Science.gov

Sample records for protein aggregation studies

  1. 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

  2. Molecular dynamics studies of protein folding and aggregation

    NASA Astrophysics Data System (ADS)

    Ding, Feng

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

  3. 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.

  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. 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.

  6. 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.

  7. 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.

  8. Why do proteins aggregate? "Intrinsically insoluble proteins" and "dark mediators" revealed by studies on "insoluble proteins" solubilized in pure water.

    PubMed

    Song, Jianxing

    2013-01-01

    In 2008, I reviewed and proposed a model for our discovery in 2005 that unrefoldable and insoluble proteins could in fact be solubilized in unsalted water. Since then, this discovery has offered us and other groups a powerful tool to characterize insoluble proteins, and we have further addressed several fundamental and disease-relevant issues associated with this discovery. Here I review these results, which are conceptualized into several novel scenarios. 1) Unlike 'misfolded proteins', which still retain the capacity to fold into well-defined structures but are misled to 'off-pathway' aggregation, unrefoldable and insoluble proteins completely lack this ability and will unavoidably aggregate in vivo with ~150 mM ions, thus designated as 'intrinsically insoluble proteins (IIPs)' here. IIPs may largely account for the 'wastefully synthesized' DRiPs identified in human cells. 2) The fact that IIPs including membrane proteins are all soluble in unsalted water, but get aggregated upon being exposed to ions, logically suggests that ions existing in the background play a central role in mediating protein aggregation, thus acting as 'dark mediators'. Our study with 14 salts confirms that IIPs lack the capacity to fold into any well-defined structures. We uncover that salts modulate protein dynamics and anions bind proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Accordingly, I modified my previous model. 3) Insoluble proteins interact with lipids to different degrees. Remarkably, an ALS-causing P56S mutation transforms the β-sandwich MSP domain into a helical integral membrane protein. Consequently, the number of membrane-interacting proteins might be much larger than currently recognized. To attack biological membranes may represent a common mechanism by which aggregated proteins initiate human diseases. 4) Our discovery also implies a solution to the 'chicken-and-egg paradox' for the origin of primitive membranes embedded

  9. Kinetics of protein aggregation

    NASA Astrophysics Data System (ADS)

    Knowles, Tuomas

    2015-03-01

    Aggregation into linear nanostructures, notably amyloid and amyloid-like fibrils, is a common form of behaviour exhibited by a range of peptides and proteins. This process was initially discovered in the context of the aetiology of a range of neurodegenerative diseases, but has recently been recognised to of general significance and has been found at the origin of a number of beneficial functional roles in nature, including as catalytic scaffolds and functional components in biofilms. This talk discusses our ongoing efforts to study the kinetics of linear protein self-assembly by using master equation approaches combined with global analysis of experimental data.

  10. 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.

  11. 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.

  12. 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.

  13. Crowding versus molecular seeding: NMR studies of protein aggregation in hen egg white

    NASA Astrophysics Data System (ADS)

    Sanfelice, D.; Adrover, M.; Martorell, G.; Pastore, A.; Temussi, P. A.

    2012-06-01

    In living systems, proteins are surrounded by many other macromolecules of different nature, at high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, such as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides or even synthetic polymers. We have recently proposed hen egg white (HEW) as a suitable, natural medium to study macromolecules in crowding conditions. Here, we show that HEW can increase dramatically the aggregation kinetics of proteins with an in-built tendency to associate. By dissecting the mechanism we demonstrate that only part of this effect is due to crowding, while another factor playing an important role is the interaction with proteins from the milieu. High molecular weight glycoproteins present in HEW act as efficient molecular seeds for aggregation. Our results bear important consequences for in-cell NMR studies and suggest a role of glycosylated proteins in aggregation.

  14. 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

  15. 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

    2016-01-01

    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

  16. Micro-Raman Study of the Ceramic Aggregations with Protein Bridging in the Optical Trap

    NASA Astrophysics Data System (ADS)

    Zavalin, Andrey; Morgan, Steve; Collins, W. Eugene

    2003-05-01

    We use a method, utilizing the one-beam optical trap, integrated with micro-Raman spectrometer for trapping the nano- and micro- scale size objects and spectroscopic studies of the created dynamic and static aggregations. Our previous presented results show, that under the certain conditions dynamic photon bonding among the objects is converted to the chemical bonding and the aggregation, formed in the trap, is stable without optical field. This method has many promising features for the applications, such as on-chip assembling tool and photon crystal growing environment. In the presented experiments dynamic aggregations up to 300 um size were created after 5-30 minutes of stable trapping conditions in the CTP glass-ceramic bead/water suspension. With the additional bonding bridging protein components, such as Bovine serum albumin (BSA) molecules, a higher (near 30been shown. Micro-Raman spectra for the permanent aggregations, assembled in the suspensions, containing CTP glass-ceramic beads and protein molecules show several new peaks, possibly associated with creation of chemical bridging bonds in the aggregations. Research supported by the MBRS program, funded by the NIH/NIGMS, grant 1S06-GM62813-01.

