an analysis of the amino acid content of the protein.

  8. Role of water in Protein Aggregation and Amyloid Polymorphism

    PubMed Central

    Thirumalai, D.; Reddy, Govardhan; Straub, John E.

    2011-01-01

    Conspectus The link between oligomers and amyloid fibrils and a variety of neurodegenerative diseases raises the need to decipher the principles governing protein aggregation. Mechanisms of in vivo amyloid formation involve a number of coconspirators and complex interactions with membranes. Nevertheless, it is believed that understanding the biophysical basis of in vitro amyloid formation in well-defined systems is important in discovering ligands that preferentially bind to regions that harbor amyloidogenic tendencies. Determination of structures of fibrils of a variety of peptides has set the stage for probing the dynamics of oligomer formation and amyloid growth using computer simulations. Most experimental and simulation studies have been interpreted largely from the perspective of proteins without much consideration of the role of solvent in enabling or inhibiting oligomer formation and assembly to protofilaments and amyloid fibrils. Here, we provide a perspective on how interactions with water affect folding landscapes of Aβ monomers, oligomer formation in Aβ16–22 fragment, protofilament formation in a peptide from yeast prion Sup35. Explicit molecular dynamics simulations of these systems illustrate how water controls the self-assembly of higher order structures and provide a structural basis for understanding the kinetics of oligomer and fibril growth. Simulations show that monomers of Aβ-peptides sample a number of compact conformations. Population of aggregation-prone structures (N*) with salt-bridge, which bear a striking similarity to the peptide structure in the fibril, requires overcoming a high desolvation barrier. In general, sequences for which N* structures are not significantly populated are unlikely to aggregate. Generically oligomers and fibrils form in two steps. In the first stage water is expelled from the region between peptides rich in hydrophobic residues (for example Aβ16–22) resulting in the disordered oligomers. In the second

  9. Modification of proteins with cyclodextrins prevents aggregation and surface adsorption and increases thermal stability.

    PubMed

    Prashar, Deepali; Cui, DaWei; Bandyopadhyay, Debjyoti; Luk, Yan-Yeung

    2011-11-01

    This work describes a general approach for preventing protein aggregation and surface adsorption by modifying proteins with β-cyclodextrins (βCD) via an efficient water-driven ligation. As compared to native unmodified proteins, the cyclodextrin-modified proteins (lysozyme and RNase A) exhibit significant reduction in aggregation, surface adsorption and increase in thermal stability. These results reveal a new chemistry for preventing protein aggregation and surface adsorption that is likely of different mechanisms than that by modifying proteins with poly(ethylene glycol).

  10. Supersaturation is a major driving force for protein aggregation in neurodegenerative diseases

    PubMed Central

    Ciryam, Prajwal; Kundra, Rishika; Morimoto, Richard I.; Dobson, Christopher M.; Vendruscolo, Michele

    2015-01-01

    The solubility of proteins is an essential requirement for their function. Yet these ubiquitous molecules can undergo aggregation when the protein homeostasis system becomes impaired. Here we ask which is the driving force for protein aggregation in the cellular environment. Emerging evidence suggests that this phenomenon arises because the native states of many proteins are inherently metastable as their cellular concentrations exceed their critical values. Such `supersaturated' proteins are strongly driven towards aggregation, and are over-represented in specific biochemical pathways associated with neurodegenerative conditions. These observations suggest that effective therapeutic approaches to combat neurodegenerative diseases could be aimed at enhancing the ability of the cell to maintain protein solubility. PMID:25636813

  11. Protein lipoxidation: Detection strategies and challenges

    PubMed Central

    Aldini, Giancarlo; Domingues, M. Rosário; Spickett, Corinne M.; Domingues, Pedro; Altomare, Alessandra; Sánchez-Gómez, Francisco J.; Oeste, Clara L.; Pérez-Sala, Dolores

    2015-01-01

    Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets. PMID:26072467

  12. Protein lipoxidation: Detection strategies and challenges.

    PubMed

    Aldini, Giancarlo; Domingues, M Rosário; Spickett, Corinne M; Domingues, Pedro; Altomare, Alessandra; Sánchez-Gómez, Francisco J; Oeste, Clara L; Pérez-Sala, Dolores

    2015-08-01

    Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

  13. LPS-protein aggregation influences protein partitioning in aqueous two-phase micellar systems.

    PubMed

    Lopes, André Moreni; Santos-Ebinuma, Valéria de Carvalho; Novaes, Leticia Celia de Lencastre; Molino, João Vitor Dutra; Barbosa, Leandro Ramos Souza; Pessoa, Adalberto; Rangel-Yagui, Carlota de Oliveira

    2013-07-01

    Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS-protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM(LPS) > 98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K GFP(uv) < 1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein-LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein-LPS aggregation.

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

    PubMed

    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.

  15. Protein misfolding and aggregation research: some thoughts on improving quality and utility.

    PubMed

    Murphy, Regina M; Roberts, Christopher J

    2013-01-01

    Once misfolded and aggregated proteins were as interesting as yesterday's trash, just a bothersome byproduct of productive activities. Today, they attract sustained interest from both basic researchers and practicing engineers. In the burgeoning biopharmaceutical industry, protein misfolding and aggregation pose significant challenges to the economic manufacture of safe and effective protein products. In the clinic, protein aggregates are believed to be pathological agents in a number of serious neurodegenerative disorders, such as Alzheimer's and Parkinson's. Over the past few years, the quantity of research into biotechnological aspects of protein misfolding and aggregation has skyrocketed. However, the quality of the published work is quite variable. In this brief opinion piece, we describe what we believe are some key features of high-quality publications in protein aggregation. We focus on experimental studies that may also have a kinetic modeling component. © 2013 American Institute of Chemical Engineers.

  16. Size effects in models for mechanically-stressed protein crystals and aggregates

    NASA Technical Reports Server (NTRS)

    Noever, David A.

    1992-01-01

    As protein aggregates increase in size, they become easier to disrupt mechanically. Using the scaling properties of models proposed to govern protein aggregation, the effect of thermal vibrations and gravity are investigated as deforming forces. For typical protein assemblies made of 30 A proteins, the assembled diameter must remain less than 100-10,000 times the molecular radius to survive in finite thermal and gravity fields. The analysis predicts the following experimental outcomes: (1) reductions in gravitational strain should favor larger protein aggregates; (2) in comparing the aggregate stability of different proteins, the addition of peptide chains should stabilize against thermal strain, but should not affect gravitational strain; (3) critical aggregate sizes should show significant (exponential) sensitivity to cluster geometry, solution preparation and growth conditions. The analysis is extended to consider qualitative size effects in crystal damage during X-ray exposure.

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

    PubMed

    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.

  18. Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes

    PubMed Central

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Gao, Yuan; Yu, Li-Rong; Alla, Ramani; Shmookler Reis, Robert

    2015-01-01

    Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10−6). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly

  19. Modulus enhancement of natural rubber through the dispersion size reduction of protein/fiber aggregates

    USDA-ARS?s Scientific Manuscript database

    Improved mechanical properties of natural rubber are required for various rubber applications. Aggregates of protein and fiber that constitute soy protein concentrate were shear-reduced and used to enhance the tensile modulus of natural rubber. The aqueous dispersion of the shear-reduced aggregates ...

  20. The ER Stress Surveillance (ERSU) pathway regulates daughter cell ER protein aggregate inheritance

    PubMed Central

    Piña, Francisco J; Niwa, Maho

    2015-01-01

    Stress induced by cytoplasmic protein aggregates can have deleterious consequences for the cell, contributing to neurodegeneration and other diseases. Protein aggregates are also formed within the endoplasmic reticulum (ER), although the fate of ER protein aggregates, specifically during cell division, is not well understood. By simultaneous visualization of both the ER itself and ER protein aggregates, we found that ER protein aggregates that induce ER stress are retained in the mother cell by activation of the ER Stress Surveillance (ERSU) pathway, which prevents inheritance of stressed ER. In contrast, under conditions of normal ER inheritance, ER protein aggregates can enter the daughter cell. Thus, whereas cytoplasmic protein aggregates are retained in the mother cell to protect the functional capacity of daughter cells, the fate of ER protein aggregates is determined by whether or not they activate the ERSU pathway to impede transmission of the cortical ER during the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.06970.001 PMID:26327697

  1. Impact of additives on the formation of protein aggregates and viscosity in concentrated protein solutions.

    PubMed

    Bauer, Katharina Christin; Suhm, Susanna; Wöll, Anna Katharina; Hubbuch, Jürgen

    2017-01-10

    In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregation. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Influence of denatured and intermediate states of folding on protein aggregation.

    PubMed

    Fawzi, Nicolas L; Chubukov, Victor; Clark, Louis A; Brown, Scott; Head-Gordon, Teresa

    2005-04-01

    We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.

  3. Biomolecule detection via target mediated nanoparticle aggregation and dielectrophoretic impedance measurement.

    PubMed

    Costanzo, Philip J; Liang, Enzhu; Patten, Timothy E; Collins, Scott D; Smith, Rosemary L

    2005-06-01

    A new biosensing system is described that is based on the aggregation of nanoparticles by a target biological molecule and dielectrophoretic impedance measurement of these aggregates. The aggregation process was verified within a microchannel via fluorescence microscopy, demonstrating that this process can be used in a real time sensor application. Positive dielectrophoresis is employed to capture the nanoparticle aggregates at the edge of thin film electrodes, where their presence is detected either by optical imaging via fluorescence microscopy or by measuring the change in electrical impedance between adjacent electrodes. The electrical detection mechanism demonstrates the potential for this method as a micro total analysis system (microTAS).

  4. Protein aggregates are associated with replicative aging without compromising protein quality control

    PubMed Central

    Saarikangas, Juha; Barral, Yves

    2015-01-01

    Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells. In budding yeast, various aging factors segregate to the aging (mother)-lineage, with poorly understood consequences. In this study, we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single, asymmetrically inherited deposit over the remaining lifespan. Surprisingly, deposit formation was not associated with stress or general decline in proteostasis. Rather, the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates. Deposit formation was dependent on Hsp42, which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery, including the prion Sup35. Importantly, loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell. Together, these data suggest that protein aggregation is an early aging-associated differentiation event in yeast, having a two-faceted role in organismal fitness. DOI: http://dx.doi.org/10.7554/eLife.06197.001 PMID:26544680

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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/. PMID:25883144

  7. Biophysical Insights into How Surfaces, Including Lipid Membranes, Modulate Protein Aggregation Related to Neurodegeneration

    PubMed Central

    Burke, Kathleen A.; Yates, Elizabeth A.; Legleiter, Justin

    2013-01-01

    There are a vast number of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), associated with the rearrangement of specific proteins to non-native conformations that promotes aggregation and deposition within tissues and/or cellular compartments. These diseases are commonly classified as protein-misfolding or amyloid diseases. The interaction of these proteins with liquid/surface interfaces is a fundamental phenomenon with potential implications for protein-misfolding diseases. Kinetic and thermodynamic studies indicate that significant conformational changes can be induced in proteins encountering surfaces, which can play a critical role in nucleating aggregate formation or stabilizing specific aggregation states. Surfaces of particular interest in neurodegenerative diseases are cellular and subcellular membranes that are predominately comprised of lipid components. The two-dimensional liquid environments provided by lipid bilayers can profoundly alter protein structure and dynamics by both specific and non-specific interactions. Importantly for misfolding diseases, these bilayer properties can not only modulate protein conformation, but also exert influence on aggregation state. A detailed understanding of the influence of (sub)cellular surfaces in driving protein aggregation and/or stabilizing specific aggregate forms could provide new insights into toxic mechanisms associated with these diseases. Here, we review the influence of surfaces in driving and stabilizing protein aggregation with a specific emphasis on lipid membranes. PMID:23459674

  8. The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient.

    PubMed

    Ho, Jason G S; Middelberg, Anton P J; Ramage, Paul; Kocher, Hans P

    2003-04-01

    Protein aggregation is commonly observed during protein refolding. To better understand this phenomenon, the intermolecular interactions experienced by a protein during unfolding and refolding are inferred from second virial coefficient (SVC) measurements. It is accepted that a negative SVC is indicative of protein-protein interactions that are attractive, whereas a positive SVC indicates net repulsive interactions. Lysozyme denatured and reduced in guanidinium hydrochloride exhibited a decreasing SVC as the denaturant was diluted, and the SVC approached zero at approximately 3 M GdnHCl. Further dilution of denaturant to renaturation conditions (1.25 M GdnHCl) led to a negative SVC, and significant protein aggregation was observed. The inclusion of 500 mM L-arginine in the renaturation buffer shifted the SVC to positive and suppressed aggregation, thereby increasing refolding yield. The formation of mixed disulfides in the denatured state prior to refolding also increased protein solubility and suppressed aggregation, even without the use of L-arginine. Again, the suppression of aggregation was shown to be caused by a shift from attractive to repulsive intermolecular interactions as reflected in a shift from a negative to a positive SVC value. To the best of our knowledge, this is the first time that SVC data have been reported for renaturation studies. We believe this technique will aid in our understanding of how certain conditions promote renaturation and increase protein solubility, thereby suppressing aggregation. SVC measurements provide a useful link, for protein folding and aggregation, between empirical observation and thermodynamics.

  9. Chemical and Biological Approaches for Adapting Proteostasis to Ameliorate Protein Misfolding and Aggregation Diseases–Progress and Prognosis

    PubMed Central

    Lindquist, Susan L.; Kelly, Jeffery W.

    2011-01-01

    Maintaining the proteome to preserve the health of an organism in the face of developmental changes, environmental insults, infectious diseases, and rigors of aging is a formidable task. The challenge is magnified by the inheritance of mutations that render individual proteins subject to misfolding and/or aggregation. Maintenance of the proteome requires the orchestration of protein synthesis, folding, degradation, and trafficking by highly conserved/deeply integrated cellular networks. In humans, no less than 2000 genes are involved. Stress sensors detect the misfolding and aggregation of proteins in specific organelles and respond by activating stress-responsive signaling pathways. These culminate in transcriptional and posttranscriptional programs that up-regulate the homeostatic mechanisms unique to that organelle. Proteostasis is also strongly influenced by the general properties of protein folding that are intrinsic to every proteome. These include the kinetics and thermodynamics of the folding, misfolding, and aggregation of individual proteins. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We then introduce chemical approaches to prevent the misfolding or aggregation of specific proteins through direct binding interactions. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organismal proteostasis. PMID:21900404

  10. Chemical and biological approaches for adapting proteostasis to ameliorate protein misfolding and aggregation diseases: progress and prognosis.

    PubMed

    Lindquist, Susan L; Kelly, Jeffery W

    2011-12-01

    Maintaining the proteome to preserve the health of an organism in the face of developmental changes, environmental insults, infectious diseases, and rigors of aging is a formidable task. The challenge is magnified by the inheritance of mutations that render individual proteins subject to misfolding and/or aggregation. Maintenance of the proteome requires the orchestration of protein synthesis, folding, degradation, and trafficking by highly conserved/deeply integrated cellular networks. In humans, no less than 2000 genes are involved. Stress sensors detect the misfolding and aggregation of proteins in specific organelles and respond by activating stress-responsive signaling pathways. These culminate in transcriptional and posttranscriptional programs that up-regulate the homeostatic mechanisms unique to that organelle. Proteostasis is also strongly influenced by the general properties of protein folding that are intrinsic to every proteome. These include the kinetics and thermodynamics of the folding, misfolding, and aggregation of individual proteins. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We then introduce chemical approaches to prevent the misfolding or aggregation of specific proteins through direct binding interactions. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organismal proteostasis.

  11. Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates

    PubMed Central

    Korac, Jelena; Schaeffer, Veronique; Kovacevic, Igor; Clement, Albrecht M.; Jungblut, Benno; Behl, Christian; Terzic, Janos; Dikic, Ivan

    2013-01-01

    SUMMARY Aggregation of misfolded proteins and the associated loss of neurons are considered as a hallmark of numerous neurodegenerative diseases. Optineurin is present in protein inclusions observed in various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, Creutzfeld-Jacob disease and Pick’s disease. Optineurin deletion mutations have also been described in ALS patients. However, the role of optineurin in mechanisms of protein aggregation remains unclear. In this report, we demonstrate that optineurin recognized various protein aggregates via its C-terminal coiled-coil domain in a ubiquitin-independent manner. We also show that optineurin depletion significantly increase protein aggregation in HeLa cells and morpholino-silencing of the optineurin ortholog in zebrafish causes the motor axonopathy phenotype similar to a zebrafish model of ALS. A more severe phenotype is observed when optineurin is depleted in zebrafish carrying ALS mutations. Furthermore, TANK1 binding kinase 1 (TBK1) is co-localized with optineurin on protein aggregates and is important in clearance of protein aggregates through the autophagy-lysosome pathway. TBK1 phosphorylates optineurin at position Ser-177 and regulates its ability to interact with autophagy modifiers. This study provides evidence for a ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates as well as additional relevance for TBK1 as an upstream regulator of the autophagic pathway. PMID:23178947

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Detection and characterization of red blood cell (RBC) aggregation with photoacoustics

    NASA Astrophysics Data System (ADS)

    Hysi, Eno; Saha, Ratan K.; Rui, Min; Kolios, Michael C.

    2012-02-01

    Red blood cells (RBCs) aggregate in the presence of increased plasma fibrinogen and low shear forces during blood flow. RBC aggregation has been observed in deep vein thrombosis, sepsis and diabetes. We propose using photoacoustics (PA) as a non-invasive imaging modality to detect RBC aggregation. The theoretical and experimental feasibility of PA for detecting and characterizing aggregation was assessed. A simulation study was performed to generate PA signals from non-aggregated and aggregated RBCs using a frequency domain approach and to study the PA signals' dependence on hematocrit and aggregate size. The effect of the finite bandwidth nature of transducers on the PA power spectra was also investigated. Experimental confirmation of theoretical results was conducted using porcine RBC samples exposed to 1064 nm optical wavelength using the Imagio Small Animal PA imaging system (Seno Medical Instruments, Inc., San Antonio, TX). Aggregation was induced with Dextran-70 (Sigma-Aldrich, St. Louis, MO) and the effect of hematocrit and aggregation level was investigated. The theoretical and experimental PA signal amplitude increased linearly with increasing hematocrit. The theoretical dominant frequency content of PA signals shifted towards lower frequencies (<30 MHz) and 9 dB enhancements in spectral power were observed as the size of aggregates increased compared to non-aggregating RBCs. Calibration of the PA spectra with the transducer response obtained from a 200 nm gold film was performed to remove system dependencies. Analysis of the spectral parameters from the calibrated spectra suggested that PA can assess the degree of aggregation at multiple hematocrit and aggregation levels.