  17. Model discrimination and mechanistic interpretation of kinetic data in protein aggregation studies.

    PubMed

    Bernacki, Joseph P; Murphy, Regina M

    2009-04-01

    Given the importance of protein aggregation in amyloid diseases and in the manufacture of protein pharmaceuticals, there has been increased interest in measuring and modeling the kinetics of protein aggregation. Several groups have analyzed aggregation data quantitatively, typically measuring aggregation kinetics by following the loss of protein monomer over time and invoking a nucleated growth mechanism. Such analysis has led to mechanistic conclusions about the size and nature of the nucleus, the aggregation pathway, and/or the physicochemical properties of aggregation-prone proteins. We have examined some of the difficulties that arise when extracting mechanistic meaning from monomer-loss kinetic data. Using literature data on the aggregation of polyglutamine, a mutant beta-clam protein, and protein L, we determined parameter values for 18 different kinetic models. We developed a statistical model discrimination method to analyze protein aggregation data in light of competing mechanisms; a key feature of the method is that it penalizes overparameterization. We show that, for typical monomer-loss kinetic data, multiple models provide equivalent fits, making mechanistic determination impossible. We also define the type and quality of experimental data needed to make more definitive conclusions about the mechanism of aggregation. Specifically, we demonstrate how direct measurement of fibril size provides robust discrimination. PMID:19348769

  18. 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. PMID:25279560

  19. 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

  20. Cataract-related changes in protein aggregates of human lens studied by ultracentrifugation.

    PubMed

    Twardowski, J; Hoja, D

    1990-01-01

    In human lens, cataract development causes an increase in the amount of protein aggregates. Their specific density of 1.25 g/ml is much higher than that of 1.14 g/ml and 1.06 g/ml obtained for water-soluble proteins. During the formation of water-insoluble aggregates their size varies up to some micrometers. Infrared spectra of water-soluble protein and in the aggregates confirm that the content of bound water in aggregates decreases. PMID:2279587

  1. The second virial coefficient as a predictor of protein aggregation propensity: A self-interaction chromatography study

    PubMed Central

    Quigley, A.; Williams, D.R.

    2015-01-01

    The second osmotic virial coefficients (b2) of four proteins – lysozyme, recombinant human lactoferrin, concanavalin A and catalase were measured by self-interaction chromatography (SIC) in solutions of varying salt type, concentration and pH. Protein aggregate sizes based on the initial hydrodynamic radius of the protein solution species present were measured using dynamic light scattering, and the relationship between b2 and protein aggregate size was studied. A linear correlation was established between b2 values and protein aggregate hydrodynamic size for all proteins, and for almost all solution conditions. Aggregate sizes of <∼10 nm, indicative of non-aggregated protein systems, were consistently observed to have b2 values >0. The observed b2 trends as a function of solution conditions were very much protein dependent, with notable trends including the existence of attractive interactions (negative b2 values) at low ionic strengths for catalase and concanavalin A, and the highly positive b2 values observed for lactoferrin over a wide range of solution conditions, reflecting lactoferrin’s innately high stability. It is concluded that the quantification of protein–protein interactions using SIC based b2 data is a potentially valuable screening tool for predicting protein aggregation propensity. PMID:26259782

  2. Effects of macromolecular crowding on protein folding and aggregation studied by density functional theory: Dynamics

    NASA Astrophysics Data System (ADS)

    Kinjo, Akira R.; Takada, Shoji

    2002-11-01

    Inside the living cell is inherently crowded with proteins and other macromolecules. Thus, it is indispensable to take into account various interactions between the protein and other macromolecules for thorough understanding of protein functions in cellular contexts. Here we focus on the excluded volume interaction imposed on the protein by surrounding macromolecules or ``crowding agents.'' We have presented a theoretical framework for describing equilibrium properties of proteins in crowded solutions [A. R. Kinjo and S. Takada, Phys. Rev. E (to be published)]. In the present paper, we extend the theory to describe nonequilibrium properties of proteins in crowded solutions. Dynamics simulations exhibit qualitatively different morphologies depending on the aggregating conditions, and it was found that macromolecular crowding accelerates the onset of aggregation while stabilizing the native protein in the quasiuniform phase before the onset of aggregation. It is also observed, however, that the aggregation may be kinetically inhibited in highly crowded conditions. The effects of crowding on folding and unfolding of proteins are also examined, and the results suggest that fast folding is an important factor in preventing aggregation of denatured proteins.