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

    PubMed Central

    Stark, Marcel; Behl, Christian

    2014-01-01

    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. PMID:25002904

  16. Kinetics of Formation and Asymmetrical Distribution of Hsp104-Bound Protein Aggregates in Yeast.

    PubMed

    Paoletti, Camille; Quintin, Sophie; Matifas, Audrey; Charvin, Gilles

    2016-04-12

    Budding yeast cells have a finite replicative life span; that is, a mother cell produces only a limited number of daughter cells before it slows division and dies. Despite the gradual aging of the mother cell, all daughters are born rejuvenated and enjoy a full replicative lifespan. It has been proposed that entry of mother cells into senescence is driven by the progressive accumulation and retention of damaged material, including protein aggregates. This additionally allows the daughter cells to be born damage free. However, the mechanism underlying such asymmetrical segregation of protein aggregates by mother and daughter cells remains controversial, in part because of the difficulties inherent in tracking the dynamics and fate of protein aggregates in vivo. To overcome such limitations, we have developed single-cell real-time imaging methodology to track the formation of heat-induced protein aggregates in otherwise unperturbed dividing cells. By combining the imaging data with a simple computational model of protein aggregation, we show that the establishment of asymmetrical partitioning of protein aggregates upon division is driven by the large bud-specific dilution rate associated with polarized growth and the absence of significant mother/bud exchange of protein aggregates during the budded phase of the cell cycle. To our knowledge, this study sheds new light on the mechanism of establishment of a segregation bias, which can be accounted for by simple physical arguments.

  17. Reduced transglutaminase-catalyzed protein aggregation is observed in the presence of creatine using sedimentation velocity.

    PubMed

    Burguera, Elena F; Love, Brian J

    2006-03-01

    Transglutaminases (TGases) are enzymes that catalyze covalent isopeptide crosslinks between reactive lysine and glutamine residues in proteins. Higher than normal local concentrations of TGase have been correlated with increased protein aggregation in vivo. These insoluble protein aggregates are the hallmark of several neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, although each aggregating protein involved is disease specific. Because TGase is implicated in protein aggregation, there is evidence that its regulation may retard disease progression. Here we report on a laser light transmission technique as an in vitro tool to gauge the efficacy of creatine, a candidate inhibitor, to regulate aggregation. Sedimentation velocities of protein-coated particles in TGase-containing water-glycerol solutions were tracked with different levels of creatine. Sedimentation velocities were converted to apparent aggregate sizes using Stoke's law of sedimentation. The results indicated that creatine promoted up to a 20% reduction in protein aggregation in vitro. This technique may prove to be useful in identifying other functional TGase inhibitors.

  18. Editor’s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response

    PubMed Central

    Ratanji, Kirsty D.; Dearman, Rebecca J.; Kimber, Ian; Thorpe, Robin; Wadhwa, Meenu; Derrick, Jeremy P.

    2016-01-01

    Protein aggregation is associated with enhanced immunogenicity of biotherapeutics. As a result, regulatory guidelines recommend screening for aggregation during bioprocessing. However, the mechanisms underlying the enhanced immunogenicity of aggregates are poorly understood. In the investigations described herein, the immunogenicity in mice of a humanized single chain variable antibody fragment (scFv) purified after expression in Escherichia coli has been examined. Reproducible scFv aggregates were obtained within the subvisible particle size range (mean diameter 2 µm) using thermal and mechanical stresses. Intraperitoneal immunization of BALB/c strain mice with 1 mg/ml of aggregated or monomeric scFv induced similar IgG and IgG1 antibody responses. In contrast, aggregate preparations stimulated significantly higher levels of anti-scFv IgG2a antibody than did the monomer. In comparative studies, aggregates of ovalbumin (OVA) within the subvisible particle size range were prepared by stir stress, and their immunogenicity compared with that of monomeric OVA in mice. Aggregated and monomeric OVA induced similar anti-OVA IgG and IgG1 antibody responses, whereas IgG2a antibody levels were significantly higher in aggregate-immunized mice. Furthermore, cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were consistent with aggregated preparations inducing a T helper (Th) 1-type response. Aggregated proteins within the subvisible range were therefore shown to induce a preferential Th1 type response, whereas monomeric proteins elicited a selective Th2 response. These data indicate that protein aggregation can impact on both the vigor and quality of immune responses. PMID:27370416

  19. Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

    PubMed Central

    Rajan, Rahul S.; Li, Tiansheng; Aras, Mohini; Sloey, Christopher; Sutherland, Weston; Arai, Hiromi; Briddell, Robert; Kinstler, Olaf; Lueras, Alexis M.K.; Zhang, Yu; Yeghnazar, Heather; Treuheit, Michael; Brems, David N.

    2006-01-01

    Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface. PMID:16597829

  20. Asymmetric damage segregation at cell division via protein aggregate fusion and attachment to organelles.

    PubMed

    Coelho, Miguel; Tolić, Iva M

    2015-07-01

    The segregation of damaged components at cell division determines the survival and aging of cells. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, aggregated proteins are retained by the mother cell. Yet, where and how aggregation occurs is not known. Recent work by Zhou and collaborators shows that the birth of protein aggregates, under specific stress conditions, requires active translation, and occurs mainly at the endoplasmic reticulum. Later, aggregates move to the mitochondrial surface through fis1-dependent association. During replicative aging, aggregate association with the mother-cell mitochondria contributes to the asymmetric segregation of aggregates, because mitochondria in the daughter cell do not carry aggregates. With increasing age of mother cells, aggregates lose their connection to the mitochondria, and segregation is less asymmetric. Relating these findings to other mechanisms of aggregate segregation in different organisms, we postulate that fusion between aggregates and their tethering to organelles such as the vacuole, nucleus, ER, or mitochondria are common principles that establish asymmetric segregation during stress resistance and aging. © 2015 WILEY Periodicals, Inc.

  1. Both protein adsorption and aggregation contribute to shear yielding and viscosity increase in protein solutions.

    PubMed

    Castellanos, Maria Monica; Pathak, Jai A; Colby, Ralph H

    2014-01-07

    A combination of sensitive rotational rheometry and surface rheometry with a double-wall ring were used to identify the origins of the viscosity increase at low shear rates in protein solutions. The rheology of two high molecular weight proteins is discussed: Bovine Serum Albumin (BSA) in a Phosphate Buffered Saline solution and an IgG1 monoclonal antibody (mAb) in a formulation buffer containing small quantities of a non-ionic surfactant. For surfactant-free BSA solutions, the interfacial viscosity dominates the low shear viscosity measured in rotational rheometers, while the surfactant-laden mAb solution has an interfacial viscosity that is small compared to that from aggregation in the bulk. A viscoelastic film forms at the air/water interface in the absence of surfactant, contributing to an apparent yield stress (thus a low shear viscosity increase) in conventional bulk rheology measurements. Addition of surfactant eliminates the interfacial yield stress. Evidence of a bulk yield stress arising from protein aggregation is presented, and correlated with results from standard characterization techniques used in the bio-pharmaceutical industry. The protein film at the air/water interface and bulk aggregates both lead to an apparent viscosity increase and their contributions are quantified using a dimensionless ratio of the interfacial and total yield stress. While steady shear viscosities at shear rates below ∼1 s(-1) contain rich information about the stability of protein solutions, embodied in the measured yield stress, such low shear rate data are regrettably often not measured and reported in the literature.

  2. Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions.

    PubMed

    Patel, Hasmukh A; Singh, Harjinder; Havea, Palatasa; Considine, Thérèse; Creamer, Lawrence K

    2005-11-30

    Whey protein concentrate solutions (12% w/v, pH 6.65 +/- 0.05) were pressure treated at 800 MPa for 20-120 min and then examined using size exclusion chromatography (SEC), small deformation rheology, transmission electron microscopy, and various types of one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). The pressure-treated samples showed a time-dependent loss of native whey proteins by SEC and 1D PAGE and a corresponding increase in non-native proteins and protein aggregates of different sizes. These aggregates altered the viscosity and opacity of the samples and were shown to be cross-linked by intermolecular disulfide bonds and by noncovalent interactions using 1D PAGE [alkaline (or native), sodium dodecyl sulfate (SDS), and SDS of reduced samples (SDS(R))] and 2D PAGE (native:SDS and SDS:SDS(R)). The sensitivity of the major whey proteins to pressure was in the order beta-lactoglobulin B (beta-LG B) > beta-LG A > bovine serum albumin (BSA) > alpha-lactalbumin (alpha-LA), and the large internal hydrophobic cavity of beta-LG may have been partially responsible for its sensitivity to high-pressure treatments. It seemed likely that, at 800 MPa, the formation of a beta-LG disulfide-bonded network preceded the formation of disulfide bonds between alpha-LA or BSA and beta-LG to form multiprotein aggregates, possibly because the disulfide bonds of alpha-LA and BSA are less exposed than those of beta-LG either during or after pressure treatment. It may be possible that intermolecular disulfide bond formation occurred at high pressure and that hydrophobic association became important after the high-pressure treatment.

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

    PubMed

    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.

  4. High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington’s Disease Model

    PubMed Central

    Titus, Steven A; Southall, Noel; Marugan, Juan; Austin, Christopher P; Zheng, Wei

    2012-01-01

    A hallmark of Huntington’s disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins. Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson’s, Alzheimer’s, and prion diseases. However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear. Identification of small molecule probes that modulate protein aggregation and cytotoxicity caused by aggregated proteins may greatly facilitate the studies on pathogenesis of these diseases and potentially lead to development of new therapies. Based on a detergent insoluble property of the Huntingtin protein aggregates, we have developed a homogenous assay to rapidly quantitate the levels of protein aggregates in a cellular model of Huntington’s disease. The protein aggregation assay has also been multiplexed with a protease release assay for the measurement of cytotoxicity resulting from aggregated proteins in the same cells. Through a testing screen of a compound library, we have demonstrated that this multiplexed cytotoxicity and protein aggregation assay has ability to identify active compounds that prevent cell death and/or modulate protein aggregation in cells of the Huntington’s disease model. Therefore, this multiplexed screening approach is also useful for development of high-throughput screening assays for other neurodegenerative diseases involving protein aggregation. PMID:23346268

  5. Protein aggregation in food models: effect of. gamma. -irradiation and lipid oxidation

    SciTech Connect

    Delincee, H.; Paul, P.

    1981-09-01

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

  6. Quantitation of pH-induced aggregation in binary protein mixtures by dielectric spectroscopy.

    PubMed

    Mellor, Brett L; Wood, Stephen J; Mazzeo, Brian A

    2012-12-01

    This paper presents a quantitative approach for measuring pH-controlled protein aggregation using dielectric spectroscopy. The technique is demonstrated through two aggregation experiments, the first between β-lactoglobulin (β-Lg) and hen lysozyme (HENL) and the second between bovine serum albumin (BSA) and HENL. In both experiments, the formation of aggregates is strongly dependent on the solution pH and is clearly indicated by a decrease in the measured permittivity when the second protein is added. A quantifiable lower-bound on the ratio of proteins involved in the aggregation process is obtained from the permittivity spectra. Lower-bound aggregation ratios of 83 % for β-Lg/HENL at pH 6.0 and 48 % for BSA/HENL at pH 9.2 were consistent with turbidity measurements made on the same solutions.

  7. An online detection system for aggregate sizes and shapes based on digital image processing

    NASA Astrophysics Data System (ADS)

    Yang, Jianhong; Chen, Sijia

    2016-07-01

    Traditional aggregate size measuring methods are time-consuming, taxing, and do not deliver online measurements. A new online detection system for determining aggregate size and shape based on a digital camera with a charge-coupled device, and subsequent digital image processing, have been developed to overcome these problems. The system captures images of aggregates while falling and flat lying. Using these data, the particle size and shape distribution can be obtained in real time. Here, we calibrate this method using standard globules. Our experiments show that the maximum particle size distribution error was only 3 wt%, while the maximum particle shape distribution error was only 2 wt% for data derived from falling aggregates, having good dispersion. In contrast, the data for flat-lying aggregates had a maximum particle size distribution error of 12 wt%, and a maximum particle shape distribution error of 10 wt%; their accuracy was clearly lower than for falling aggregates. However, they performed well for single-graded aggregates, and did not require a dispersion device. Our system is low-cost and easy to install. It can successfully achieve online detection of aggregate size and shape with good reliability, and it has great potential for aggregate quality assurance.

  8. An online detection system for aggregate sizes and shapes based on digital image processing

    NASA Astrophysics Data System (ADS)

    Yang, Jianhong; Chen, Sijia

    2017-02-01

    Traditional aggregate size measuring methods are time-consuming, taxing, and do not deliver online measurements. A new online detection system for determining aggregate size and shape based on a digital camera with a charge-coupled device, and subsequent digital image processing, have been developed to overcome these problems. The system captures images of aggregates while falling and flat lying. Using these data, the particle size and shape distribution can be obtained in real time. Here, we calibrate this method using standard globules. Our experiments show that the maximum particle size distribution error was only 3 wt%, while the maximum particle shape distribution error was only 2 wt% for data derived from falling aggregates, having good dispersion. In contrast, the data for flat-lying aggregates had a maximum particle size distribution error of 12 wt%, and a maximum particle shape distribution error of 10 wt%; their accuracy was clearly lower than for falling aggregates. However, they performed well for single-graded aggregates, and did not require a dispersion device. Our system is low-cost and easy to install. It can successfully achieve online detection of aggregate size and shape with good reliability, and it has great potential for aggregate quality assurance.

  9. HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates

    PubMed Central

    Boyault, Cyril; Zhang, Yu; Fritah, Sabrina; Caron, Cécile; Gilquin, Benoit; Kwon, So Hee; Garrido, Carmen; Yao, Tso-Pang; Vourc’h, Claire; Matthias, Patrick; Khochbin, Saadi

    2007-01-01

    A cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation. PMID:17785525

  10. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.

    PubMed

    Bucciantini, Monica; Giannoni, Elisa; Chiti, Fabrizio; Baroni, Fabiana; Formigli, Lucia; Zurdo, Jesús; Taddei, Niccolò; Ramponi, Giampietro; Dobson, Christopher M; Stefani, Massimo

    2002-04-04

    A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

  11. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases

    NASA Astrophysics Data System (ADS)

    Bucciantini, Monica; Giannoni, Elisa; Chiti, Fabrizio; Baroni, Fabiana; Formigli, Lucia; Zurdo, Jesús; Taddei, Niccolò; Ramponi, Giampietro; Dobson, Christopher M.; Stefani, Massimo

    2002-04-01

    A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

  12. Recruitment into stress granules prevents irreversible aggregation of FUS protein mislocalized to the cytoplasm

    PubMed Central

    Shelkovnikova, Tatyana A; Robinson, Hannah K; Connor-Robson, Natalie; Buchman, Vladimir L

    2013-01-01

    Fused in sarcoma (FUS) belongs to the group of RNA-binding proteins implicated as underlying factors in amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. Multiple FUS gene mutations have been linked to hereditary forms, and aggregation of FUS protein is believed to play an important role in pathogenesis of these diseases. In cultured cells, FUS variants with disease-associated amino acid substitutions or short deletions affecting nuclear localization signal (NLS) and causing cytoplasmic mislocalization can be sequestered into stress granules (SGs). We demonstrated that disruption of motifs responsible for RNA recognition and binding not only prevents SG recruitment, but also dramatically increases the protein propensity to aggregate in the cell cytoplasm with formation of juxtanuclear structures displaying typical features of aggresomes. Functional RNA-binding domains from TAR DNA-binding protein of 43 kDa (TDP-43) fused to highly aggregation-prone C-terminally truncated FUS protein restored the ability to enter SGs and prevented aggregation of the chimeric protein. Truncated FUS was also able to trap endogenous FUS molecules in the cytoplasmic aggregates. Our data indicate that RNA binding and recruitment to SGs protect cytoplasmic FUS from aggregation, and loss of this protection may trigger its pathological aggregation in vivo. PMID:24013423

  13. Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

    PubMed Central

    Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

    2016-01-01

    Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

  14. [Molecular therapy targeting protein misfolding and aggregation for the polyglutamine diseases].

    PubMed

    Nagai, Yoshitaka

    2009-11-01

    Abnormal aggregation and deposition of misfolded proteins have been recognized as a common molecular pathogenesis of various neurodegenerative diseases including Alzheimer's, Parkinson's, and the polyglutamine (polyQ) diseases. The polyQ diseases, including Huntington's disease and various spinocerebellar ataxias, are caused by abnormal expansions of the polyQ stretch (> 35-40) within disease-causative proteins, which are thought to trigger their misfolding and aggregation, leading to their deposition as inclusion bodies, and eventually resulting in neurodegeneration. We found that the expanded polyQ protein undergoes a conformational transition to a beta-sheet dominant structure in the monomeric state, triggering cytotoxicity, and subsequently resulting in formation of insoluble amyloid-like fibrillar aggregates. Targeting misfolding and aggregation of the expanded polyQ protein, we demonstrated that QBP1 (PolyQ-Binding Peptide 1: SNWKWWPGIFD) prevents the toxic beta-sheet transition and aggregation of the expanded polyQ protein in vitro and suppresses polyQ-induced neurodegeneration in Drosophila. From high-throughput screening of a chemical compound library (46,000), we have identified approximately 100 polyQ aggregate inhibitors as therapeutic candidates so far. We also found that 17-AAG, an HSF1-activating compound, suppresses polyQ-induced neurodegeneration in Drosophila through induction of endogenous molecular chaperones. We propose that our therapeutic strategy targeting protein misfolding and aggregation can also be applied to other neurodegenerative diseases.

  15. Detecting protein-protein interactions using Renilla luciferase fusion proteins.

    PubMed

    Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

    2002-11-01

    We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

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

    PubMed

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

    2016-05-01

    Neurodegenerative diseases are largely defined by protein aggregates in affected tissues. Aggregates contain some shared components as well as proteins thought to be specific for each disease. Aggregation has not previously been reported in the normal, aging heart or the hypertensive heart. Detergent-insoluble protein aggregates were isolated from mouse heart and characterized on 2-dimensional gels. Their levels increased markedly and significantly with aging and after sustained angiotensin II-induced hypertension. Of the aggregate components identified by high-resolution proteomics, half changed in abundance with age (392/787) or with sustained hypertension (459/824), whereas 30% (273/901) changed concordantly in both, each P<0.05. One fifth of these proteins were previously associated with age-progressive neurodegenerative or cardiovascular diseases, or both (eg, ApoE, ApoJ, ApoAIV, clusterin, complement C3, and others involved in stress-response and protein-homeostasis pathways). Because fibrosis is a characteristic of both aged and hypertensive hearts, we posited that aging of fibroblasts may contribute to the aggregates observed in cardiac tissue. Indeed, as cardiac myofibroblasts "senesced" (approached their replicative limit) in vitro, they accrued aggregates with many of the same constituent proteins observed in vivo during natural aging or sustained hypertension. In summary, we have shown for the first time that compact (detergent-insoluble) protein aggregates accumulate during natural aging, chronic hypertension, and in vitro myofibroblast senescence, sharing many common proteins. Thus, aggregates that arise from disparate causes (aging, hypertension, and replicative senescence) may have common underlying mechanisms of accrual.

  17. Ultrafast laser-probing spectroscopy for studying molecular structure of protein aggregates.

    PubMed

    Jung, Huihun; Szwejkowski, Chester J; Pena-Francesch, Abdon; Tomko, John A; Allen, Benjamin; Özdemir, Şahin Kaya; Hopkins, Patrick; Demirel, Melik C

    2017-03-09

    We report the development of a new technique to screen protein aggregation based on laser-probing spectroscopy with sub-picosecond resolution. Protein aggregation is an important topic for materials science, fundamental biology as well as clinical studies in neurodegenerative diseases and translation studies in biomaterials engineering. However, techniques to study protein aggregation and assembly are limited to infrared spectroscopy, fluorescent assays, immunostaining, or functional assays among others. Here, we report a new technique to characterize protein structure-property relationship based on ultrafast laser-probing spectroscopy. First, we show theoretically that the temperature dependence of the refractive index of a protein is correlated to its crystallinity. Then, we performed time-domain thermo-transmission experiments on purified semi-crystalline proteins, both native and recombinant (i.e., silk and squid ring teeth), and also on intact E. coli cells bearing overexpressed recombinant protein. Our results demonstrate, for the first time, relative quantification of crystallinity in real time for protein aggregates. Our approach can potentially be used for screening an ultra-large number of proteins in vivo. Using this technique, we could answer many fundamental questions in structural protein research, such as the underlying sequence-structure relationship for protein assembly and aggregation.

  18. Accelerating the clearance of mutant huntingtin protein aggregates through autophagy induction by europium hydroxide nanorods.

    PubMed

    Wei, Peng-Fei; Zhang, Li; Nethi, Susheel Kumar; Barui, Ayan Kumar; Lin, Jun; Zhou, Wei; Shen, Yi; Man, Na; Zhang, Yun-Jiao; Xu, Jing; Patra, Chitta Ranjan; Wen, Long-Ping

    2014-01-01

    Autophagy is one of the well-known pathways to accelerate the clearance of protein aggregates, which contributes to the therapy of neurodegenerative diseases. Although there are numerous reports that demonstrate the induction of autophagy with small molecules including rapamycin, trehalose and lithium, however, there are few reports mentioning the clearance of aggregate-prone proteins through autophagy induction by nanoparticles. In the present article, we have demonstrated that europium hydroxide [Eu(III)(OH)3] nanorods can reduce huntingtin protein aggregation (EGFP-tagged huntingtin protein with 74 polyQ repeats), responsible for neurodegenerative diseases. Again, we have found that these nanorods induce authentic autophagy flux in different cell lines (Neuro 2a, PC12 and HeLa cells) through the expression of higher levels of characteristic autophagy marker protein LC3-II and degradation of selective autophagy substrate/cargo receptor p62/SQSTM1. Furthermore, depression of protein aggregation clearance through the autophagy blockade has also been observed by using specific inhibitors (wortmannin and chloroquine), indicating that autophagy is involved in the degradation of huntingtin protein aggregation. Since [Eu(III)(OH)3] nanorods can enhance the degradation of huntingtin protein aggregation via autophagy induction, we strongly believe that these nanorods would be useful for the development of therapeutic treatment strategies for various neurodegenerative diseases in near future using nanomedicine approach.

  19. Polyglutamine protein aggregation and toxicity are linked to the cellular stress response.

    PubMed

    Cowan, K J; Diamond, M I; Welch, W J

    2003-06-15

    Chronic exposure of cells to expanded polyglutamine proteins results in eventual cell demise. We constructed mouse cell lines expressing either the full-length androgen receptor (AR), or truncated forms of AR containing 25 or 65 glutamines to study the cellular consequences of chronic low-level exposure to these proteins. Expression of the polyglutamine-expanded truncated AR protein, but not the full-length expanded protein, resulted in the formation of cytoplasmic and nuclear aggregates and eventual cell death. Nuclear aggregates preferentially stained positive for heat shock protein (hsp)72, a sensitive indicator of a cellular stress response. Biochemical studies revealed that the presence of nuclear aggregates correlated with activation of the c-jun NH2-terminal kinase (JNK). Different metabolic insults, including heat shock treatment, and exposure to sodium arsenite or menadione, proved more toxic to those cells expressing the polyglutamine-expanded truncated protein than to cells expressing the non-expanded form. Cells containing cytoplasmic polyglutamine-protein aggregates exhibited a delayed expression of hsp72 after heat shock. Once expressed, hsp72 failed to localize normally and instead was sequestered within the protein aggregates. This was accompanied by an inability of the aggregate-containing cells to cease their stress response as evidenced by the continued presence of activated JNK. Finally, activation of the cellular stress response increased the overall extent of polyglutamine protein aggregation, especially within the nucleus. Inclusion of a JNK inhibitor reduced this stress-dependent increase in nuclear aggregates. Abnormal stress responses may contribute to enhanced cell vulnerability in cells expressing polyglutamine-expanded proteins and may increase the propensity of such cells to form cytoplasmic and nuclear inclusions.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Polythiophenes Inhibit Prion Propagation by Stabilizing Prion Protein (PrP) Aggregates*

    PubMed Central

    Margalith, Ilan; Suter, Carlo; Ballmer, Boris; Schwarz, Petra; Tiberi, Cinzia; Sonati, Tiziana; Falsig, Jeppe; Nyström, Sofie; Hammarström, Per; Åslund, Andreas; Nilsson, K. Peter R.; Yam, Alice; Whitters, Eric; Hornemann, Simone; Aguzzi, Adriano

    2012-01-01

    Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrPC (PrPSc) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrPSc to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23–231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23–231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrPSc by stabilizing the conformation of PrPC or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrPSc deposits. PMID:22493452

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

    PubMed

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

    2015-05-19

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

  3. Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant.

    PubMed

    Ignatova, Zoya; Gierasch, Lila M

    2006-09-05

    Small organic molecules termed osmolytes are harnessed by a variety of cell types in a wide range of organisms to counter unfavorable physiological conditions that challenge protein stability and function. Using a well characterized reporter system that we developed to allow in vivo observations, we have explored how the osmolyte proline influences the stability and aggregation of a model aggregation-prone protein, P39A cellular retinoic acid-binding protein. Strikingly, we find that the natural osmolyte proline abrogates aggregation both in vitro and in vivo (in an Escherichia coli expression system). Importantly, proline also prevented aggregation of constructs containing exon 1 of huntingtin with extended polyglutamine tracts. Although compatible osmolytes are known to stabilize the native state, our results point to a destabilizing effect of proline on partially folded states and early aggregates and a solubilizing effect on the native state. Because proline is believed to act through a combination of solvophobic backbone interactions and favorable side-chain interactions that are not specific to a particular sequence or structure, the observed effect is likely to be general. Thus, the osmolyte proline may be protective against biomedically important protein aggregates that are hallmarks of several late-onset neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. In addition, these results should be of practical importance because they may enable protein expression at higher efficiency under conditions where aggregation competes with proper folding.

  4. Protein aggregation propensity is a crucial determinant of intracellular inclusion formation and quality control degradation.

    PubMed

    Villar-Piqué, Anna; Ventura, Salvador

    2013-12-01

    Protein aggregation is linked to many pathological conditions, including several neurodegenerative diseases. The aggregation propensities of proteins are thought to be controlled to a large extent by the physicochemical properties encoded in the primary sequence. We have previously exploited a set of amyloid β peptide (Aβ42) variants exhibiting a continuous gradient of intrinsic aggregation propensities to demonstrate that this rule applies in vivo in bacteria. In the present work we have characterized the behavior of these Aβ42 mutants when expressed in yeast. In contrast to bacteria, the intrinsic aggregation propensity is gated by yeast, in such a way that this property correlates with the formation of intracellular inclusions only above a specific aggregation threshold. Proteins displaying solubility levels above this threshold escape the inclusion formation pathway. In addition, the most aggregation-prone variants are selectively cleared by the yeast quality control degradation machinery. Thus, both inclusion formation and proteolysis target the same aggregation-prone variants and cooperate to minimize the presence of these potentially dangerous species in the cytosol. The demonstration that sorting to these pathways in eukaryotes is strongly influenced by protein primary sequence should facilitate the development of rational approaches to predict and hopefully prevent in vivo protein deposition. © 2013.

  5. Protein Aggregates Are Recruited to Aggresome by Histone Deacetylase 6 via Unanchored Ubiquitin C Termini

    SciTech Connect

    Ouyang, Hui; Ali, Yousuf O.; Ravichandran, Mani; Dong, Aiping; Qiu, Wei; MacKenzie, Farrell; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.; Zhai, R. Grace

    2012-07-11

    The aggresome pathway is activated when proteasomal clearance of misfolded proteins is hindered. Misfolded polyubiquitinated protein aggregates are recruited and transported to the aggresome via the microtubule network by a protein complex consisting of histone deacetylase 6 (HDAC6) and the dynein motor complex. The current model suggests that HDAC6 recognizes protein aggregates by binding directly to polyubiquitinated proteins. Here, we show that there are substantial amounts of unanchored ubiquitin in protein aggregates with solvent-accessible C termini. The ubiquitin-binding domain (ZnF-UBP) of HDAC6 binds exclusively to the unanchored C-terminal diglycine motif of ubiquitin instead of conjugated polyubiquitin. The unanchored ubiquitin C termini in the aggregates are generated in situ by aggregate-associated deubiquitinase ataxin-3. These results provide structural and mechanistic bases for the role of HDAC6 in aggresome formation and further suggest a novel ubiquitin-mediated signaling pathway, where the exposure of ubiquitin C termini within protein aggregates enables HDAC6 recognition and transport to the aggresome.

  6. A review on protein misfolding, aggregation and strategies to prevent related ailments.

    PubMed

    Shamsi, Tooba Naz; Athar, Teeba; Parveen, Romana; Fatima, Sadaf

    2017-07-23

    This review aims to highlight the fundamental mechanism of protein misfolding leading to protein aggregation and associated diseases. It also aims to anticipate novel therapeutic strategies with which to prevent or treat these highly debilitating conditions linked to these pathologies. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. The core cause of over 20 different human diseases which have now been designated as 'conformational diseases' including neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD) and Parkinson's disease (PD) etc. A comprehensive study on protein misfolding, aggregation, and the outcomes of the effects of cytotoxic aggregates will lead to understand the aggregation-mediated cell toxicity and serves as a foundation for future research in development of promising therapies and drugs. This review has also shed light on the mechanism of protein misfolding which leads to its aggregation and hence the neurodegeneration. From these considerations, one could also envisage the possibility that protein aggregation may be exploited by nature to perform specific physiological functions in differing biological contexts. Copyright © 2017. Published by Elsevier B.V.

  7. Connecting high-temperature and low-temperature protein stability and aggregation.

    PubMed

    Rosa, Mónica; Roberts, Christopher J; Rodrigues, Miguel A

    2017-01-01

    Protein aggregation is a long-standing problem for preservation of proteins in both laboratory settings and for commercial biotechnology products. It is well established that heating (cooling) can accelerate (slow) aggregation by populating (depopulating) unfolded or partially unfolded monomer states that are key intermediates in aggregation processes. However, there is a long-standing question of whether the same mechanism(s) that lead to aggregation under high-temperature stress are relevant for low-temperature stress such as in refrigerated or supercooled liquids. This report shows the first direct comparison of "hot" and "cold" aggregation kinetics and folding/unfolding thermodynamics, using bovine hemoglobin as a model system. The results suggest that the same mechanism for non-native aggregation holds from "hot" to "cold" temperatures, with an aggregation temperature-of-maximum-stability slightly below 0°C. This highlights that sub-zero temperatures can induce cold-mediated aggregation, even in the absence of freezing stresses. From a practical perspective, the results suggests the possibility that cold-stress may be a useful alternative to heat-stress for extrapolating predictions of protein shelf life at refrigerated conditions, as well as providing a foundation for more mechanistic studies of cold-stress conditions in future work. A comparison between isochoric and isobaric methods is also briefly discussed.

  8. Connecting high-temperature and low-temperature protein stability and aggregation

    PubMed Central

    Rosa, Mónica; Roberts, Christopher J.

    2017-01-01

    Protein aggregation is a long-standing problem for preservation of proteins in both laboratory settings and for commercial biotechnology products. It is well established that heating (cooling) can accelerate (slow) aggregation by populating (depopulating) unfolded or partially unfolded monomer states that are key intermediates in aggregation processes. However, there is a long-standing question of whether the same mechanism(s) that lead to aggregation under high-temperature stress are relevant for low-temperature stress such as in refrigerated or supercooled liquids. This report shows the first direct comparison of “hot” and “cold” aggregation kinetics and folding/unfolding thermodynamics, using bovine hemoglobin as a model system. The results suggest that the same mechanism for non-native aggregation holds from “hot” to “cold” temperatures, with an aggregation temperature-of-maximum-stability slightly below 0°C. This highlights that sub-zero temperatures can induce cold-mediated aggregation, even in the absence of freezing stresses. From a practical perspective, the results suggests the possibility that cold-stress may be a useful alternative to heat-stress for extrapolating predictions of protein shelf life at refrigerated conditions, as well as providing a foundation for more mechanistic studies of cold-stress conditions in future work. A comparison between isochoric and isobaric methods is also briefly discussed. PMID:28472066

  9. Particle Formation and Aggregation of a Therapeutic Protein in Nanobubble Suspensions.

    PubMed

    Snell, Jared R; Zhou, Chen; Carpenter, John F; Randolph, Theodore W

    2016-10-01

    The generation of nanobubbles following reconstitution of lyophilized trehalose formulations has recently been reported. Here, we characterize particle formation and aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in reconstituted formulations of lyophilized trehalose. Particle characterization methods including resonant mass measurement and nanoparticle tracking analysis were used to count and size particles generated upon reconstitution of lyophilized trehalose formulations. In addition, accelerated degradation studies were conducted to monitor rhIL-1ra aggregation in solutions containing various concentrations of suspended nanobubbles. Reconstitution of lyophilized trehalose formulations with solutions containing rhIL-1ra reduced nanobubble concentrations and generated negatively buoyant particles attributed to aggregated rhIL-1ra. Furthermore, levels of rhIL-1ra aggregation following incubation in aqueous solution correlated with concentrations of suspended nanobubbles. The results of this study suggest that nanobubbles may be a contributor to protein aggregation and particle formation in reconstituted, lyophilized therapeutic protein formulations.

  10. Simple Model Study of Phase Transition Properties of Isolated and Aggregated Protein

    NASA Astrophysics Data System (ADS)

    Ji, Yong-Yun; Yi, Wei-Qi; Zhang, Lin-Xi

    2011-03-01

    We investigate the phase transition properties of isolated and aggregated protein by exhaustive numerical study in the confined conformation space with maximally compact lattice model. The study within the confined conformation space shows some general folding properties. Various sequences show different folding properties: two-state folding, three-state folding and prion-like folding behavior. We find that the aggregated protein holds a more evident transition than isolated one and the transition temperature is generally lower than that in isolated case.

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

    PubMed

    Dasmeh, Pouria; Kepp, Kasper P

    2017-08-01

    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 stability and net charge and changes at the dimer interface were selectively introduced upon separation from old world monkeys and lesser apes (gibbons). Consequently, human, chimpanzee and gorilla SOD1s have a net charge of -6 at physiological pH, whereas the closely related gibbons and macaques have -3. These features consistently point towards selection against the malicious aggregation effects of elevated SOD1 levels in long-living great apes. The findings mirror the impact of human SOD1 mutations that reduce net charge and/or stability and cause ALS, a motor neuron disease characterized by oxidative stress and SOD1 aggregates and triggered by aging. Our study thus marks an example of direct selection for a particular chemical phenotype (high net charge and stability) in a single human protein with possible implications for the evolution of aging.

  12. Detecting internally symmetric protein structures.

    PubMed

    Kim, Changhoon; Basner, Jodi; Lee, Byungkook

    2010-06-03

    Many functional proteins have a symmetric structure. Most of these are multimeric complexes, which are made of non-symmetric monomers arranged in a symmetric manner. However, there are also a large number of proteins that have a symmetric structure in the monomeric state. These internally symmetric proteins are interesting objects from the point of view of their folding, function, and evolution. Most algorithms that detect the internally symmetric proteins depend on finding repeating units of similar structure and do not use the symmetry information. We describe a new method, called SymD, for detecting symmetric protein structures. The SymD procedure works by comparing the structure to its own copy after the copy is circularly permuted by all possible number of residues. The procedure is relatively insensitive to symmetry-breaking insertions and deletions and amplifies positive signals from symmetry. It finds 70% to 80% of the TIM barrel fold domains in the ASTRAL 40 domain database and 100% of the beta-propellers as symmetric. More globally, 10% to 15% of the proteins in the ASTRAL 40 domain database may be considered symmetric according to this procedure depending on the precise cutoff value used to measure the degree of perfection of the symmetry. Symmetrical proteins occur in all structural classes and can have a closed, circular structure, a cylindrical barrel-like structure, or an open, helical structure. SymD is a sensitive procedure for detecting internally symmetric protein structures. Using this procedure, we estimate that 10% to 15% of the known protein domains may be considered symmetric. We also report an initial, overall view of the types of symmetries and symmetric folds that occur in the protein domain structure universe.

  13. Detecting internally symmetric protein structures

    PubMed Central

    2010-01-01

    Background Many functional proteins have a symmetric structure. Most of these are multimeric complexes, which are made of non-symmetric monomers arranged in a symmetric manner. However, there are also a large number of proteins that have a symmetric structure in the monomeric state. These internally symmetric proteins are interesting objects from the point of view of their folding, function, and evolution. Most algorithms that detect the internally symmetric proteins depend on finding repeating units of similar structure and do not use the symmetry information. Results We describe a new method, called SymD, for detecting symmetric protein structures. The SymD procedure works by comparing the structure to its own copy after the copy is circularly permuted by all possible number of residues. The procedure is relatively insensitive to symmetry-breaking insertions and deletions and amplifies positive signals from symmetry. It finds 70% to 80% of the TIM barrel fold domains in the ASTRAL 40 domain database and 100% of the beta-propellers as symmetric. More globally, 10% to 15% of the proteins in the ASTRAL 40 domain database may be considered symmetric according to this procedure depending on the precise cutoff value used to measure the degree of perfection of the symmetry. Symmetrical proteins occur in all structural classes and can have a closed, circular structure, a cylindrical barrel-like structure, or an open, helical structure. Conclusions SymD is a sensitive procedure for detecting internally symmetric protein structures. Using this procedure, we estimate that 10% to 15% of the known protein domains may be considered symmetric. We also report an initial, overall view of the types of symmetries and symmetric folds that occur in the protein domain structure universe. PMID:20525292

  14. Optical tweezers study of red blood cell aggregation and disaggregation in plasma and protein solutions.

    PubMed

    Lee, Kisung; Kinnunen, Matti; Khokhlova, Maria D; Lyubin, Evgeny V; Priezzhev, Alexander V; Meglinski, Igor; Fedyanin, Andrey A

    2016-03-01

    Kinetics of optical tweezers (OT)-induced spontaneous aggregation and disaggregation of red blood cells (RBCs) were studied at the level of cell doublets to assess RBC interaction mechanics. Measurements were performed under in vitro conditions in plasma and fibrinogen and fibrinogen + albumin solutions. The RBC spontaneous aggregation kinetics was found to exhibit different behavior depending on the cell environment. In contrast, the RBC disaggregation kinetics was similar in all solutions qualitatively and quantitatively, demonstrating a significant contribution of the studied proteins to the process. The impact of the study on assessing RBC interaction mechanics and the protein contribution to the reversible RBC aggregation process is discussed.