  3. Immunogenicity of Therapeutic Protein Aggregates.

    PubMed

    Moussa, Ehab M; Panchal, Jainik P; Moorthy, Balakrishnan S; Blum, Janice S; Joubert, Marisa K; Narhi, Linda O; Topp, Elizabeth M

    2016-02-01

    Therapeutic proteins have a propensity for aggregation during manufacturing, shipping, and storage. The presence of aggregates in protein drug products can induce adverse immune responses in patients that may affect safety and efficacy, and so it is of concern to both manufacturers and regulatory agencies. In this vein, there is a lack of understanding of the physicochemical determinants of immunological responses and a lack of standardized analytical methods to survey the molecular properties of aggregates associated with immune activation. In this review, we provide an overview of the basic immune mechanisms in the context of interactions with protein aggregates. We then critically examine the literature with emphasis on the underlying immune mechanisms as they relate to aggregate properties. Finally, we highlight the gaps in our current understanding of this issue and offer recommendations for future research. PMID:26869409

  4. 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).

  5. Structural Evolution of Metastable Protein Aggregates in the Presence of Trivalent Salt Studied by (V)SANS and SAXS.

    PubMed

    Sauter, Andrea; Zhang, Fajun; Szekely, Noemi K; Pipich, Vitaliy; Sztucki, Michael; Schreiber, Frank

    2016-06-23

    We present a study of the structural evolution of protein aggregates formed in solutions of a globular protein, β-lactoglobulin (BLG), in the presence of YCl3. These aggregates are often observed before crystallization starts and they are metastable with respect to the crystalline phase. Here we focus on the characterization of the hierarchical structure of this intermediate phase and its temperature dependent structure evolution using a combination of (very) small angle neutron and X-ray scattering (VSANS, SANS, and SAXS). Results show that the hierarchical structure ranges from nanometer scale protein monomer, dimer and compact protein clusters to micrometer scale fractal protein aggregates. Upon cooling, the overall hierarchical structure is preserved, but the evolution of the internal structure within the aggregates is clearly visible: the monomer-monomer correlation peak reduces its intensity and disappears completely at lower temperatures, whereas the cluster-cluster correlation is enhanced. At a larger length scale, the fractal dimension of protein aggregates increases. The kinetics of the structure change during a temperature ramp was further investigated using time-resolved SAXS. The time dependent SAXS profiles show clear isosbestic points and the kinetics of the structural evolution can be well described using a two-state model. These dynamic properties of protein aggregates on a broad length scale may be essential for being the precursors of nucleation.

  6. [The actions of nucleating proteins in vesicle aggregation and fusion: a preliminary study].

    PubMed

    Chen, Y; Zhang, Y; Cai, D

    1997-03-01

    After investigation of the effects of Con A-binding proteins, the major biliary pronucleating effectors system on the figures and lipid composition of model biliary vesicles, the authors found that Con A-binding proteins accelerate aggregation and fusion of vesicles as a result of increased vesicular cholesterol and decreased vesicular phospholipids as well as cholesterol-saturated vesicles. We also found that vesicular proteins though in small quantity are potent in pronucleating effects. The distribution of several known nucleating proteins between the vesicles and micelles is quite different, and there are higher content and more potent pronucleating effectors in patients with cholesterol gallstone than those with pigment stone. By affinity staining method, con A-binding proteins were shown in native biliary vesicles as lipid-protein complex. These results suggest that the existence and changes in their quantity and quality of vesicular nucleating proteins play an important role in vesicle aggregation and fusion.

  7. Aggregation analysis of Con A binding proteins of human seminal plasma: a dynamic light scattering study.

    PubMed

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Singh, Sarman; Yadav, Savita

    2013-02-01

    Concanavalin A (Con A) binding fraction of human seminal plasma is vital as it shows decapacitating activity and contains proteins which have critical roles in fertility related processes. Con A binding proteins were isolated by lectin affinity chromatography. These proteins form high molecular weight aggregates at near physiological pH (7.0) as inferred by gel filtration. Aggregation analysis was performed by dynamic light scattering (DLS). DLS analysis was also performed at different pH values and in presence of various additives including NaCl, EDTA, cholesterol and sugars, such as d-glucose, d-fructose and d-mannose to identify their effect on aggregation size. The results indicate that degree of aggregation was highly reduced in presence of d-fructose, EDTA and at lower and higher pH values as depicted by lowering of hydrodynamic radii. This aggregation behaviour might be decisive for fertility related events with a suggestive role towards inhibition of premature capacitation.

  8. Protein aggregation in salt solutions.

    PubMed

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

    2015-05-26

    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.

  9. 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

  10. 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.

  11. Proline inhibits aggregation during protein refolding.

    PubMed Central

    Samuel, D.; Kumar, T. K.; Ganesh, G.; Jayaraman, G.; Yang, P. W.; Chang, M. M.; Trivedi, V. D.; Wang, S. L.; Hwang, K. C.; Chang, D. K.; Yu, C.