  15. Aggregation of Whey Protein Hydrolysate Using Alcalase 2.4 L

    PubMed Central

    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. PMID:25290460

  16. Optical tweezers study of red blood cell aggregation and disaggregation in plasma and protein solutions

    NASA Astrophysics Data System (ADS)

    Lee, Kisung; Kinnunen, Matti; Khokhlova, Maria D.; Lyubin, Evgeny V.; Priezzhev, Alexander V.; Meglinski, Igor; Fedyanin, Andrey A.

    2016-03-01

    Kinetics of optical tweezers (OT)-induced spontaneous aggregation and disaggregation of red blood cells (RBCs) were studied at the level of cell doublets to assess RBC interaction mechanics. Measurements were performed under in vitro conditions in plasma and fibrinogen and fibrinogen + albumin solutions. The RBC spontaneous aggregation kinetics was found to exhibit different behavior depending on the cell environment. In contrast, the RBC disaggregation kinetics was similar in all solutions qualitatively and quantitatively, demonstrating a significant contribution of the studied proteins to the process. The impact of the study on assessing RBC interaction mechanics and the protein contribution to the reversible RBC aggregation process is discussed.

  17. Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition

    PubMed Central

    Yu, Anan; Shibata, Yoko; Shah, Bijal; Calamini, Barbara; Lo, Donald C.; Morimoto, Richard I.

    2014-01-01

    Protein conformational diseases exhibit complex pathologies linked to numerous molecular defects. Aggregation of a disease-associated protein causes the misfolding and aggregation of other proteins, but how this interferes with diverse cellular pathways is unclear. Here, we show that aggregation of neurodegenerative disease-related proteins (polyglutamine, huntingtin, ataxin-1, and superoxide dismutase-1) inhibits clathrin-mediated endocytosis (CME) in mammalian cells by aggregate-driven sequestration of the major molecular chaperone heat shock cognate protein 70 (HSC70), which is required to drive multiple steps of CME. CME suppression was also phenocopied by HSC70 RNAi depletion and could be restored by conditionally increasing HSC70 abundance. Aggregation caused dysregulated AMPA receptor internalization and also inhibited CME in primary neurons expressing mutant huntingtin, showing direct relevance of our findings to the pathology in neurodegenerative diseases. We propose that aggregate-associated chaperone competition leads to both gain-of-function and loss-of-function phenotypes as chaperones become functionally depleted from multiple clients, leading to the decline of multiple cellular processes. The inherent properties of chaperones place them at risk, contributing to the complex pathologies of protein conformational diseases. PMID:24706768

  18. Time-resolved FT-IR microspectroscopy of protein aggregation induced by heat-shock in live cells.

    PubMed

    Mitri, Elisa; Kenig, Saša; Coceano, Giovanna; Bedolla, Diana E; Tormen, Massimo; Grenci, Gianluca; Vaccari, Lisa

    2015-04-07

    Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein folding, and protein aggregation are indeed involved in several diseases including cancer, aging-associated, and neurodegenerative disorders. Accumulation of protein aggregates can also be induced from a variety of stressful conditions, such as temperature increase or oxidative stress. In this work, we monitored by Fourier transform-infrared (FT-IR) microspectroscopy the response of live breast cancer MCF-7 and mammary breast adenocarcinoma MDA-MB 231 cell lines to severe heat-shock (HS), caused by the rise of the cellular medium temperature from 37 ± 0.5 °C to 42 ± 0.5 °C. Through the study of the time-evolution of the second derivatives of the spectra and by the 2D correlation analysis of FT-IR absorbance data, we were able to identify a common sudden heat-shock response (HSR) among the two cell lines. The hyperfluidization of mammalian cell membranes, the transient increment of the signal lipids, as well as the alteration of proteome profile were all monitored within the first 40 min of stress application, while the persistent intracellular accumulation of extended β-folded protein aggregates was detected after 40 min up to 2 h. As a whole, this paper offers a further prove of the diagnostic capabilities of FT-IR microspectroscopy for monitoring in real-time the biochemical rearrangements undergone by live cells upon external stimulation.

  19. SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter.

    PubMed Central

    Sagné, C; Isambert, M F; Henry, J P; Gasnier, B

    1996-01-01

    The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation. PMID:8670158

  20. Fractal dimensions of soy protein nanoparticle aggregates determined by dynamic mechanical method

    USDA-ARS?s Scientific Manuscript database

    Soy protein isolate (SPI) is obtained from soybeans by removing soybean oil and soy carbohydrates. Soy protein nanoparticles were prepared by alkaline hydrolysis of SPI and centrifugal separation process. Structurally, SPI is a globular protein and its aggregates in water consist of sphere-like pr...

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

    SciTech Connect

    Wang Hongmin; Monteiro, Mervyn J. . E-mail: monteiro@umbi.umd.edu

    2007-08-01

    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.

  2. Emerging ideas on the molecular basis of protein and peptide aggregation.

    PubMed

    Thirumalai, D; Klimov, D K; Dima, R I

    2003-04-01

    Several neurodegenerative diseases are associated with the unfolding and subsequent fibrillization of proteins. Although neither the assembly mechanism nor the atomic structures of the amyloid fibrils are known, recent experimental and computational studies suggest that a few general principles that govern protein aggregation may exist. Analysis of the results of several important recent studies has led to a set of tentative ideas concerning the oligomerization of proteins and peptides. General rules have been described that may be useful in predicting regions of known proteins (prions and transthyretin) that are susceptible to fluctuations, which give rise to structures that can aggregate by the nucleation-growth mechanism. Despite large variations in the sequence-dependent polymerization kinetics of several structurally unrelated proteins, there appear to be only a few plausible scenarios for protein and peptide aggregation.

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

    PubMed

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

    2017-03-14

    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.

  4. Insights into the molecular mechanism of protein native-like aggregation upon glycation.

    PubMed

    Oliveira, Luis M A; Gomes, Ricardo A; Yang, Dennis; Dennison, Sarah R; Família, Carlos; Lages, Ana; Coelho, Ana V; Murphy, Regina M; Phoenix, David A; Quintas, Alexandre

    2013-06-01

    Several human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Familial Amyloidotic Polyneuropathy, have long been associated with, structural and functional changes in disease related proteins leading to aggregation into amyloid fibrils. Such changes can be triggered by post-translational modifications. Methylglyoxal modifications have been shown to induce the formation of small and stable native-like aggregates in the case of the amyloidogenic proteins insulin and α-synuclein. However, the fundamental biophysical mechanism underlying such methylglyoxal-induced protein aggregation is not yet fully understood. In this work cytochrome c (Cyt c) was used as a model protein for the characterization of specific glycation targets and to study their impact on protein structure, stability, and ability to form native-like aggregates. Our results show that methylglyoxal covalently modifies Cyt c at a single residue and induces early conformational changes that lead to the formation of native-like aggregates. Furthermore, partially unfolded species are formed, but do not seem to be implicated in the aggregation process. This shows a clear difference from the amyloid fibril mechanisms which involve partially or totally unfolded intermediates. Equilibrium-unfolding experiments show that glycation strongly decreases Cyt c conformational stability, which is balanced with an increase of conformational stability upon aggregation. Data collected from analytical and spectroscopic techniques, along with kinetic analysis based on least-squares parameter fitting and statistical model discrimination are used to help to understand the driving force underlying glycation-induced native-like aggregation, and enable the proposal of a comprehensive thermodynamic and kinetic model for native-like aggregation of methylglyoxal glycated Cyt c. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Biophysical approaches for the study of interactions between molecular chaperones and protein aggregates.

    PubMed

    Wright, Maya A; Aprile, Francesco A; Arosio, Paolo; Vendruscolo, Michele; Dobson, Christopher M; Knowles, Tuomas P J

    2015-10-04

    Molecular chaperones are key components of the arsenal of cellular defence mechanisms active against protein aggregation. In addition to their established role in assisting protein folding, increasing evidence indicates that molecular chaperones are able to protect against a range of potentially damaging aspects of protein behaviour, including misfolding and aggregation events that can result in the generation of aberrant protein assemblies whose formation is implicated in the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The interactions between molecular chaperones and different amyloidogenic protein species are difficult to study owing to the inherent heterogeneity of the aggregation process as well as the dynamic nature of molecular chaperones under physiological conditions. As a consequence, understanding the detailed microscopic mechanisms underlying the nature and means of inhibition of aggregate formation remains challenging yet is a key objective for protein biophysics. In this review, we discuss recent results from biophysical studies on the interactions between molecular chaperones and protein aggregates. In particular, we focus on the insights gained from current experimental techniques into the dynamics of the oligomerisation process of molecular chaperones, and highlight the opportunities that future biophysical approaches have in advancing our understanding of the great variety of biological functions of this important class of proteins.

  6. Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells

    PubMed Central

    Ho, Bosco; Au, Amanda E.; Schoenwaelder, Simone M.; Smyth, Mark J.; Bottomley, Stephen P.; Kleifeld, Oded; Medcalf, Robert L.

    2016-01-01

    Amyloidogenic protein aggregation impairs cell function and is a hallmark of many chronic degenerative disorders. Protein aggregation is also a major event during acute injury; however, unlike amyloidogenesis, the process of injury-induced protein aggregation remains largely undefined. To provide this insight, we profiled the insoluble proteome of several cell types after acute injury. These experiments show that the disulfide-driven process of nucleocytoplasmic coagulation (NCC) is the main form of injury-induced protein aggregation. NCC is mechanistically distinct from amyloidogenesis, but still broadly impairs cell function by promoting the aggregation of hundreds of abundant and essential intracellular proteins. A small proportion of the intracellular proteome resists NCC and is instead released from necrotic cells. Notably, the physicochemical properties of NCC-resistant proteins are contrary to those of NCC-sensitive proteins. These observations challenge the dogma that liberation of constituents during necrosis is anarchic. Rather, inherent physicochemical features including cysteine content, hydrophobicity and intrinsic disorder determine whether a protein is released from necrotic cells. Furthermore, as half of the identified NCC-resistant proteins are known autoantigens, we propose that physicochemical properties that control NCC also affect immune tolerance and other host responses important for the restoration of homeostasis after necrotic injury. PMID:27810968

  7. Controlling Agglomeration of Protein Aggregates for Structure Formation in Liquid Oil: A Sticky Business

    PubMed Central

    2017-01-01

    Proteins are known to be effective building blocks when it comes to structure formation in aqueous environments. Recently, we have shown that submicron colloidal protein particles can also be used to provide structure to liquid oil and form so-called oleogels (de VriesA.J. Colloid Interface Sci.2017, 486, 75−83)27693552. To prevent particle agglomeration, a solvent exchange procedure was used to transfer the aggregates from water to the oil phase. The aim of the current paper was to elucidate on the enhanced stability against agglomeration of heat-set whey protein isolate (WPI) aggregates to develop an alternative for the solvent exchange procedure. Protein aggregates were transferred from water to several solvents differing in polarity to investigate the effect on agglomeration and changes in protein composition. We show that after drying protein aggregates by evaporation from solvents with a low polarity (e.g., hexane), the protein powder shows good dispersibility in liquid oil compared to powders dried from solvents with a high polarity. This difference in dispersibility could not be related to changes in protein composition or conformation but was instead related to the reduction of attractive capillary forces between the protein aggregates during drying. Following another route, agglomeration was also prevented by applying high freezing rates prior to freeze-drying. The rheological properties of the oleogels prepared with such freeze-dried protein aggregates were shown to be similar to that of oleogels prepared using a solvent exchange procedure. This Research Article provides valuable insights in how to tune the drying process to control protein agglomeration to allow for subsequent structure formation of proteins in liquid oil. PMID:28225592

  8. Controlling Agglomeration of Protein Aggregates for Structure Formation in Liquid Oil: A Sticky Business.

    PubMed

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

    2017-03-22

    Proteins are known to be effective building blocks when it comes to structure formation in aqueous environments. Recently, we have shown that submicron colloidal protein particles can also be used to provide structure to liquid oil and form so-called oleogels ( de Vries , A. J. Colloid Interface Sci. 2017 , 486 , 75 - 83 ) . To prevent particle agglomeration, a solvent exchange procedure was used to transfer the aggregates from water to the oil phase. The aim of the current paper was to elucidate on the enhanced stability against agglomeration of heat-set whey protein isolate (WPI) aggregates to develop an alternative for the solvent exchange procedure. Protein aggregates were transferred from water to several solvents differing in polarity to investigate the effect on agglomeration and changes in protein composition. We show that after drying protein aggregates by evaporation from solvents with a low polarity (e.g., hexane), the protein powder shows good dispersibility in liquid oil compared to powders dried from solvents with a high polarity. This difference in dispersibility could not be related to changes in protein composition or conformation but was instead related to the reduction of attractive capillary forces between the protein aggregates during drying. Following another route, agglomeration was also prevented by applying high freezing rates prior to freeze-drying. The rheological properties of the oleogels prepared with such freeze-dried protein aggregates were shown to be similar to that of oleogels prepared using a solvent exchange procedure. This Research Article provides valuable insights in how to tune the drying process to control protein agglomeration to allow for subsequent structure formation of proteins in liquid oil.

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

    PubMed Central

    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 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. PMID:24865853

  10. Gold nanoparticle aggregation-based colorimetric assay for β-casein detection in bovine milk samples.

    PubMed

    Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Song, F; Lu, S Y; Ren, H L; Hu, P; Liu, Z S; Zhang, J H

    2014-11-01

    Traditional Kjeldahl method, used for quality evaluation of bovine milk, has intrinsic defects of time-consuming sample preparation and two analyses to determine the difference between non-protein nitrogen content and total protein nitrogen content. Herein, based upon antibody functionalized gold nanoparticles (AuNPs), we described a colorimetric method for β-casein (β-CN) detection in bovine milk samples. The linear dynamic range and the LOD were 0.08-250 μg mL(-1), and 0.03 μg mL(-1) respectively. In addition, the real content of β-CN in bovine milk was measured by using the developed assay. The results are closely correlated with those from Kjeldahl method. The advantages of β-CN triggered AuNP aggregation-based colorimetric assay are simple signal generation, the high sensitivity and specificity as well as no need of complicated sample preparation, which make it for on-site detection of β-CN in bovine milk samples.

  11. Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography-multiangle laser light scattering.

    PubMed

    Ye, Hongping

    2006-09-01

    The feasibility of size exclusion chromotography (SEC)-multiangle laser-light scattering as a technique to investigate aggregation and degradation of glycosylated and nonglycosylated proteins, and antibodies under various conditions such as addition of detergent, changes in pH, and variation of protein concentration and heat stress temperature was examined. Separation of proteins and their aggregates was performed using SEC-high-performance liquid chromatography. Detection of analytes was carried out with on-line UV, refractive index, and multiangle laser light-scattering detectors. Quantification and molecular weight determination were performed using commercial software. Aggregation and degradation were examined under various conditions and quantitative results are presented for bovine serum albumin, choriogonadotropin, glyceraldehyde-3-phosphate dehydrogenase, Herceptin, and ReoPro. This method can simultaneously determine both the quantities and the molecular weights of macromolecules from a single injection. The determination of molecular weight is absolute which avoids misleading results caused by molecular shape or interactions with the column matrix. This technique is valuable not only for assessing the extent of aggregation but also for effectively monitoring molecule degradation as evidenced by molecular weight reduction and change in monomer amount.

  12. Endoplasmic reticulum stress caused by aggregate-prone proteins containing homopolymeric amino acids.

    PubMed

    Uchio, Naohiro; Oma, Yoko; Toriumi, Kazuya; Sasagawa, Noboru; Tanida, Isei; Fujita, Eriko; Kouroku, Yoriko; Kuroda, Reiko; Momoi, Takashi; Ishiura, Shoichi

    2007-11-01

    Many human proteins have homopolymeric amino acid (HPAA) tracts, but their physiological functions or cellular effects are not well understood. Previously, we expressed 20 HPAAs in mammalian cells and showed characteristic intracellular localization, in that hydrophobic HPAAs aggregated strongly and caused high cytotoxicity in proportion to their hydrophobicity. In the present study, we investigated the cytotoxicity of these aggregate-prone hydrophobic HPAAs, assuming that the ubiquitin proteasome system is impaired in the same manner as other well-known aggregate-prone polyglutamine-containing proteins. Some highly hydrophobic HPAAs caused a deficiency in the ubiquitin proteasome system and excess endoplasmic reticulum stress, leading to apoptosis. These results indicate that the property of causing excess endoplasmic reticulum stress by proteasome impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress is not a common cytotoxic effect of aggregate-prone proteins such as polyglutamine.

  13. Protein aggregation activates erratic stress response in dietary restricted yeast cells

    PubMed Central

    Bhadra, Ankan Kumar; Das, Eshita; Roy, Ipsita

    2016-01-01

    Chronic stress and prolonged activation of defence pathways have deleterious consequences for the cell. Dietary restriction is believed to be beneficial as it induces the cellular stress response machinery. We report here that although the phenomenon is beneficial in a wild-type cell, dietary restriction leads to an inconsistent response in a cell that is already under proteotoxicity-induced stress. Using a yeast model of Huntington’s disease, we show that contrary to expectation, aggregation of mutant huntingtin is exacerbated and activation of the unfolded protein response pathway is dampened under dietary restriction. Global proteomic analysis shows that when exposed to a single stress, either protein aggregation or dietary restriction, the expression of foldases like peptidyl-prolyl isomerase, is strongly upregulated. However, under combinatorial stress, this lead is lost, which results in enhanced protein aggregation and reduced cell survival. Successful designing of aggregation-targeted therapeutics will need to take additional stressors into account. PMID:27633120

  14. Acid-induced cold gelation of globular proteins: effects of protein aggregate characteristics and disulfide bonding on rheological properties.

    PubMed

    Alting, Arno C; Weijers, Mireille; de Hoog, Els H A; van de Pijpekamp, Anne M; Cohen Stuart, Martien A; Hamer, Rob J; de Kruif, Cornelis G; Visschers, Ronald W

    2004-02-11

    The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked monomers. Both types of protein aggregates had comparable numbers of thiol groups exposed at their surfaces but had clearly different shapes. During acid-induced gelation, the characteristic ordering caused by the repulsive character disappeared and was replaced by a random distribution. This process did not depend on aggregate characteristics and probably applies to any type of protein aggregate. Covalent bonds are the main determinants of the gel hardness. The formation of additional disulfide bonds during gelation depended on the number and accessibility of thiol groups and disulfide bonds in the molecule and was found to clearly differ between the proteins studied. However, upon blocking of the thiol groups, long fibrillar structures of ovalbumin contribute significantly to gel hardness, demonstrating the importance of aggregate shape.

  15. Characterization of Sizes of Aggregates of Insulin Analogs and the Conformations of the Constituent Protein Molecules: A Concomitant Dynamic Light Scattering and Raman Spectroscopy Study.

    PubMed

    Zhou, Chen; Qi, Wei; Lewis, E Neil; Carpenter, John F

    2016-02-01

    To generate aggregates, 3 insulin analogs, lispro, aspart, and glulisine, were incubated without phenolic preservatives for 30 days at 37 °C. As a function of incubation time, aggregation was quantified with size exclusion chromatography, and the sizes of aggregates and the conformations of the constituent molecules were characterized with concomitant dynamic light scattering and Raman spectroscopy. During incubation, lispro was progressively converted into soluble aggregates with hydrodynamic diameters of circa 15 nm, and 95% of the native protein had aggregated at day 30. Raman spectroscopy documented that aggregation resulted in conversion of a large fraction of native alpha helix into nonnative beta sheet structure and a distortion of disulfide bonds. In contrast, for aspart and glulisine only 20% of the native proteins aggregated after 30 days, and minimal structural perturbations were detected. In addition, consistent with the relative aggregation rates during isothermal incubation, Raman spectroscopy showed that during heating the onset temperature for secondary structural perturbations of lispro occurred 7 °C-10 °C lower than those for aspart or glulisine. Overall the results of this study demonstrated that-as in the case during formation of amyloid fibrils from insulin-formation of soluble aggregates of lispro resulted in a high level of conversion of alpha helix into beta sheet.

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

    PubMed

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

    2016-04-06

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

  17. Revealing linear aggregates of light harvesting antenna proteins in photosynthetic membranes.

    PubMed

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

    2010-01-05

    How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions by using atomic force microscopy (AFM) imaging and spectroscopic analysis. Our results suggest that the light harvesting protein, LH2, can exist as linear aggregates of 4 +/- 2 proteins in the photosynthetic membranes and that the protein distributions are highly heterogeneous. In the photosynthetic membranes examined in our measurements, the ratio of the aggregated to the nonaggregated LH2 proteins is about 3:1 to 5:1 depending on the intensity of the illumination used during sample incubation and on the bacterial species. AFM images further identify that the LH2 proteins in the linear aggregates are monotonically tilted at an angle 4 +/- 2 degrees from the plane of the photosynthetic membranes. The aggregates result in red-shifted absorption and emission spectra that are measured using various mutant membranes, including an LH2 knockout, LH1 knockout, and LH2 at different population densities. Measuring the fluorescence lifetimes of purified LH2 and LH2 in membranes, we have observed that the LH2 proteins in membranes exhibit biexponential lifetime decays whereas the purified LH2 proteins gave single exponential lifetime decays. We attribute that the two lifetime components originate from the existence of both aggregated and nonaggregated LH2 proteins in the photosynthetic membranes.

  18. Transmission electron microscopy as an orthogonal method to characterize protein aggregates

    PubMed Central

    Sung, Joyce J.; Pardeshi, Neha N.; Mulder, Anke M.; Mulligan, Sean K.; Quispe, Joel; On, Kathy; Carragher, Bridget; Potter, Clinton S.; Carpenter, John F.; Schneemann, Anette

    2015-01-01

    Aggregation of protein-based therapeutics is a challenging problem in the biopharmaceutical industry. Of particular concern are implications for product efficacy and clinical safety due to potentially increased immunogenicity of the aggregates. We used transmission electron microscopy (TEM) to characterize biophysical and morphological features of antibody aggregates formed upon controlled environmental stresses. TEM results were contrasted with results obtained in parallel by independent methods, including size exclusion chromatography, dynamic light scattering, microflow imaging and nanoparticle tracking. For TEM, stressed samples were imaged by negative staining and in the frozen-hydrated state. In both cases, aggregates appeared amorphous but differed in fine structural detail. Specifically, negatively stained aggregates were compact and consisted of smaller globular structures that had a notable three dimensional character. Elements of the native IgG structure were retained, suggesting that the aggregates were not assembled from denatured protein. In contrast, aggregates in frozen-hydrated samples appeared as extended, branched protein networks with large surface area. Using multiple scales of magnification, a wide range of particle sizes was observed and semi-quantitatively characterized. The detailed information provided by TEM extended observations obtained with the independent methods, demonstrating the suitability of TEM as a complementary approach to submicron particle analysis. PMID:25231267

  19. Are exosomes the vehicle for protein aggregate propagation in neurodegenerative diseases?

    PubMed

    Lim, Yoon-Ju; Lee, Seung-Jae

    2017-08-29

    Abnormal protein aggregation has been implicated in neurodegenerative processes in human neurological disorders, such as Alzheimer's disease and Parkinson's disease. Recently, studies have established a novel concept that protein aggregates are transmitted among neuronal cells. By extension, such interneuronal aggregate transmission has been hypothesized to be the underlying mechanism for the pathological and clinical disease progression. However, the precise mechanism of the interneuronal aggregate transmission remains ill-defined. Recent reports have suggested that exosomes, a specific group of extracellular vesicles that are involved in intercellular transfer of cellular macromolecules such as proteins and RNAs, could play an important role in the aggregate transmission among neurons. Here, we review various types of extracellular vesicles and critically evaluate the evidence supporting the role of exosomes in interneuronal aggregate transmission and neurodegeneration. We also discuss the competing mechanisms other than the exosome-mediated transmission. By doing so, we aim to assess the current state of knowledge on the mechanism of interneuronal aggregate transmission and suggest the future directions of research towards understanding the mechanism.

  20. Protein aggregation, cardiovascular diseases, and exercise training: Where do we stand?

    PubMed

    Gouveia, Marisol; Xia, Ke; Colón, Wilfredo; Vieira, Sandra I; Ribeiro, Fernando

    2017-07-28

    Cells ensure their protein quality control through the proteostasis network. Aging and age-related diseases, such as neurodegenerative and cardiovascular diseases, have been associated to the reduction of proteostasis network efficiency and, consequently, to the accumulation of protein misfolded aggregates. The decline in protein homeostasis has been associated with the development and progression of atherosclerotic cardiovascular disease, cardiac hypertrophy, cardiomyopathies, and heart failure. Exercise training is a key component of the management of patients with cardiovascular disease, consistently improving quality of life and prognosis. In this review, we give an overview on age-related protein aggregation, the role of the increase of misfolded protein aggregates on cardiovascular pathophysiology, and describe the beneficial or deleterious effects of the proteostasis network on the development of cardiovascular disease. We subsequently discuss how exercise training, a key lifestyle intervention in those with cardiovascular disease, could restore proteostasis and improve disease status. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

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

    2004-09-01

    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°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°C.

  2. Amyloidogenicity at a Distance: How Distal Protein Regions Modulate Aggregation in Disease.

    PubMed

    Lucato, Christina M; Lupton, Christopher J; Halls, Michelle L; Ellisdon, Andrew M

    2017-03-22

    The misfolding of proteins to form amyloid is a key pathological feature of several progressive, and currently incurable, diseases. A mechanistic understanding of the pathway from soluble, native protein to insoluble amyloid is crucial for therapeutic design, and recent efforts have helped to elucidate the key molecular events that trigger protein misfolding. Generally, either global or local structural perturbations occur early in amyloidogenesis to expose aggregation-prone regions of the protein that can then self-associate to form toxic oligomers. Surprisingly, these initiating structural changes are often caused or influenced by protein regions distal to the classically amyloidogenic sequences. Understanding the importance of these distal regions in the pathogenic process has highlighted many remaining knowledge gaps regarding the precise molecular events that occur in classic aggregation pathways. In this review, we discuss how these distal regions can influence aggregation in disease and the recent technical and conceptual advances that have allowed this insight.

  3. Effects of electric fields on protein unfolding and aggregation: influence on edible films formation.

    PubMed

    Pereira, Ricardo N; Souza, Bartolomeu W S; Cerqueira, Miguel A; Teixeira, José A; Vicente, António A

    2010-11-08

    Electric fields application is receiving increased attention because of its uniform heating of liquids. The mechanisms of unfolding and aggregation of whey proteins during ohmic heating may influence properties of edible films made thereof. The aim of this work was to evaluate the effects of ohmic heating on physical and structural properties of whey protein edible films and compare them with those obtained by conventional heating. The results showed that ohmic heating determined less aggregation and lower concentration of free sulphydryls in film-forming solutions. Ohmic films were thinner, less permeable to water vapor and presented nearly the same mechanical properties of conventional films. Ohmic heating induced protein conformational changes by increasing the contents of β-sheet structures in the film network. This work emphasized the effects of ohmic heating in unfolding and aggregation mechanisms of whey proteins during heat denaturation, which determined the production of protein edible films with distinctive properties.

  4. Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau

    PubMed Central

    Pavlova, Anna; Cheng, Chi-Yuan; Kinnebrew, Maia; Lew, John; Dahlquist, Frederick W.; Han, Songi

    2016-01-01

    Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255–441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on site-specific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable β-sheet structured species and maintain their stacking order without exchanging their subunits. The population of β-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50–70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth. PMID:26712030

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

    PubMed

    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

  6. Protein Detection with Aptamer Biosensors

    PubMed Central

    Strehlitz, Beate; Nikolaus, Nadia; Stoltenburg, Regina

    2008-01-01

    Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors) will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers. PMID:27879936

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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

    2016-08-01

    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.

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

    NASA Astrophysics Data System (ADS)

    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

  10. Colorimetric sensor array for protein discrimination based on different DNA chain length-dependent gold nanoparticles aggregation.

    PubMed

    Wei, Xiangcong; Wang, Yuxian; Zhao, Yingxin; Chen, Zhengbo

    2017-11-15

    A facile colorimetric sensor array for detection of proteins was demonstrated using DNA as nonspecific receptors. We found that different proteins could trigger the DNA-protected gold nanoparticles (AuNPs) to show different aggregation behaviors in the presence of salt with high concentrations along with various color changes. As a proof-of-concept application, a multi-protein discrimination array was fabricated with two ssDNA strands (15A and 30A bases) as a receptor array. The combinatorial colorimetric response of this sensor array can be analyzed by linear discriminant analysis (LDA) and hierarchical cluster analysis (HCA). 12 proteins have been well distinguished with the naked eye at the 50nM level. The proteins in human serum have also been discriminated. Furthermore, the accuracies of discrimination of the similar mixtures of proteins and unknown samples were all 100%. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    SciTech Connect

    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.

  12. Enhancing Nanoparticle-Based Visible Detection by Controlling the Extent of Aggregation

    NASA Astrophysics Data System (ADS)

    Lim, Seokwon; Koo, Ok Kyung; You, Young Sang; Lee, Yeong Eun; Kim, Min-Sik; Chang, Pahn-Shick; Kang, Dong Hyun; Yu, Jae-Hyuk; Choi, Young Jin; Gunasekaran, Sundaram

    2012-06-01

    Visible indication based on the aggregation of colloidal nanoparticles (NPs) is highly advantageous for rapid on-site detection of biological entities, which even untrained persons can perform without specialized instrumentation. However, since the extent of aggregation should exceed a certain minimum threshold to produce visible change, further applications of this conventional method have been hampered by insufficient sensitivity or certain limiting characteristics of the target. Here we report a signal amplification strategy to enhance visible detection by introducing switchable linkers (SLs), which are designed to lose their function to bridge NPs in the presence of target and control the extent of aggregation. By precisely designing the system, considering the quantitative relationship between the functionalized NPs and SLs, highly sensitive and quantitative visible detection is possible. We confirmed the ultrahigh sensitivity of this method by detecting the presence of 20 fM of streptavidin and fewer than 100 CFU/mL of Escherichia coli.

  13. Real-time protein aggregation monitoring based on a simultaneous light scattering investigation and a Bloch surface wave-based approach

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

    We present a study of the dynamics of protein aggregation using a Bloch surface wave (BSW) label-free sensing scheme. In a previous work, we demonstrated the ability to detect the early dynamic events of fibrillogenesis of amyloid betapeptides (Aβ), linked to Alzheimer's Disease. Here, we demonstrate the efficacy of the BSW sensor by describing a simultaneous light scattering measurement, with the purpose of real-time monitoring the size change of the Aβ aggregates, throughout fibrillization.

  14. Aggregation of FET Proteins as a Pathological Change in Amyotrophic Lateral Sclerosis.

    PubMed

    Furukawa, Yoshiaki; Tokuda, Eiichi

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease that is characterized by the formation of abnormal inclusions in neurons. While the pathomechanism of ALS remains obscure, a number of proteins have been identified in the inclusion bodies, and the pathological roles of RNA-binding proteins have been increasingly emphasized. Among those, the FET proteins (FUS, EWSR1, TAF15) were recently identified as RNA-binding proteins in pathological inclusions of ALS and other neurodegenerative diseases; moreover, mutations in the genes encoding the FET proteins were found to be associated with familial forms of ALS. FET proteins are normally localized in the nucleus, but the introduction of pathogenic mutations in FET proteins leads to their abnormal redistribution to the cytoplasm, where they form aggregates. While further investigation will be required to understand the intracellular factors controlling the aggregation propensities of FET proteins, they are thought to lose their physiological functions and become toxic through their misfolding/aggregation. Here, we will briefly review recent advances of our understanding of the physiological functions and aggregation behavior of FET proteins in vivo as well as in vitro.

  15. Aggregation-induced changes in the chemical exchange saturation transfer (CEST) signals of proteins.

    PubMed

    Goerke, Steffen; Milde, Katharina S; Bukowiecki, Raul; Kunz, Patrick; Klika, Karel D; Wiglenda, Thomas; Mogk, Axel; Wanker, Erich E; Bukau, Bernd; Ladd, Mark E; Bachert, Peter; Zaiss, Moritz

    2017-01-01

    Chemical exchange saturation transfer (CEST) is an MRI technique that allows mapping of biomolecules (small metabolites, proteins) with nearly the sensitivity of conventional water proton MRI. In living organisms, several tissue-specific CEST effects have been observed and successfully applied to diagnostic imaging. In these studies, particularly the signals of proteins showed a distinct correlation with pathological changes. However, as CEST effects depend on various properties that determine and affect the chemical exchange processes, the origins of the observed signal changes remain to be understood. In this study, protein aggregation was identified as an additional process that is encoded in the CEST signals of proteins. Investigation of distinct proteins that are involved in pathological disorders, namely amyloid beta and huntingtin, revealed a significant decrease of all protein CEST signals upon controlled aggregation. This finding is of particular interest with regard to diagnostic imaging of patients with neurodegenerative diseases that involve amyloidogenesis, such as Alzheimer's or Huntington's disease. To investigate whether the observed CEST signal decrease also occurs in heterogeneous mixtures of aggregated cellular proteins, and thus prospectively in tissue, heat-shocked yeast cell lysates were employed. Additionally, investigation of different cell compartments verified the assignment of the protein CEST signals to the soluble part of the proteome. The results of in vitro experiments demonstrate that aggregation affects the CEST signals of proteins. This observation can enable hypotheses for CEST imaging as a non-invasive diagnostic tool for monitoring pathological alterations of the proteome in vivo.

  16. Impact of Proteins on Aggregation Kinetics and Adsorption Ability of Hematite Nanoparticles in Aqueous Dispersions.

    PubMed

    Sheng, Anxu; Liu, Feng; Xie, Nan; Liu, Juan

    2016-03-01

    The initial aggregation kinetics of hematite nanoparticles (NPs) that were conjugated with two model globular proteins-cytochrome c from bovine heart (Cyt) and bovine serum albumin (BSA)-were investigated over a range of monovalent (NaCl) and divalent (CaCl2) electrolyte concentrations at pH 5.7 and 9. The aggregation behavior of Cyt-NP conjugates was similar to that of bare hematite NPs, but the additional electrosteric repulsion increased the critical coagulation concentration (CCC) values from 69 mM to 113 mM in NaCl at pH 5.7. An unsaturated layer of BSA, a protein larger than Cyt, on hematite NPs resulted in fast aggregation at low salt concentrations and pH 5.7, due to the strong attractive patch-charge interaction. However, the BSA-NP conjugates could be stabilized simply by elevating salt concentrations, owing to the screening of the attractive patch-charge force and the increasing contribution from steric force. This study showed that the aggregation state of protein-conjugated NPs is proved to be completely switchable via ionic strength, pH, protein size, and protein coverage. Macroscopic Cu(II) sorption experiments further established that reducing aggregation of hematite NPs via tailoring ionic strength and protein conjugation could promote the metal uptake by hematite NPs under harsh conditions.

  17. Protein aggregation as a mechanism of adaptive cellular responses.

    PubMed

    Saarikangas, Juha; Barral, Yves

    2016-11-01

    Coalescence of proteins into different types of intracellular bodies has surfaced as a widespread adaptive mechanism to re-organize cells and cellular functions in response to specific cues. These structures, composed of proteins or protein-mRNA-complexes, regulate cellular processes through modulating enzymatic activities, gene expression or shielding macromolecules from damage. Accordingly, such bodies are associated with a wide-range of processes, including meiosis, memory-encoding, host-pathogen interactions, cancer, stress responses, as well as protein quality control, DNA replication stress and aneuploidy. Importantly, these distinct coalescence responses are controlled, and in many cases regulated by chaperone proteins. While cells can tolerate and proficiently coordinate numerous distinct types of protein bodies, some of them are also intimately linked to diseases or the adverse effects of aging. Several protein bodies that differ in composition, packing, dynamics, size, and localization were originally discovered in budding yeast. Here, we provide a concise and comparative review of their nature and nomenclature.

  18. Modulation of prion-dependent polyglutamine aggregation and toxicity by chaperone proteins in the yeast model.

    PubMed

    Gokhale, Kavita C; Newnam, Gary P; Sherman, Michael Y; Chernoff, Yury O

    2005-06-17

    In yeast, aggregation and toxicity of the expanded polyglutamine fragment of human huntingtin strictly depend on the presence of the endogenous self-perpetuating aggregated proteins (prions), which contain glutamine/asparagine-rich domains. Some chaperones of the Hsp100/70/40 complex, modulating propagation of yeast prions, were also reported to influence polyglutamine aggregation in yeast, but it was not clear whether they do it directly or via affecting prions. Our data show that although some chaperone alterations indeed act on polyglutamines via curing endogenous prions, other alterations decrease size and ameliorate toxicity of polyglutamine aggregates without affecting prion propagation. Therefore, the role of yeast chaperones in polyglutamine aggregation and toxicity is not restricted only to their effects on the endogenous prions. Moreover, chaperone interactions with prion and polyglutamine aggregates appear to be of a highly specific nature. One and the same chaperone alteration, substitution A503V in the middle region of the chaperone Hsp104, exhibited opposite effects on one of the endogenous prions ([PSI(+)], the prion form of Sup35) and on polyglutamines, increasing aggregate size and toxicity in the former case and decreasing them in the latter case. On the other hand, different members of a single chaperone family exhibited opposite effects on one and the same type of aggregates: excess of the Hsp40 chaperone Ydj1 increased polyglutamine aggregate size and toxicity, whereas excess of the other Hsp40 chaperone, Sis1, decreased them. As many stress-defense proteins are conserved between yeast and mammals, these data shed light on possible mechanisms modulating polyglutamine aggregation and toxicity in mammalian cells.