    2000-01-01

    The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone. PMID:10716186

  12. Protein sequences encode safeguards against aggregation.

    PubMed

    Reumers, Joke; Maurer-Stroh, Sebastian; Schymkowitz, Joost; Rousseau, Fréderic

    2009-03-01

    Functional requirements shaped proteins into globular structures. Under these structural constraints, which require both regular secondary structure and a hydrophobic core, protein aggregation is an unavoidable corollary to protein structure. However, as aggregation results in reduced fitness, natural selection will tend to eliminate strongly aggregating sequences. The analysis of distribution and variation of aggregation patterns in the human proteome using the TANGO algorithm confirms the findings of a previous study on several proteomes: the flanks of aggregation-prone regions are enriched with charged residues and proline, the so-called gatekeeper-residues. Moreover, in this study, we observed a widespread redundancy in gatekeeper usage. Interestingly, aggregating regions from key proteins such as p53 or huntingtin are among the most extensive "gatekept" sequences. As a consequence, mutations that remove gatekeepers could therefore result in a strong increase in disease-susceptibility. In a set of disease-associated mutations from the UniProt database, we find a strong enrichment of mutations that disrupt gatekeeper motifs. Closer inspection of a number of case studies indicates clearly that removing gatekeepers may play a determining role in widely varying disorders, such as van der Woude syndrome (VWS), X-linked Fabry disease (FD), and limb-girdle muscular dystrophy. PMID:19156839

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

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

  19. 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.

  20. Therapeutic Protein Aggregation: Mechanisms, Design, and Control

    PubMed Central

    Roberts, Christopher J.

    2014-01-01

    While it is well known that proteins are only marginally stable in their folded states, it is often less well appreciated that most proteins are inherently aggregation-prone in their unfolded or partially unfolded states, and the resulting aggregates can be extremely stable and long-lived. For therapeutic proteins, aggregates are a significant risk factor for deleterious immune responses in patients, and can form via a variety of mechanisms. Controlling aggregation using a mechanistic approach may allow improved design of therapeutic protein stability, as a complement to existing design strategies that target desired protein structures and function. Recent results highlight the importance of balancing protein environment with the inherent aggregation propensities of polypeptide chains. PMID:24908382

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

    PubMed Central

    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. PMID:26989481

  2. 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.

  3. 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

  4. Immunogenicity of therapeutic proteins: influence of aggregation.

    PubMed

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

    2014-01-01

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

  5. A novel method for study of the aggregation of protein induced by metal ion aluminum(III) using resonance Rayleigh scattering technique

    NASA Astrophysics Data System (ADS)

    Long, Xiufen; Zhang, Caihua; Cheng, Jiongjia; Bi, Shuping

    2008-01-01

    We present a novel method for the study of the aggregation of protein induced by metal ion aluminum(III) using resonance Rayleigh scattering (RRS) technique. In neutral Tris-HCl medium, the effect of this aggregation of protein results in the enhancement of RRS intensity and the relationship between the enhancement of the RRS signal and the Al concentration is nonlinear. On this basis, we established a new method for the determination of the critical induced-aggregation concentrations ( CCIAC) of metal ion Al(III) inducing the protein aggregation. Our results show that many factors, such as, pH value, anions, salts, temperature and solvents have obvious effects. We also studied the extent of aggregation and structural changes using ultra-violet spectrometry, protein intrinsic fluorescence and circular dichroism to further understand the exact mechanisms of the aggregation characteristics of proteins induced by metal ion Al(III) at the molecular level, to help us to develop effective methods to investigate the toxicity of metal ion Al, and to provide theoretical and quantitative evidences for the development of appropriate treatments for neurodementia such as Parkinson's disease, Alzheimer's disease and dementia related to dialysis.

  6. 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.

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

    PubMed

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

    2015-06-01

    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). PMID:25965109

  8. Production of prone-to-aggregate proteins.

    PubMed

    Lebendiker, Mario; Danieli, Tsafi

    2014-01-21

    Expression of recombinant proteins in Escherichia coli (E. coli) remains the most popular and cost-effective method for producing proteins in basic research and for pharmaceutical applications. Despite accumulating experience and methodologies developed over the years, production of recombinant proteins prone to aggregate in E. coli-based systems poses a major challenge in most research applications. The challenge of manufacturing these proteins for pharmaceutical applications is even greater. This review will discuss effective methods to reduce and even prevent the formation of aggregates in the course of recombinant protein production. We will focus on important steps along the production path, which include cloning, expression, purification, concentration, and storage. PMID:24211444

  9. 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.

  10. 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

  11. 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. PMID:27043825

  12. Functional protein aggregates: just the tip of the iceberg.

    PubMed

    Villaverde, Antonio; Corchero, José Luis; Seras-Franzoso, Joaquin; Garcia-Fruitós, Elena

    2015-01-01

    An increasing number of both prokaryotic and eukaryotic cell types are being adapted as platforms for recombinant protein production. The overproduction of proteins in such expression systems leads to the formation of insoluble protein-based aggregates. Although these protein clusters have been poorly studied in most of the eukaryotic systems, aggregates formed in E. coli, named inclusion bodies (IBs), have been deeply characterized in the last decades. Contrary to the general belief, an important fraction of the protein embedded in IB is functional, showing promise in biocatalysis, regenerative medicine and cell therapy. Thus, the exploration of all these functional protein clusters would largely expand their potential in both pharma and biotech industry.

  13. Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET(2).

    PubMed

    Hübner, Gerda M; Larsen, Jane Nøhr; Guerra, Barbara; Niefind, Karsten; Vrecl, Milka; Issinger, Olaf-Georg

    2014-12-01

    Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET(2) pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP(2). This BRET(2) sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET(2) sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate β-arrestin2, decreased the BRET(2) signal by up to 50%. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50%. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET(2) sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.

  14. Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.

    PubMed

    Rodríguez-Escudero, María; Cid, Víctor J; Molina, María; Schulze-Luehrmann, Jan; Lührmann, Anja; Rodríguez-Escudero, Isabel

    2016-01-01

    Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.

  15. Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation

    PubMed Central

    Rodríguez-Escudero, María; Cid, Víctor J.; Molina, María; Schulze-Luehrmann, Jan; Lührmann, Anja; Rodríguez-Escudero, Isabel

    2016-01-01

    Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors

  16. Protein aggregation and lyophilization: Protein structural descriptors as predictors of aggregation propensity

    PubMed Central

    Roughton, Brock C.; Iyer, Lavanya K.; Bertelsen, Esben; Topp, Elizabeth M.; Camarda, Kyle V.

    2014-01-01

    Lyophilization can induce aggregation in therapeutic proteins, but the relative importance of protein structure, formulation and processing conditions are poorly understood. To evaluate the contribution of protein structure to lyophilization-induced aggregation, fifteen proteins were co-lyophilized with each of five excipients. Extent of aggregation following lyophilization, measured using size-exclusion chromatography, was correlated with computational and biophysical protein structural descriptors via multiple linear regression. Descriptor selection was performed using exhaustive search and forward selection. The results demonstrate that, for a given excipient, extent of aggregation is highly correlated by eight to twelve structural descriptors. Leave-one-out cross validation showed that the correlations were able to successfully predict the aggregation for a protein “left out” of the data set. Selected descriptors varied with excipient, indicating both protein structure and excipient type contribute to lyophilization-induced aggregation. The results show some descriptors used to predict protein aggregation in solution are useful in predicting lyophilized protein aggregation. PMID:24516290

  17. Effect of additives on protein aggregation.

    PubMed

    Hamada, Hiroyuki; Arakawa, Tsutomu; Shiraki, Kentaro

    2009-06-01

    This paper overviews solution additives that affect protein stability and aggregation during refolding, heating, and freezing processes. Solution additives are mainly grouped into two classes, i.e., protein denaturants and stabilizers. The former includes guanidine, urea, strong ionic detergents, and certain chaotropic salts; the latter includes certain amino acids, sugars, polyhydric alcohols, osmolytes, and kosmotropic salts. However, there are solution additives that are not unambiguously placed into these two classes, including arginine, certain divalent cation salts (e.g., MgCl(2)) and certain polyhydric alcohols (e.g., ethylene glycol). Certain non-ionic or non-detergent surfactants, ionic liquids, amino acid derivatives, polyamines, and certain amphiphilic polymers may belong to this class. They have marginal effects on protein structure and stability, but are able to disrupt protein interactions. Information on additives that do not catalyze chemical reactions nor affect protein functions helps us to design protein solutions for increased stability or reduced aggregation. PMID:19519415

  18. 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.

  19. Protein-peptide interaction: study of heat-induced aggregation and gelation of β-lactoglobulin in the presence of two peptides from its own hydrolysate.

    PubMed

    Kosters, Hans A; Wierenga, Peter A; de Vries, Renko; Gruppen, Harry

    2013-05-01

    Two peptides, [f135-158] and [f135-162]-SH, were used to study the binding of the peptides to native β-lactolobulin, as well as the subsequent effects on aggregation and gelation of β-lactoglobulin. The binding of the peptide [f135-158] to β-lactoglobulin at room temperature was confirmed by SELDI-TOF-MS. It was further illustrated by increased turbidity of mixed solutions of peptide and protein (at pH 7), indicating association of proteins and peptides in larger complexes. At pH below the isoelectric point of the protein, the presence of peptides did not lead to an increased turbidity, showing the absence of complexation. The protein-peptide complexes formed at pH 7 were found to dissociate directly upon heating. After prolonged heating, extensive aggregation was observed, whereas no aggregation was seen for the pure protein or pure peptide solutions. The presence of the free sulfhydryl group in [f135-162]-SH resulted in a 10 times increase in the amount of aggregation of β-lactoglobulin upon heating, illustrating the additional effect of the free sulfhydryl group. Subsequent studies on the gel strength of heat-induced gels also showed a clear difference between these two peptides. The replacement of additional β-lactoglobulin by [f135-158] resulted in a decrease in gel strength, whereas replacement by peptide [f135-162]-SH increased gel strength.