  19. Structural Basis for Protein anti-Aggregation Activity of the Trigger Factor Chaperone*

    PubMed Central

    Saio, Tomohide; Guan, Xiao; Rossi, Paolo; Economou, Anastassios; Kalodimos, Charalampos G.

    2014-01-01

    Molecular chaperones prevent aggregation and misfolding of proteins but scarcity of structural data has impeded an understanding of the recognition and anti-aggregation mechanisms. Here we report the solution structure, dynamics and energetics of three Trigger Factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. NMR relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion but as the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and how by acting as unfoldases and holdases prevent the aggregation and premature (mis)folding of unfolded proteins. PMID:24812405

  20. Metacaspase Yca1 is required for clearance of insoluble protein aggregates.

    PubMed

    Lee, Robin E C; Brunette, Steve; Puente, Lawrence G; Megeney, Lynn A

    2010-07-27

    In complex organisms, caspase proteases mediate a variety of cell behaviors, including proliferation, differentiation, and programmed cell death/apoptosis. Structural homologs to the caspase family (termed metacaspases) engage apoptosis in single-cell eukaryotes, yet the molecular mechanisms that contribute to nondeath roles are currently undefined. Here, we report an unexpected role for the Saccharomyces cerevisiae metacaspase Yca1 in protein quality control. Quantitative proteomic analysis of Deltayca1 cells identified significant alterations to vacuolar catabolism and stress-response proteins in the absence of induced stress. Yca1 protein complexes are enriched for aggregate-remodeling chaperones that colocalize with Yca1-GFP fusions. Finally, deletion and inactivation mutants of Yca1 accrue protein aggregates and autophagic bodies during log-phase growth. Together, our results show that Yca1 contributes to the fitness and adaptability of growing yeast through an aggregate remodeling activity.

  1. Prion-like transmission of pathogenic protein aggregates in genetic models of neurodegenerative disease.

    PubMed

    Pearce, Margaret Mp

    2017-06-01

    A key pathological hallmark of most neurodegenerative diseases is the misfolding of a particular protein, leading to deposition of toxic protein aggregates in brain tissue. Recent data provide compelling evidence that pathogenic protein aggregates have prion-like properties-they self-replicate by templated misfolding of monomeric proteins and spread between individual cells. Studies in genetic model organisms have expanded our understanding of how prion-like pathogenic aggregates propagate in vivo, revealing potential roles for spreading along neural networks and key cellular processes in both neurons and glial cells. These findings and future studies in genetic models will help guide the development of novel therapeutic strategies that directly target the molecular mechanisms underlying these devastating diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. The role of proline in the prevention of aggregation during protein folding in vitro.

    PubMed

    Kumar, T K; Samuel, D; Jayaraman, G; Srimathi, T; Yu, C

    1998-10-01

    Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.

  3. Nanoelectrochemical Immunosensors for Protein Detection

    NASA Astrophysics Data System (ADS)

    Carpentiero, Alessandro; de Leo, Manuela; Garcia Romero, Ivan; Pozzi Mucelli, Stefano; Reuther, Freimut; Stanta, Giorgio; Tormen, Massimo; Ugo, Paolo; Zamuner, Martina

    Nanoelectrochemical immunosensors fabricated by templated electrodeposition of gold nanoelectrodes inside the pores of polycarbonate (PC) track-etched membranes, followed by the immobilization of the biorecognition elements on the surrounding PC, have proven high sensitivity and specificity for protein detection. The signal transduction scheme involves a suitable redox mediator added to the sample solution to shuttle electrons from the gold nanoelectrodes to the biorecognition layer, both elements being in strict spatial proximity. Highly improved signal-to-background current ratio, which are peculiar of NEEs with respect to other electrochemical transducers, can be exploited in this way. Two detection schemes were tested: one based on the direct immobilization of the target protein on the PC of the NEE (approach A) and the other based on the immobilisation on PC of an antibody to capture the target protein (approach B). The biorecognition process was completed by adding a primary antibody and a secondary antibody with horse radish peroxidase (HRP) as enzyme label; methylene blue was the redox mediator added to the electrolyte solution. Typical target analytes were single chain fragment variable proteins, for approach A, and trastuzumab (also known as Herceptin®), for approach B. NEE-based capture sensors were tested successfully to detect small amounts of the receptor protein HER2 in biological samples. Finally, motivated by the target of a better control of the geometrical characteristics of ensembles of nanoelectrodes (size, density, geometrical arrangement, and degree of recession), and by the positive results obtained with track-etch membranes of PC from the standpoint of protein immobilization, we demonstrated the fabrication of nanobiosensors by patterning ordered arrays of nanoelectrodes (NEAs) by electron beam lithography (EBL) on polycarbonate. EBL results perfectly suitable for the top-down fabrication of arrays of nanobiosensors on thin PC films

  4. Electrostatically-controlled protein adsorption onto lipid bilayer: modeling adsorbate aggregation behavior.

    PubMed

    Trusova, Valeriya M; Gorbenko, Galyna P

    2008-03-01

    Using adsorption models based on scaled particle (SPT) and double layer theories the electrostatically-controlled protein adsorption onto membrane surface has been simulated for non-associating and self-associating ligands. The binding isotherms of monomeric and oligomeric protein species have been calculated over a range of variable parameters including lipid and protein concentrations, protein and membrane charges, pH and ionic strength. Adsorption behavior of monomers appeared to be the most sensitive to the changes in the protein aggregation state. The hallmarks of the protein oligomerization are identified. The practical guides for optimal design of binding experiments focused on obtaining proofs of protein self-association are suggested.

  5. Folding and aggregation of export-defective mutants of the maltose-binding protein.

    PubMed

    Betton, Jean-Michel; Phichith, Denis; Hunke, Sabine

    2002-09-01

    We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31. To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.

  6. High-pressure NMR techniques for the study of protein dynamics, folding and aggregation

    NASA Astrophysics Data System (ADS)

    Nguyen, Luan M.; Roche, Julien

    2017-04-01

    High-pressure is a well-known perturbation method used to destabilize globular proteins and dissociate protein complexes or aggregates. The heterogeneity of the response to pressure offers a unique opportunity to dissect the thermodynamic contributions to protein stability. In addition, pressure perturbation is generally reversible, which is essential for a proper thermodynamic characterization of a protein equilibrium. When combined with NMR spectroscopy, hydrostatic pressure offers the possibility of monitoring at an atomic resolution the structural transitions occurring upon unfolding and determining the kinetic properties of the process. The recent development of commercially available high-pressure sample cells greatly increased the potential applications for high-pressure NMR experiments that can now be routinely performed. This review summarizes the recent applications and future directions of high-pressure NMR techniques for the characterization of protein conformational fluctuations, protein folding and the stability of protein complexes and aggregates.

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

    PubMed Central

    Zurawel, Ashley A.; Kabeche, Ruth; DiGregorio, Sonja E.; Deng, Lin; Menon, Kartikeya M.; Opalko, Hannah

    2016-01-01

    ABSTRACT 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. PMID:27677791

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

    PubMed

    Caughey, Byron; Lansbury, Peter T

    2003-01-01

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

  9. Principles, approaches, and challenges for predicting protein aggregation rates and shelf life.

    PubMed

    Weiss, William F; Young, Teresa M; Roberts, Christopher J

    2009-04-01

    Control and prevention of unwanted aggregation for therapeutic proteins is a ubiquitous hurdle during biopharmaceutical product manufacture, storage, shipping, and administration. Methods to predict the relative or absolute rates of aggregation are therefore of great practical interest in biopharmaceutical research and development. Aggregation is often well-described as a multi-stage process involving unfolding or misfolding of free monomers, along with one or more assembly steps to form soluble or insoluble oligomers or higher-molecular-weight species. This report reviews the current state of the art in experimental and practical theoretical approaches that attempt to predict in vitro protein aggregation rates or propensities relevant to pharmaceutical proteins. Most available approaches fall within four primary categories. The principles and assumptions underlying each category are reviewed, along with advantages and limitations in each case. The importance of appropriate experimental techniques and models to probe and quantify the thermodynamics and/or dynamics of multiple steps or stages within the overall aggregation process is stressed. The primary focus is on aggregation in solution, relevant to parenteral dosage forms. Additional challenges are briefly reviewed.

  10. Spinocerebellar ataxia type 6 protein aggregates cause deficits in motor learning and cerebellar plasticity.

    PubMed

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

    2015-06-10

    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 a CT fragment of the Cav2.1 channel, which is detected specifically in cytosolic and nuclear fractions in SCA6 patients, is associated with the SCA6 pathogenesis. To test this hypothesis, we expressed P/Q-type channel protein fragments from two different human CT splice variants, as predicted from SCA6 patients, in PCs of mice using viral and transgenic approaches. These splice variants represent a short (CT-short without polyQs) and a long (CT-long with 27 polyQs) CT fragment. Our results show that the different splice variants of the CTs differentially distribute within PCs, i.e., the short CTs reveal predominantly nuclear inclusions, whereas the long CTs prominently reveal both nuclear and cytoplasmic aggregates. Postnatal expression of CTs in PCs in mice reveals that only CT-long causes SCA6-like symptoms, i.e., deficits in eyeblink conditioning (EBC), ataxia, and PC degeneration. The physiological phenotypes associated specifically with the long CT fragment can be explained by an impairment of LTD and LTP at the parallel fiber-to-PC synapse and alteration in spontaneous PC activity. Thus, our results suggest that the polyQ carrying the CT fragment of the P/Q-type channel is sufficient to cause SCA6 pathogenesis in mice and identifies EBC as a new diagnostic strategy to evaluate Ca(2+) channel-mediated human diseases.

  11. Homogeneous immunoassay based on aggregation of antibody-functionalized gold nanoparticles coupled with light scattering detection.

    PubMed

    Du, Baoan; Li, Zhengping; Cheng, Yongqiang

    2008-05-30

    A universal platform of homogeneous noncompetitive immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. The assay is based on aggregation of antibody-functionalized gold nanoparticles directed by the immunoreaction coupled with light scattering detection with a common spectrofluorimeter. In phosphate buffer (pH 7.0) solution, the light scattering intensity of the gold nanoparticles functionalized with goat-anti-human IgG can be greatly enhanced by addition of the human IgG. Based on this phenomenon, a wide dynamic range of 0.05-10 microg ml(-1) for determination of human IgG can be obtained, and the detection limit can reach 10 ng ml(-1). The proposed immunoassay can be accomplished in a homogeneous solution with one-step operation within 10 min and has been successfully applied to the determination of human IgG in serum samples, in which the results are well consistent with those of the enzyme-linked immunosorbent assay (ELISA), indicating its high selectivity and practicality. Therefore, the gold nanoparticle-based light scattering method can be used as a model to establish the general methods for protein assay in the fields of molecular biology and clinical diagnostics.

  12. Network Intrusion Detection and Visualization using Aggregations in a Cyber Security Data Warehouse

    SciTech Connect

    Czejdo, Bogdan; Ferragut, Erik M; Goodall, John R; Laska, Jason A

    2012-01-01

    The challenge of achieving situational understanding is a limiting factor in effective, timely, and adaptive cyber-security analysis. Anomaly detection fills a critical role in network assessment and trend analysis, both of which underlie the establishment of comprehensive situational understanding. To that end, we propose a cyber security data warehouse implemented as a hierarchical graph of aggregations that captures anomalies at multiple scales. Each node of our pro-posed graph is a summarization table of cyber event aggregations, and the edges are aggregation operators. The cyber security data warehouse enables domain experts to quickly traverse a multi-scale aggregation space systematically. We describe the architecture of a test bed system and a summary of results on the IEEE VAST 2012 Cyber Forensics data.

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

    PubMed

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

    2015-02-01

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

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

    PubMed

    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

  15. A consensus method for the prediction of 'aggregation-prone' peptides in globular proteins.

    PubMed

    Tsolis, Antonios C; Papandreou, Nikos C; Iconomidou, Vassiliki A; Hamodrakas, Stavros J

    2013-01-01

    The purpose of this work was to construct a consensus prediction algorithm of 'aggregation-prone' peptides in globular proteins, combining existing tools. This allows comparison of the different algorithms and the production of more objective and accurate results. Eleven (11) individual methods are combined and produce AMYLPRED2, a publicly, freely available web tool to academic users (http://biophysics.biol.uoa.gr/AMYLPRED2), for the consensus prediction of amyloidogenic determinants/'aggregation-prone' peptides in proteins, from sequence alone. The performance of AMYLPRED2 indicates that it functions better than individual aggregation-prediction algorithms, as perhaps expected. AMYLPRED2 is a useful tool for identifying amyloid-forming regions in proteins that are associated with several conformational diseases, called amyloidoses, such as Altzheimer's, Parkinson's, prion diseases and type II diabetes. It may also be useful for understanding the properties of protein folding and misfolding and for helping to the control of protein aggregation/solubility in biotechnology (recombinant proteins forming bacterial inclusion bodies) and biotherapeutics (monoclonal antibodies and biopharmaceutical proteins).

  16. Use of dielectric properties to detect egg protein denaturation.

    PubMed

    Bircan, C; Barringer, S A

    2002-01-01

    Changes in water and ion binding that occur during egg protein denaturation can be detected by measuring the dielectric properties. The dielectric properties of egg yolk, egg white and egg ovalbumin were tested from 25 to 105 degrees C at 11 frequencies from 300-2450 MHz. DSC was used to determine the temperature of protein denaturation. Both the dielectric constant and loss factor of egg yolk decreased due to denaturation of the protein lipovitellin. The dielectric constant increased at the initial denaturation and decreased after the complete denaturation and aggregation of egg ovalbumin in both egg white and ovalbumin.

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

    PubMed

    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®

  18. Membrane Remodeling by Surface-Bound Protein Aggregates: Insights from Coarse-Grained Molecular Dynamics Simulation

    PubMed Central

    2015-01-01

    The mechanism of curvature generation in membranes has been studied for decades due to its important role in many cellular functions. However, it is not clear if, or how, aggregates of lipid-anchored proteins might affect the geometry and elastic property of membranes. As an initial step toward addressing this issue, we performed structural, geometrical, and stress field analyses of coarse-grained molecular dynamics trajectories of a domain-forming bilayer in which an aggregate of lipidated proteins was asymmetrically bound. The results suggest a general mechanism whereby asymmetric incorporation of lipid-modified protein aggregates curve multidomain membranes primarily by expanding the surface area of the monolayer in which the lipid anchor is inserted. PMID:24803997

  19. Extended polyglutamine tracts cause aggregation and structural perturbation of an adjacent beta barrel protein.

    PubMed

    Ignatova, Zoya; Gierasch, Lila M

    2006-05-05

    Formation of fibrillar intranuclear inclusions and related neuropathologies of the CAG-repeat disorders are linked to the expansion of a polyglutamine tract. Despite considerable effort, the etiology of these devastating diseases remains unclear. Although polypeptides with glutamine tracts recapitulate many of the observed characteristics of the gene products with CAG repeats, such as in vitro and in vivo aggregation and toxicity in model organisms, extended polyglutamine segments have also been reported to structurally perturb proteins into which they are inserted. Additionally, the sequence context of a polyglutamine tract has recently been shown to modulate its propensity to aggregate. These findings raise the possibility that indirect influences of the repeat tract on adjacent protein domains are contributory to pathologies. Destabilization of an adjacent domain may lead to loss of function, as well as favoring non-native structures in the neighboring domain causing them to be prone to intermolecular association and consequent aggregation. To explore these phenomena, we have used chimeras of a well studied globular protein and exon 1 of huntingtin. We find that expansion of the polyglutamine segment beyond the pathological threshold (>35 glutamines) results in structural perturbation of the neighboring protein whether the huntingtin exon is N- or C-terminal. Elongation of the polyglutamine region also substantially increases the propensity of the chimera to aggregate, both in vitro and in vivo, and in vitro aggregation kinetics of a chimera with a 53-glutamine repeat follow a nucleation polymerization mechanism with a monomeric nucleus.

  20. Synergistic effects of detergents and organic solvents on protein refolding: control of aggregation and folding rates.

    PubMed

    Yamamoto, Etsushi; Yamaguchi, Satoshi; Nagamune, Teruyuki

    2011-01-01

    This paper presents the synergistic enhancement of the refolding yield of denatured and reduced lysozyme by using detergents as aggregation inhibitors and water-miscible organic cosolvents as modulators for the detergents. Adding only cetyltrimethylammonium bromide (CTAB) led to a slight increase in the refolding yield (up to 13%). Further addition of dimethylsulfoxide (DMSO) with CTAB drastically increased the refolding yield up to 35%, a value which was higher than the simple sum of the refolding yields in the presence of only CTAB or DMSO. The synergistic enhancement was also observed in the coexistence of other detergents, such as polyethylene glycol monooleyl ether (n = 50) and N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and cosolvents, such as N,N-dimethylformamide and N,N-dimethylacetamide. Experimental data and a kinetic analysis revealed the guideline for selecting a couple of additives; detergents which can adequately inhibit the aggregation of proteins by binding to hydrophobic surfaces of refolding intermediates should be employed as an aggregation inhibitor, and cosolvents which can properly prevent both protein-protein and protein-detergent interactions act as effective modulators for the aggregation inhibitor, resulting in a desirable balance between folding and aggregation rates. Copyright © 2010. Published by Elsevier B.V.

  1. Understanding the frustration arising from the competition between function, misfolding, and aggregation in a globular protein.

    PubMed

    Gianni, Stefano; Camilloni, Carlo; Giri, Rajanish; Toto, Angelo; Bonetti, Daniela; Morrone, Angela; Sormanni, Pietro; Brunori, Maurizio; Vendruscolo, Michele

    2014-09-30

    Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron-sulfur clusters but whose mutations are associated with Friedreich's ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.

  2. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit

    SciTech Connect

    Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

    2010-07-23

    Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and they require chaperones to keep them soluble and translocation competent. Here we show that a novel targeting factor in the chloroplast signal recognition particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to 'ATPases associated with various cellular activities' (AAA{sup +}) chaperones, cpSRP43 uses specific binding interactions with its substrate to mediate its 'disaggregase' activity. This disaggregase capability can allow targeting machineries to more effectively capture their protein substrates and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example to our knowledge of an ATP-independent disaggregase and shows that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate.