  20. 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

    2016-01-01

    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. PMID:26877167

  1. 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

    2016-01-01

    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.

  2. 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

  3. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study

    PubMed Central

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  4. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study.

    PubMed

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  5. 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. PMID:26312418

  6. Revealing different aggregation pathways of amyloidogenic proteins by ultrasound velocimetry.

    PubMed

    Smirnovas, Vytautas; Winter, Roland

    2008-04-15

    In this work, we performed a detailed thermodynamic study, including ultrasound velocimetry, densimetry, calorimetry, and FTIR spectroscopy, of an aggregation-prone protein (insulin) under different salt-screening conditions to gain a deeper insight into the scenario of physicochemical events during its temperature-induced unfolding and aggregation reactions. Differences in aggregation and fibrillization pathways are reflected in changes of the partial molar volume, the coefficients of thermal expansion and compressibility, and the infrared spectral properties of the protein. Combining all experimental data allows setting up a scheme for the temperature-dependent insulin aggregation reaction in the presence and absence of NaCl. As revealed by complementary atomic force microscopy studies, under charge-screening conditions, a process involving structural reorganization, ripening, and formation of more compact nuclei from amorphous oligomers is involved in the formation of mature fibrillar morphologies. In this work, our focus was to put forward a comprehensive discussion of the use of ultrasound velocimetry in disentangling different aggregation pathways. In fact, ultrasound velocimetry proved to be very sensitive to changes in aggregation pathway, highlighting the importance of density and compressibility changes in the different aggregation and fibrillization reactions of the protein.

  7. Zinc significantly changes the aggregation pathway and the conformation of aggregates of human prion protein.

    PubMed

    Pan, Kai; Yi, Chuan-Wei; Chen, Jie; Liang, Yi

    2015-08-01

    Prion diseases are caused by the conformational change of cellular prion protein PrP(C) into pathological prion protein PrP(Sc). Here we study the effect of zinc on the aggregation and conformational change of human prion protein (PrP). As revealed by thioflavin T binding assays, Sarkosyl-soluble SDS-PAGE, and transmission electron microscopy, aggregation of wild-type PrP in the absence of Zn(2+) undergoes four steps: amorphous aggregates, profibrils, mature fibrils, and fragmented fibrils. When the molar ratio of Zn(2+) to PrP was 9:1, however, aggregation of wild-type PrP undergoes another pathway in which wild-type PrP forms oligomers quickly and then forms short-rod aggregates. Unlike wild-type PrP, the octarepeats deletion mutant PrPΔocta forms typical mature fibrils either with or without zinc. As evidenced by isothermal titration calorimetry, Fourier transform infrared spectroscopy, and proteinase K digestion assays, Zn(2+) strongly binds to wild-type PrP monomers with the first binding constant exceeding 10(7)M(-1) under denaturing conditions, and changes the conformation of wild-type PrP aggregates remarkably, but weakly binds to PrPΔocta with binding affinity around 10(4)M(-1) and has no obvious effects on the conformation of PrPΔocta aggregates. Our data demonstrate that zinc significantly changes the aggregation pathway and the conformation of wild-type PrP aggregates mainly via interaction with its octarepeat region. Our findings could explain how zinc modifies pathological PrP conformation associated with prion diseases.

  8. Neurocognitive derivation of protein surface property from protein aggregate parameters

    PubMed Central

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as protein marker. The protocol used multiple ILMFD inputs obtained for a protein to produce a set of mapped outputs as possible SRI candidates. The outputs were further clustered and largest cluster centre after normalization was found to be a close approximation of expected SRI that was calculated from known PDB structure. The outcome showed that faster derivation of individual protein’s surface property might be possible using its bulk form, heat denatured aggregates. PMID:21572883

  9. 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. PMID:23168257

  10. 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

  11. 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

  12. Computational approaches to understanding protein aggregation in neurodegeneration

    PubMed Central

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

    2014-01-01

    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. PMID:24620031

  13. 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.

  14. Orientational Alignment of Amyloidogenic Proteins in Pre-Aggregated Solutions

    NASA Astrophysics Data System (ADS)

    Schröder, C.; Steinhauser, O.; Sasisanker, P.; Weingärtner, H.

    2015-03-01

    In the present study we combine dielectric relaxation spectroscopy with generalized Born simulations to explore the role of orientational order for protein aggregation in solutions of bovine pancreatic insulin at various p H conditions. Under aggregation-prone conditions at low p H , insulin monomers prefer antiparallel dipole alignments, which are consistent with the orientation of the monomeric subunits in the dimer structure. This alignment is also true for two dimers, suggesting that already at moderate protein concentrations the species assemble in equilibrium clusters, in which the molecules adopt preferred orientations also found for the protomers of the corresponding oligomers.

  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. 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.

  17. 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

  18. 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 %).

  19. A method to rapidly create protein aggregates in living cells

    PubMed Central

    Miyazaki, Yusuke; Mizumoto, Kota; Dey, Gautam; Kudo, Takamasa; Perrino, John; Chen, Ling-chun; Meyer, Tobias; Wandless, Thomas J.

    2016-01-01

    The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates. PMID:27229621

  20. 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.

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

    PubMed Central

    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. PMID:26776680

  2. Mechanism of Suppression of Protein Aggregation by α-Crystallin

    PubMed Central

    Markossian, Kira A.; Yudin, Igor K.; Kurganov, Boris I.

    2009-01-01

    This review summarizes experimental data illuminating the mechanism of suppression of heat-induced protein aggregation by α-crystallin, one of the small heat shock proteins. The dynamic light scattering data show that the initial stage of thermal aggregation of proteins is the formation of the initial aggregates involving hundreds of molecules of the denatured protein. Further sticking of the starting aggregates proceeds in a regime of diffusion-limited cluster-cluster aggregation. The protective effect of α-crystallin is due to transition of the aggregation process to the regime of reaction-limited cluster-cluster aggregation, wherein the sticking probability for the colliding particles becomes lower than unity. PMID:19399251

  3. Specificity in intracellular protein aggregation and inclusion body formation

    PubMed Central

    Rajan, Rahul S.; Illing, Michelle E.; Bence, Neil F.; Kopito, Ron R.

    2001-01-01

    Protein aggregation is widely considered to be a nonspecific coalescence of misfolded proteins, driven by interactions between solvent-exposed hydrophobic surfaces that are normally buried within a protein's interior. Accordingly, abnormal interactions between misfolded proteins with normal cellular constituents has been proposed to underlie the toxicity associated with protein aggregates in many neurodegenerative disorders. Here we have used fluorescence resonance energy transfer and deconvolution microscopy to investigate the degree to which unrelated misfolded proteins expressed in the same cells coaggregate with one another. Our data reveal that in cells, protein aggregation exhibits exquisite specificity even among extremely hydrophobic substrates expressed at very high levels. PMID:11687604

  4. 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

  5. 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

  6. Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.

    PubMed

    O'Brien, C J; Blanco, M A; Costanzo, J A; Enterline, M; Fernandez, E J; Robinson, A S; Roberts, C J

    2016-06-01

    Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms. PMID:27160179

  7. 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.

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

    PubMed Central

    O’Meally, Robert; Sonnenberg, Jason L.; Cole, Robert N.; Shewmaker, Frank P.

    2015-01-01

    Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases. PMID:26317359

  9. Cis-Suppression to Arrest Protein Aggregation in Mammalian Cells

    PubMed Central

    Gregoire, Simpson; Zhang, Shaojie; Costanzo, Joseph; Wilson, Kelly; Fernandez, Erik J.; Kwon, Inchan

    2015-01-01

    Protein misfolding and aggregation are implicated in numerous human diseases and significantly lower production yield of proteins expressed in mammalian cells. Despite the importance of understanding and suppressing protein aggregation in mammalian cells, a protein design and selection strategy to modulate protein misfolding/aggregation in mammalian cells has not yet been reported. In this work, we address the particular challenge presented by mutation-induced protein aggregation in mammalian cells. We hypothesize that an additional mutation(s) can be introduced in an aggregation-prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation (cis-suppression). As a model protein, we chose human copper, zinc superoxide dismutase mutant (SOD1A4V) containing an alanine to valine mutation at residue 4, associated with the familial form of amyotrophic lateral sclerosis. We used the program RosettaDesign to identify Phe20 in SOD1A4V as a key residue responsible for SOD1A4V conformational destabilization. This information was used to rationally develop a pool of candidate mutations at the Phe20 site. After two rounds of mammalian-cell based screening of the variants, three novel SOD1A4V variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1A4V variants were verified using cell fractionation and in vitro stability assays. PMID:24114411

  10. Surface characterization of proteins using multi-fractal property of heat-denatured aggregates

    PubMed Central

    Lahiri, Tapobrata; Mishra, Hrishikesh; Sarkar, Subrata; Misra, Krishna

    2008-01-01

    Multi-fractal property of heat-denatured protein aggregates (HDPA) is characteristic of its individual form. The visual similarity between digitally generated microscopic images of HDPA with that of surface-image of its individual X-ray structures in protein databank (PDB) displayed using Visual Molecular Dynamics (VMD) viewer is the basis of the study. We deigned experiments to view the fractal nature of proteins at different aggregate scales. Intensity based multi-fractal dimensions (ILMFD) extracted from various planes of digital microscopic images of protein aggregates were used to characterize HDPA into different classes. Moreover, the ILMFD parameters extracted from aggregates show similar classification pattern to digital images of protein surface displayed by VMD viewer using PDB entry. We discuss the use of irregular patterns of heat-denatured aggregate proteins to understand various surface properties in native proteins. PMID:18795110