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

    PubMed Central

    2012-01-01

    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 vivo, and can be

  4. Detection of hydrogen sulfide through photoluminescence quenching of penicillamine-copper nanocluster aggregates

    NASA Astrophysics Data System (ADS)

    Ma, Jia-Ying; Chen, Po-Cheng; Chang, Huan-Tsung

    2014-05-01

    We have developed a one-pot, inexpensive, simple and rapid method to synthesize photoluminescent copper nanocluster (Cu NC) aggregates from Cu2+ ions in 65% (v v-1) dimethylformamide aqueous solution containing penicillamine (PA) as a capping and reducing agent. As-prepared PA-Cu NC aggregates emit at 580 nm when excited at 326 nm, with a quantum yield of 2.0%. The photoluminescence of PA-Cu NC aggregates originate from ligand-to-metal charge transfer, which is supported by a long lifetime (126.5 ns) and a large Stokes shift (254 nm). As-prepared PA-Cu NC aggregates have different emission wavelengths with the same excitation wavelength in various organic-aqueous solutions. The PA-Cu NC aggregates are highly selective and sensitive to the detection of hydrogen sulfide (H2S), based on analyte-induced photoluminescence quenching through the formation of CuS nanoparticles. The probe allows the detection of H2S, with a linear range of 1-100 μM and a limit of detection (signal-to-noise ratio = 3) of 500 nM. The practicality of this probe has been validated through the analysis of hot spring water samples.

  5. Detection of hydrogen sulfide through photoluminescence quenching of penicillamine-copper nanocluster aggregates.

    PubMed

    Ma, Jia-Ying; Chen, Po-Cheng; Chang, Huan-Tsung

    2014-05-16

    We have developed a one-pot, inexpensive, simple and rapid method to synthesize photoluminescent copper nanocluster (Cu NC) aggregates from Cu(2+) ions in 65% (v v(-1)) dimethylformamide aqueous solution containing penicillamine (PA) as a capping and reducing agent. As-prepared PA-Cu NC aggregates emit at 580 nm when excited at 326 nm, with a quantum yield of 2.0%. The photoluminescence of PA-Cu NC aggregates originate from ligand-to-metal charge transfer, which is supported by a long lifetime (126.5 ns) and a large Stokes shift (254 nm). As-prepared PA-Cu NC aggregates have different emission wavelengths with the same excitation wavelength in various organic-aqueous solutions. The PA-Cu NC aggregates are highly selective and sensitive to the detection of hydrogen sulfide (H₂S), based on analyte-induced photoluminescence quenching through the formation of CuS nanoparticles. The probe allows the detection of H₂S, with a linear range of 1-100 μM and a limit of detection (signal-to-noise ratio = 3) of 500 nM. The practicality of this probe has been validated through the analysis of hot spring water samples.

  6. Effect of Excipients on Liquid-Liquid Phase Separation and Aggregation in Dual Variable Domain Immunoglobulin Protein Solutions.

    PubMed

    Raut, Ashlesha S; Kalonia, Devendra S

    2016-03-07

    Liquid-liquid phase separation (LLPS) and aggregation can reduce the physical stability of therapeutic protein formulations. On undergoing LLPS, the protein-rich phase can promote aggregation during storage due to high concentration of the protein. Effect of different excipients on aggregation in protein solution is well documented; however data on the effect of excipients on LLPS is scarce in the literature. In this study, the effect of four excipients (PEG 400, Tween 80, sucrose, and hydroxypropyl beta-cyclodextrin (HPβCD)) on liquid-liquid phase separation and aggregation in a dual variable domain immunoglobulin protein solution was investigated. Sucrose suppressed both LLPS and aggregation, Tween 80 had no effect on either, and PEG 400 increased LLPS and aggregation. Attractive protein-protein interactions and liquid-liquid phase separation decreased with increasing concentration of HPβCD, indicating its specific binding to the protein. However, HPβCD had no effect on the formation of soluble aggregates and fragments in this study. LLPS and aggregation are highly temperature dependent; at low temperature protein exhibits LLPS, at high temperature protein exhibits aggregation, and at an intermediate temperature both phenomena occur simultaneously depending on the solution conditions.

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

    PubMed Central

    Yin, Liusong; Chen, Xiaoying; Tiwari, Abhinav; Vicini, Paolo; Hickling, Timothy P.

    2015-01-01

    Therapeutic protein products (TPP) have been widely used to treat a variety of human diseases, including cancer, hemophilia, and autoimmune diseases. However, TPP can induce unwanted immune responses that can impact both drug efficacy and patient safety. The presence of aggregates is of particular concern as they have been implicated in inducing both T cell-independent and T cell-dependent immune responses. We used mathematical modeling to evaluate several mechanisms through which aggregates of TPP could contribute to the development of immunogenicity. Modeling interactions between aggregates and B cell receptors demonstrated that aggregates are unlikely to induce T cell-independent immune responses by cross-linking B cell receptors because the amount of signal transducing complex that can form under physiologically relevant conditions is limited. We systematically evaluate the role of aggregates in inducing T cell-dependent immune responses using a recently developed multiscale mechanistic mathematical model. Our analysis indicates that aggregates could contribute to T cell-dependent immune response by inducing high affinity epitopes which may not be present in the nonaggregated TPP and/or by enhancing danger signals to break tolerance. In summary, our computational analysis is suggestive of novel insights into the mechanisms underlying aggregate-induced immunogenicity, which could be used to develop mitigation strategies. PMID:26682236

  8. Shape matters: the complex relationship between aggregation and toxicity in protein-misfolding diseases.

    PubMed

    Ries, Heidrun Maja; Nussbaum-Krammer, Carmen

    2016-10-15

    A particular subgroup of protein-misfolding diseases, comprising Alzheimer's and Parkinson's disease, involves amyloidogenic proteins that can form alternative pathogenic conformations with a high tendency to self-assemble into oligomeric and fibrillar species. Although misfolded proteins have been clearly linked to disease, the exact nature of the toxic species remains highly controversial. Increasing evidence suggests that there is little correlation between the occurrence of macroscopic protein deposits and toxic phenotypes in affected cells and tissues. In this article, we recap amyloid aggregation pathways, describe prion-like propagation, elaborate on detrimental interactions of protein aggregates with the cellular protein quality control system and discuss why some aggregates are toxic, whereas others seem to be beneficial. On the basis of recent studies on prion strains, we reason that the specific aggregate conformation and the resulting individual interaction with the cellular environment might be the major determinant of toxicity. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  9. DSF method optimization and its application in predicting protein thermal aggregation kinetics.

    PubMed

    Shi, Shuai; Semple, Andrew; Cheung, Jason; Shameem, Mohammed

    2013-08-01

    Differential scanning fluorimetry (DSF) has gained wide acceptance in the therapeutic protein development. However, the effects of dyes and surfactants that may affect structural transitions have not been studied thoroughly to date. We therefore first optimized the DSF method by studying surfactant-containing formulations and found that the presence of surfactants generally required medium-to-high protein concentrations and that high SYPRO® Orange concentration in a DSF experiment may lower protein thermal transitions. We also benchmarked DSF against differential scanning calorimetry (DSC) and evaluated the capability of thermal parameters (from DSF/DSC) to predict real-time thermal aggregation kinetics monitored by size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) in different scenarios. For monoclonal antibody (MAb) fragment, both DSF and DSC were predictive of thermal aggregation rate. For MAb3, a good correlation was observed between DSF and DSC, none of which was, however, indicative of protein aggregation kinetics. In a surfactant ranging study, DSF did not agree with DSC and was not predictive of the aggregation kinetics of the MAb fragment. The concentration-dependent thermal behavior was also studied by DSF. Although higher concentration, in general, tends to lower protein transition temperature, case where it was independent of protein concentration was also presented. Copyright © 2013 Wiley Periodicals, Inc.

  10. The aggregation behavior and interactions of yak milk protein under thermal treatment.

    PubMed

    Wang, T T; Guo, Z W; Liu, Z P; Feng, Q Y; Wang, X L; Tian, Q; Ren, F Z; Mao, X Y

    2016-08-01

    The aggregation behavior and interactions of yak milk protein were investigated after heat treatments. Skim yak milk was heated at temperatures in the range of 65 to 95°C for 10 min. The results showed that the whey proteins in yak milk were denatured after heat treatment, especially at temperatures higher than 85°C. Sodium dodecyl sulfate-PAGE analysis indicated that heat treatment induced milk protein denaturation accompanied with aggregation to a certain extent. When the heating temperature was 75 and 85°C, the aggregation behavior of yak milk proteins was almost completely due to the formation of disulfide bonds, whereas denatured α-lactalbumin and β-lactoglobulin interacted with κ-casein. When yak milk was heated at 85 and 95°C, other noncovalent interactions were found between proteins including hydrophobic interactions. The particle size distributions and microstructures demonstrated that the heat stability of yak milk proteins was significantly lowered by heat treatment. When yak milk was heated at 65 and 75°C, no obvious changes were found in the particle size distribution and microstructures in yak milk. When the temperature was 85 and 95°C, the particle size distribution shifted to larger size trend and aggregates were visible in the heated yak milk.

  11. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  12. Detection of significant protein coevolution.

    PubMed

    Ochoa, David; Juan, David; Valencia, Alfonso; Pazos, Florencio

    2015-07-01

    The evolution of proteins cannot be fully understood without taking into account the coevolutionary linkages entangling them. From a practical point of view, coevolution between protein families has been used as a way of detecting protein interactions and functional relationships from genomic information. The most common approach to inferring protein coevolution involves the quantification of phylogenetic tree similarity using a family of methodologies termed mirrortree. In spite of their success, a fundamental problem of these approaches is the lack of an adequate statistical framework to assess the significance of a given coevolutionary score (tree similarity). As a consequence, a number of ad hoc filters and arbitrary thresholds are required in an attempt to obtain a final set of confident coevolutionary signals. In this work, we developed a method for associating confidence estimators (P values) to the tree-similarity scores, using a null model specifically designed for the tree comparison problem. We show how this approach largely improves the quality and coverage (number of pairs that can be evaluated) of the detected coevolution in all the stages of the mirrortree workflow, independently of the starting genomic information. This not only leads to a better understanding of protein coevolution and its biological implications, but also to obtain a highly reliable and comprehensive network of predicted interactions, as well as information on the substructure of macromolecular complexes using only genomic information. The software and datasets used in this work are freely available at: http://csbg.cnb.csic.es/pMT/. pazos@cnb.csic.es Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. Analysis and characterization of aggregation of a therapeutic Fc-fusion protein.

    PubMed

    Wang, Tian; Fodor, Szilan; Hapuarachchi, Suminda; Jiang, Xinzhao Grace; Chen, Ken; Apostol, Izydor; Huang, Gang

    2013-01-01

    Protein aggregation was observed in a purification intermediate of a therapeutic Fc-fusion protein stored at -30 °C, even though the protein was stable at 4 and -80 °C. The protein was expressed in Escherichia coli as an inclusion body, refolded, and purified using chromatography columns. To study the nature of this aggregation, a series of experiments were conducted to investigate factors that contributed to the protein instability during freezing. We found that the presence of free thiols in the protein is the intrinsic cause. The free thiol cross-linking sites were determined to be at the peptide moiety of the Fc-fusion protein using LC-MS. Partially frozen accompanied by the elevated pH and increased salt and protein concentrations were identified as extrinsic factors that facilitated the aggregation. These results provided important insights into purification process improvement and solution storage of this Fc-fusion protein. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Prion Aggregates Are Recruited to the Insoluble Protein Deposit (IPOD) via Myosin 2-Based Vesicular Transport

    PubMed Central

    Nawroth, Peter P.; Tyedmers, Jens

    2016-01-01

    Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed “Insoluble PrOtein Deposit (IPOD)”, but nothing is known about the mechanism of substrate recruitment to this site. The IPOD is located directly adjacent to the Phagophore Assembly Site (PAS) where the cell initiates autophagy and the Cytoplasm-to-Vacuole Targeting (CVT) pathway destined for delivery of precursor peptidases to the vacuole. Recruitment of CVT substrates to the PAS was proposed to occur via vesicular transport on Atg9 vesicles and requires an intact actin cytoskeleton and “SNAP (Soluble NSF Attachment Protein) Receptor Proteins (SNARE)” protein function. It is, however, unknown how this vesicular transport machinery is linked to the actin cytoskeleton. We demonstrate that recruitment of model amyloid PrD-GFP and the CVT substrate precursor-aminopeptidase 1 (preApe1) to the IPOD or PAS, respectively, is disturbed after genetic impairment of Myo2-based actin cable transport and SNARE protein function. Rather than accumulating at the respective deposition sites, both substrates reversibly accumulated often together in the same punctate structures. Components of the CVT vesicular transport machinery including Atg8 and Atg9 as well as Myo2 partially co-localized with the joint accumulations. Thus we propose a model where vesicles, loaded with preApe1 or PrD-GFP, are recruited to tropomyosin coated actin cables via the Myo2 motor protein for delivery to the PAS and IPOD, respectively. We discuss that deposition at the IPOD is not an integrated mandatory part of the degradation pathway for amyloid aggregates, but more likely stores excess aggregates until downstream degradation pathways have the capacity to turn them over after liberation by the Hsp104

  15. Prion Aggregates Are Recruited to the Insoluble Protein Deposit (IPOD) via Myosin 2-Based Vesicular Transport.

    PubMed

    Kumar, Rajesh; Nawroth, Peter P; Tyedmers, Jens

    2016-09-01

    Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed "Insoluble PrOtein Deposit (IPOD)", but nothing is known about the mechanism of substrate recruitment to this site. The IPOD is located directly adjacent to the Phagophore Assembly Site (PAS) where the cell initiates autophagy and the Cytoplasm-to-Vacuole Targeting (CVT) pathway destined for delivery of precursor peptidases to the vacuole. Recruitment of CVT substrates to the PAS was proposed to occur via vesicular transport on Atg9 vesicles and requires an intact actin cytoskeleton and "SNAP (Soluble NSF Attachment Protein) Receptor Proteins (SNARE)" protein function. It is, however, unknown how this vesicular transport machinery is linked to the actin cytoskeleton. We demonstrate that recruitment of model amyloid PrD-GFP and the CVT substrate precursor-aminopeptidase 1 (preApe1) to the IPOD or PAS, respectively, is disturbed after genetic impairment of Myo2-based actin cable transport and SNARE protein function. Rather than accumulating at the respective deposition sites, both substrates reversibly accumulated often together in the same punctate structures. Components of the CVT vesicular transport machinery including Atg8 and Atg9 as well as Myo2 partially co-localized with the joint accumulations. Thus we propose a model where vesicles, loaded with preApe1 or PrD-GFP, are recruited to tropomyosin coated actin cables via the Myo2 motor protein for delivery to the PAS and IPOD, respectively. We discuss that deposition at the IPOD is not an integrated mandatory part of the degradation pathway for amyloid aggregates, but more likely stores excess aggregates until downstream degradation pathways have the capacity to turn them over after liberation by the Hsp104

  16. Colorimetric detection of biothiols based on aggregation of chitosan-stabilized silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Mohammadi, Somayeh; Khayatian, Gholamreza

    2017-10-01

    We have described a simple and reliable colorimetric method for the sensing of biothiols such as cysteine, homocysteine, and glutathione in biological samples. The selective binding of chitosan capped silver nanoparticles to biothiols induced aggregation of the chitosan-Ag NPs. But the other amino acids that do not have thiol group cannot aggregate the chitosan-Ag NPs. Aggregation of chitosan-Ag NPs has been confirmed with UV-vis absorption spectra, zeta potential and transmission electron microscopy images. Under optimum conditions, good linear relationships existed between the absorption ratios (at A500/A415) and the concentrations of cysteine, homocysteine, and glutathione in the range of 0.1-10.0 μM with detection limits of 15.0, 84.6 and 40.0 nM, respectively. This probe was successfully applied to detect these biothiols in biological samples (urine and plasma).

  17. Effect of protein glycation in the presence or absence of wheat proteins on detection of soybean proteins by commercial ELISA.

    PubMed

    Platteau, C; Cucu, T; De Meulenaer, B; Devreese, B; De Loose, M; Taverniers, I

    2011-02-01

    Soybean (Glycine max) is the world's primary provider of protein and oil and is widely used in foodstuffs. However, the use of soybean in foodstuffs might pose a serious threat to allergic consumers since some proteins can cause allergic reactions. To date mostly ELISA methods are used for testing contamination of foodstuffs with soybean. In view of the complexity regarding allergen detection in foodstuffs and appropriate food product labelling, the aim of this study was to investigate the impact of the Maillard reaction on the detectability of soybean proteins using commercial ELISA kits. Accumulation of protein-bound carbonyls, modification of reactive lysine residues and severe aggregation as a result of incubation with glucose, in the presence or absence of soluble wheat proteins, were recorded. Moreover, detection of soybean proteins by means of three commercial ELISA kits was strongly altered and was highly dependent on the type of kit used.

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

    PubMed

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

    2013-06-26

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

  19. A network of ubiquitin ligases is important for the dynamics of misfolded protein aggregates in yeast.

    PubMed

    Theodoraki, Maria A; Nillegoda, Nadinath B; Saini, Jagdeep; Caplan, Avrom J

    2012-07-06

    Quality control ubiquitin ligases promote degradation of misfolded proteins by the proteasome. If the capacity of the ubiquitin/proteasome system is exceeded, then misfolded proteins accumulate in aggregates that are cleared by the autophagic system. To identify components of the ubiquitin/proteasome system that protect against aggregation, we analyzed a GFP-tagged protein kinase, Ste11ΔN(K444R)-GFP, in yeast strains deleted for 14 different ubiquitin ligases. We show that deletion of almost all of these ligases affected the proteostatic balance in untreated cells such that Ste11ΔN(K444R)-GFP aggregation was changed significantly compared with the levels found in wild type cells. By contrast, aggregation was increased significantly in only six E3 deletion strains when Ste11ΔN(K444R)-GFP folding was impaired due to inhibition of the molecular chaperone Hsp90 with geldanamycin. The increase in aggregation of Ste11ΔN(K444R)-GFP due to deletion of UBR1 and UFD4 was partially suppressed by deletion of UBR2 due to up-regulation of Rpn4, which controls proteasome activity. Deletion of UBR1 in combination with LTN1, UFD4, or DOA10 led to a marked hypersensitivity to azetidine 2-carboxylic acid, suggesting some redundancy in the networks of quality control ubiquitin ligases. Finally, we show that Ubr1 promotes clearance of protein aggregates when the autophagic system is inactivated. These results provide insight into the mechanics by which ubiquitin ligases cooperate and provide feedback regulation in the clearance of misfolded proteins.

  20. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease

    PubMed Central

    Filimonenko, Maria; Stuffers, Susanne; Raiborg, Camilla; Yamamoto, Ai; Malerød, Lene; Fisher, Elizabeth M.C.; Isaacs, Adrian; Brech, Andreas; Stenmark, Harald; Simonsen, Anne

    2007-01-01

    The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations. PMID:17984323

  1. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease.

    PubMed

    Filimonenko, Maria; Stuffers, Susanne; Raiborg, Camilla; Yamamoto, Ai; Malerød, Lene; Fisher, Elizabeth M C; Isaacs, Adrian; Brech, Andreas; Stenmark, Harald; Simonsen, Anne

    2007-11-05

    The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.

  2. Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor.

    PubMed

    Diamond, M I; Robinson, M R; Yamamoto, K R

    2000-01-18

    Spinobulbar muscular atrophy and Huntington's disease are caused by polyglutamine expansion in the androgen receptor and huntingtin, respectively, and their pathogenesis has been associated with abnormal nuclear localization and aggregation of truncated forms of these proteins. Here we show, in diverse cell types, that glucocorticoids can up- or down-modulate aggregation and nuclear localization of expanded polyglutamine polypeptides derived from the androgen receptor and huntingtin through specific regulation of gene expression. Wild-type glucocorticoid receptor (GR), as well as C-terminal deletion derivatives, suppressed the aggregation and nuclear localization of these polypeptides, whereas mutations within the DNA binding domain and N terminus of GR abolished this activity. Surprisingly, deletion of a transcriptional regulatory domain within the GR N terminus markedly increased aggregation and nuclear localization of the expanded polyglutamine proteins. Thus, aggregation and nuclear localization of expanded polyglutamine proteins are regulated cellular processes that can be modulated by a well-characterized transcriptional regulator, the GR. Our findings suggest approaches to study the molecular pathogenesis and selective neuronal degeneration of polyglutamine expansion diseases.

  3. Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor

    PubMed Central

    Diamond, Marc I.; Robinson, Melissa R.; Yamamoto, Keith R.

    2000-01-01

    Spinobulbar muscular atrophy and Huntington's disease are caused by polyglutamine expansion in the androgen receptor and huntingtin, respectively, and their pathogenesis has been associated with abnormal nuclear localization and aggregation of truncated forms of these proteins. Here we show, in diverse cell types, that glucocorticoids can up- or down-modulate aggregation and nuclear localization of expanded polyglutamine polypeptides derived from the androgen receptor and huntingtin through specific regulation of gene expression. Wild-type glucocorticoid receptor (GR), as well as C-terminal deletion derivatives, suppressed the aggregation and nuclear localization of these polypeptides, whereas mutations within the DNA binding domain and N terminus of GR abolished this activity. Surprisingly, deletion of a transcriptional regulatory domain within the GR N terminus markedly increased aggregation and nuclear localization of the expanded polyglutamine proteins. Thus, aggregation and nuclear localization of expanded polyglutamine proteins are regulated cellular processes that can be modulated by a well-characterized transcriptional regulator, the GR. Our findings suggest approaches to study the molecular pathogenesis and selective neuronal degeneration of polyglutamine expansion diseases. PMID:10639135

  4. Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates

    PubMed Central

    Malinovska, Liliana; Kroschwald, Sonja; Munder, Matthias C.; Richter, Doris; Alberti, Simon

    2012-01-01

    Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system. PMID:22718905

  5. Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates.

    PubMed

    Malinovska, Liliana; Kroschwald, Sonja; Munder, Matthias C; Richter, Doris; Alberti, Simon

    2012-08-01

    Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.

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

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

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

  8. In vivo aggregation properties of the nuclear poly(A)-binding protein PABPN1.

    PubMed

    Tavanez, João Paulo; Calado, Patricia; Braga, José; Lafarga, Miguel; Carmo-Fonseca, Maria

    2005-05-01

    A broad range of degenerative diseases is associated with intracellular inclusions formed by toxic, aggregation-prone mutant proteins. Intranuclear inclusions constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD), a dominantly inherited disease caused by (GCG) repeat expansions in the gene that encodes for nuclear poly(A) binding protein (PABPN1). The mutation results in an extended polyalanine stretch that has been proposed to induce protein aggregation and formation of intranuclear inclusions. Here we show that normal PABPN1 is inherently aggregation-prone when exogenously expressed in either HeLa or myogenic C2 cells. Similar deposits of insoluble PABPN1 are formed by variant forms of the protein containing either a polyalanine expansion or a complete deletion of the polyalanine tract, indicating that the mutation responsible for OPMD is not essential for formation of PABPN1 inclusions. In contrast, interfering with any of the protein domains required for stimulation of poly(A) polymerase prevents the formation of inclusions. Most surprisingly, photobleaching experiments reveal that both normal and expanded PABPN1 molecules are not irreversibly sequestered into aggregates, but rather move rapidly in and out of the inclusions. These findings have important implications for the interpretation of OPMD model systems based on exogenous expression of PABPN1.

  9. Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques.

    PubMed

    Kitamura, Akira; Nagata, Kazuhiro; Kinjo, Masataka

    2015-03-16

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

  10. Protein aggregation and neurodegeneration in prototypical neurodegenerative diseases: Examples of amyloidopathies, tauopathies and synucleinopathies.

    PubMed

    Bourdenx, Mathieu; Koulakiotis, Nikolaos Stavros; Sanoudou, Despina; Bezard, Erwan; Dehay, Benjamin; Tsarbopoulos, Anthony

    2015-07-21

    Alzheimer's and Parkinson's diseases are the most prevalent neurodegenerative diseases that generate important health-related direct and indirect socio-economic costs. They are characterized by severe neuronal losses in several disease-specific brain regions associated with deposits of aggregated proteins. In Alzheimer's disease, β-amyloid peptide-containing plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein tau are the two main neuropathological lesions, while Parkinson's disease is defined by the presence of Lewy Bodies that are intraneuronal proteinaceous cytoplasmic inclusions. α-Synuclein has been identified as a major protein component of Lewy Bodies and heavily implicated in the pathogenesis of Parkinson's disease. In the past few years, evidence has emerged to explain how these aggregate-prone proteins can undergo spontaneous self-aggregation, propagate from cell to cell, and mediate neurotoxicity. Current research now indicates that oligomeric forms are probably the toxic species. This article discusses recent progress in the understanding of the pathogenesis of these diseases, with a focus on the underlying mechanisms of protein aggregation, and emphasizes the pathophysiological molecular mechanisms leading to cellular toxicity. Finally, we present the putative direct link between β-amyloid peptide and tau in causing toxicity in Alzheimer's disease as well as α-synuclein in Parkinson's disease, along with some of the most promising therapeutic strategies currently in development for those incurable neurodegenerative disorders.

  11. Reversible aggregation of HIV-1 Gag proteins mediated by nucleic acids.

    PubMed

    Chen, Zhilin; Cheng, Wei

    2017-01-22

    HIV-1 Gag protein is the major structural protein for the assembly of virion particles. Although studies have been carried out using partially purified Gag proteins to investigate the mechanisms of viral particle assembly, the outcomes of an assembly reaction remain controversial. Here we have developed an improved procedure for purification of several untagged retroviral Gag proteins from E. coli to more than 95% purity and characterized Gag assembly in solution. We found that HIV-1 Gag proteins can undergo nucleic acid-dependent aggregation with several unexpected features: (1) they form spherical particles that are as large as microns in diameter; (2) the size of the aggregates vary with the molar ratio between nucleic acids and proteins, with the average size of these particles reaching maximal at a molar ratio of 1:2 between nucleic acids and proteins; and (3) these particles can be efficiently disassembled simply upon addition of excess nucleic acids into the solution, suggesting the presence of an ordered assembly. Single-stranded DNA oligos that are 10 nucleotides or shorter do not support the formation of these particles. Furthermore, the matrix domain of the Gag protein dramatically facilitates the formation of these aggregates. These studies uncover a previously uncharacterized pathway of HIV Gag assembly in vitro, and have implications for HIV-1 Gag assembly and pathogenesis in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Discrete Molecular Dynamics Approach to the Study of Disordered and Aggregating Proteins.

    PubMed

    Emperador, Agustí; Orozco, Modesto

    2017-03-14

    We present a refinement of the Coarse Grained PACSAB force field for Discrete Molecular Dynamics (DMD) simulations of proteins in aqueous conditions. As the original version, the refined method provides good representation of the structure and dynamics of folded proteins but provides much better representations of a variety of unfolded proteins, including some very large, impossible to analyze by atomistic simulation methods. The PACSAB/DMD method also reproduces accurately aggregation properties, providing good pictures of the structural ensembles of proteins showing a folded core and an intrinsically disordered region. The combination of accuracy and speed makes the method presented here a good alternative for the exploration of unstructured protein systems.

  13. Competition between protein folding and aggregation: A three-dimensional lattice-model simulation

    NASA Astrophysics Data System (ADS)

    Bratko, D.; Blanch, H. W.

    2001-01-01

    Aggregation of protein molecules resulting in the loss of biological activity and the formation of insoluble deposits represents a serious problem for the biotechnology and pharmaceutical industries and in medicine. Considerable experimental and theoretical efforts are being made in order to improve our understanding of, and ability to control, the process. In the present work, we describe a Monte Carlo study of a multichain system of coarse-grained model proteins akin to lattice models developed for simulations of protein folding. The model is designed to examine the competition between intramolecular interactions leading to the native protein structure, and intermolecular association, resulting in the formation of aggregates of misfolded chains. Interactions between the segments are described by a variation of the Go potential [N. Go and H. Abe, Biopolymers 20, 1013 (1981)] that extends the recognition between attracting types of segments to pairs on distinct chains. For the particular model we adopt, the global free energy minimum of a pair of protein molecules corresponds to a dimer of native proteins. When three or more molecules interact, clusters of misfolded chains can be more stable than aggregates of native folds. A considerable fraction of native structure, however, is preserved in these cases. Rates of conformational changes rapidly decrease with the size of the protein cluster. Within the timescale accessible to computer simulations, the folding-aggregation balance is strongly affected by kinetic considerations. Both the native form and aggregates can persist in metastable states, even if conditions such as temperature or concentration favor a transition to an alternative form. Refolding yield can be affected by the presence of an additional polymer species mimicking the function of a molecular chaperone.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

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

    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.

  16. A Multistage Pathway for Human Prion Protein Aggregation in Vitro: From Multimeric Seeds to β-Oligomers and Nonfibrillar Structures

    PubMed Central

    Cho, Kang R.; Yu, Shuiliang; Yin, Shaoman; Plomp, Marco; Qiu, S. Roger; Lakshminarayanan, Rajamani; Moradian-Oldak, Janet; Sy, Man-Sun

    2015-01-01

    Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt—Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3–4 monomers quickly appeared. Oligomers of ∼11–12 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation. PMID:21534611

  17. Silk protein aggregation kinetics revealed by Rheo-IR.

    PubMed

    Boulet-Audet, Maxime; Terry, Ann E; Vollrath, Fritz; Holland, Chris

    2014-02-01

    The remarkable mechanical properties of silk fibres stem from a multi-scale hierarchical structure created when an aqueous protein "melt" is converted to an insoluble solid via flow. To directly relate a silk protein's structure and function in response to flow, we present the first application of a Rheo-IR platform, which couples cone and plate rheology with attenuated total reflectance infrared spectroscopy. This technique provides a new window into silk processing by linking shear thinning to an increase in molecular alignment, with shear thickening affecting changes in the silk protein's secondary structure. Additionally, compared to other static characterization methods for silk, Rheo-IR proved particularly useful at revealing the intrinsic difference between natural (native) and reconstituted silk feedstocks. Hence Rheo-IR offers important novel insights into natural silk processing. This has intrinsic academic merit, but it might also be useful when designing reconstituted silk analogues alongside other polymeric systems, whether natural or synthetic. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

    PubMed

    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

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

    PubMed Central

    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 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. PMID:24970215

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

    PubMed

    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.

  1. Structural coalescence underlies the aggregation propensity of a β-barrel protein motif.

    PubMed

    Angelani, Carla R; Caramelo, Julio J; Curto, Lucrecia M; Delfino, José M

    2017-01-01

    A clear understanding of the structural foundations underlying protein aggregation is an elusive goal of central biomedical importance. A step toward this aim is exemplified by the β-barrel motif represented by the intestinal fatty acid binding protein (IFABP) and two abridged all-β sheet forms (Δ98Δ and Δ78Δ). At odds with the established notion that a perturbation of the native fold should necessarily favor a buildup of intermediate forms with an enhanced tendency to aggregate, the intrinsic stability (ΔG°H2O) of these proteins does not bear a straightforward correlation with their trifluoroethanol (TFE)-induced aggregation propensity. In view of this fact, we found it more insightful to delve into the connection between structure and stability under sub-aggregating conditions (10% TFE). In the absence of the co-solvent, the abridged variants display a common native-like region decorated with a disordered C-terminal stretch. Upon TFE addition, an increase in secondary structure content is observed, assimilating them to the parent protein. In this sense, TFE perturbs a common native like region while exerting a global compaction effect. Importantly, in all cases, fatty acid binding function is preserved. Interestingly, energetic as well as structural diversity in aqueous solution evolves into a common conformational ensemble more akin in stability. These facts reconcile apparent paradoxical findings related to stability and rates of aggregation. This scenario likely mimics the accrual of aggregation-prone species in the population, an early critical event for the development of fibrillation.

  2. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

    PubMed

    Davis, Marie Y; Trinh, Kien; Thomas, Ruth E; Yu, Selina; Germanos, Alexandre A; Whitley, Brittany N; Sardi, Sergio Pablo; Montine, Thomas J; Pallanck, Leo J

    2016-03-01

    Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity.

  3. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration

    PubMed Central

    Thomas, Ruth E.; Yu, Selina; Germanos, Alexandre A.; Whitley, Brittany N.; Sardi, Sergio Pablo; Montine, Thomas J.; Pallanck, Leo J.

    2016-01-01

    Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease, and are the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity. PMID:27019408

  4. Structural coalescence underlies the aggregation propensity of a β-barrel protein motif

    PubMed Central

    Angelani, Carla R.; Caramelo, Julio J.; Curto, Lucrecia M.; Delfino, José M.

    2017-01-01

    A clear understanding of the structural foundations underlying protein aggregation is an elusive goal of central biomedical importance. A step toward this aim is exemplified by the β-barrel motif represented by the intestinal fatty acid binding protein (IFABP) and two abridged all-β sheet forms (Δ98Δ and Δ78Δ). At odds with the established notion that a perturbation of the native fold should necessarily favor a buildup of intermediate forms with an enhanced tendency to aggregate, the intrinsic stability (ΔG°H2O) of these proteins does not bear a straightforward correlation with their trifluoroethanol (TFE)-induced aggregation propensity. In view of this fact, we found it more insightful to delve into the connection between structure and stability under sub-aggregating conditions (10% TFE). In the absence of the co-solvent, the abridged variants display a common native-like region decorated with a disordered C-terminal stretch. Upon TFE addition, an increase in secondary structure content is observed, assimilating them to the parent protein. In this sense, TFE perturbs a common native like region while exerting a global compaction effect. Importantly, in all cases, fatty acid binding function is preserved. Interestingly, energetic as well as structural diversity in aqueous solution evolves into a common conformational ensemble more akin in stability. These facts reconcile apparent paradoxical findings related to stability and rates of aggregation. This scenario likely mimics the accrual of aggregation-prone species in the population, an early critical event for the development of fibrillation. PMID:28187186

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

    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.

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

    PubMed Central

    Karakani, Ali Mohammadi; Riazi, Gholamhossein; Mahmood Ghaffari, Seyed; Ahmadian, Shahin; Mokhtari, Farzad; Jalili Firuzi, Mahshad; Zahra Bathaie, Seyedeh

    2015-01-01

    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. PMID:26124935

  7. Chapter 3: A fluorescent window into protein folding and aggregation in cells.

    PubMed

    Ignatova, Zoya; Gierasch, Lila M

    2008-01-01

    Evolutionary selective pressures have tuned the efficiency of the protein-folding reaction in the crowded complex environment in the cell. Nevertheless, the fidelity of folding is imperfect, leading to off-pathway intermolecular interactions that compete with proper folding and to consequent formation of thermodynamically stable aggregates. Such aggregates constitute the histopathological hallmarks of many neurodegenerative pathologies. Yet, most of the approaches to characterize protein folding and/or misfolding are limited to in vitro conditions. Here, we describe a strategy to directly monitor the behavior of a protein in prokaryotic and eukaryotic cells. The method is based on incorporation of structurally non-perturbing, specific binding motifs for a bis-arsenical fluoroscein dye, FlAsH, in sites that result in distinct dye fluorescence signals for the folded and unfolded states of the protein under study. Our approach has been developed using as a case study the predominantly beta-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein, alone or as a chimera fused to the exon 1-encoded fragment of huntingtin, which harbors a polyglutamine repeat tract. We have designed protocols to label this protein in vivo and to monitor the resulting fluorescence signal, which reports on any misfolding transition and formation of aggregates, yielding quantitatively interpretable data.

  8. Inhibition of protein aggregation by zwitterionic polymer-based core-shell nanogels

    PubMed Central

    Rajan, Robin; Matsumura, Kazuaki

    2017-01-01

    Protein aggregation is a process by which misfolded proteins polymerizes into aggregates and forms fibrous structures with a β-sheet conformation, known as amyloids. It is an undesired outcome, as it not only causes numerous neurodegenerative diseases, but is also a major deterrent in the development of protein biopharmaceuticals. Here, we report a rational design for the synthesis of novel zwitterionic polymer-based core-shell nanogels via controlled radical polymerization. Nanogels with different sizes and functionalities in the core and shell were prepared. The nanogels exhibit remarkable efficiency in the protection of lysozyme against aggregation. Addition of nanogels suppresses the formation of toxic fibrils and also enables lysozyme to retain its enzymatic activity. Increasing the molecular weight and degree of hydrophobicity markedly increases its overall efficiency. Investigation of higher order structures revealed that lysozyme when heated without any additive loses its secondary structure and transforms into a random coil conformation. In contrast, presence of nanogels facilitates the retention of higher order structures by acting as molecular chaperones, thereby reducing molecular collisions. The present study is the first to show that it is possible to design zwitterionic nanogels using appropriate polymerization techniques that will protect proteins under conditions of extreme stress and inhibit aggregation. PMID:28374820

  9. Oligomeric Neuronal Protein Aggregates as Biomarkers for Traumatic Brain Injury (TBI) and Alzheimer Disease (AD)

    DTIC Science & Technology

    2013-10-01

    as Biomarkers for Traumatic Brain Injury (TBI) and Alzheimer Disease (AD) PRINCIPAL INVESTIGATOR: Michael Sierks CONTRACTING...Oligomeric Neuronal Protein Aggregates as Biomarkers for Traumatic Brain Injury (TBI) and Alzheimer Disease (AD) 5b. GRANT NUMBER 12109023 5c...AD so treatment can begin early and the effectiveness of the treatments can be monitored. Major Findings: We have essentially finished development

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

    PubMed

    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 < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and 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.

  11. Impact of Sterilization Method on Protein Aggregation and Particle Formation in Polymer-Based Syringes.

    PubMed

    Kiminami, Hideaki; Krueger, Aaron B; Abe, Yoshihiko; Yoshino, Keisuke; Carpenter, John F

    2017-04-01

    The effects of sterilization methods on the storage stability of erythropoietin (EPO) in polymer-based syringes were assessed by quantifying protein oxidation, aggregation, and particle formation. Micro-particle counting and size exclusion chromatography coupled with a multi-angle light scattering detector demonstrated much lower levels of protein particles and aggregates for EPO stored for 12 weeks in steam-sterilized than in radiation (Rad)-sterilized syringes. Intermediate levels