  11. Studies on recycled aggregates-based concrete.

    PubMed

    Rakshvir, Major; Barai, Sudhirkumar V

    2006-06-01

    Reduced extraction of raw materials, reduced transportation cost, improved profits, reduced environmental impact and fast-depleting reserves of conventional natural aggregates has necessitated the use of recycling, in order to be able to conserve conventional natural aggregate. In this study various physical and mechanical properties of recycled concrete aggregates were examined. Recycled concrete aggregates are different from natural aggregates and concrete made from them has specific properties. The percentages of recycled concrete aggregates were varied and it was observed that properties such as compressive strength showed a decrease of up to 10% as the percentage of recycled concrete aggregates increased. Water absorption of recycled aggregates was found to be greater than natural aggregates, and this needs to be compensated during mix design.

  12. 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

  13. Misfolded Proteins Induce Aggregation of the Lectin Yos9*

    PubMed Central

    Smith, Melanie H.; Rodriguez, Edwin H.; Weissman, Jonathan S.

    2014-01-01

    A substantial fraction of nascent proteins delivered into the endoplasmic reticulum (ER) never reach their native conformations. Eukaryotes use a series of complementary pathways to efficiently recognize and dispose of these terminally misfolded proteins. In this process, collectively termed ER-associated degradation (ERAD), misfolded proteins are retrotranslocated to the cytosol, polyubiquitinated, and degraded by the proteasome. Although there has been great progress in identifying ERAD components, how these factors accurately identify substrates remains poorly understood. The targeting of misfolded glycoproteins in the ER lumen for ERAD requires the lectin Yos9, which recognizes the glycan species found on terminally misfolded proteins. In a role that remains poorly characterized, Yos9 also binds the protein component of ERAD substrates. Here, we identified a 45-kDa domain of Yos9, consisting of residues 22–421, that is proteolytically stable, highly structured, and able to fully support ERAD in vivo. In vitro binding studies show that Yos9(22–421) exhibits sequence-specific recognition of linear peptides from the ERAD substrate, carboxypeptidase Y G255R (CPY*), and binds a model unfolded peptide ΔEspP and protein Δ131Δ in solution. Binding of Yos9 to these substrates results in their cooperative aggregation. Although the physiological consequences of this substrate-induced aggregation remain to be seen, it has the potential to play a role in the regulation of ERAD. PMID:25086047

  14. Small angle x-ray scattering: Instrument development and studies of protein aggregation, cellulose hydrolysis, and the production of nanoporous metals using surfactact templates

    NASA Astrophysics Data System (ADS)

    Banuelos, Jose Leobardo

    Small angle x-ray scattering (SAXS) was used to obtain structural insights into protein aggregation, the enzymatic hydrolysis of cellulose, and the structural evolution of surfactant-templated nanoporous palladium and platinum systems during their synthesis. SAXS is bulk technique that allows probing the nanometer-scale morphology, interactions, density, and distribution of a variety of nonperiodic systems in the solid, liquid, or gaseous state. A 10-meter Small Angle Scattering camera, originally at ORNL, was assembled. During its re-commissioning, several upgrades were made including new data acquisition software built using National Instrument's Labview development environment, as well as portability to use analysis tools in wide use in scattering community. The Multiple Energy Diffractometer Using Small, medium and wide Angles (MEDUSA) was designed and built, its development will be discussed. The ability of proteins to change their conformation in response to changes in pressure, temperature, the presence of other molecular species, and ionic concentration in the solvents they are found, is a remarkable phenomenon that allows living cells to function properly. When proteins irreversibly unfold or mis-fold and aggregate this gives rise to severely debilitating diseases such as Alzheimer's and prion diseases. Protein aggregation was measured using SAXS on aqueous solutions of bovine serum albumin, myoglobin, and cellulase enzymes. Understanding how cellulose can be broken down into fermentable sugars is an important step in the development of strategies for producing alternative energy from biomass. The enzymatic hydrolysis of cellulose was studied using both small angle neutron scattering and SAXS. One result from these investigations was finding supporting evidence that nanopores within the cellulose fibril matrix allow biologically active enzymes access to digest parts of the fibers. The production of mesoporous materials for hydrogen storage applications was

  15. 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

  16. Electrostatics Controls the Formation of Amyloid Superstructures in Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Foderà, Vito; Zaccone, Alessio; Lattuada, Marco; Donald, Athene M.

    2013-09-01

    The possibility for proteins to aggregate in different superstructures, i.e. large-scale polymorphism, has been widely observed, but an understanding of the physicochemical mechanisms behind it is still out of reach. Here we present a theoretical model for the description of a generic aggregate formed from an ensemble of charged proteins. The model predicts the formation of multifractal structures with the geometry of the growth determined by the electrostatic interactions between single proteins. The model predictions are successfully verified in comparison with experimental curves for aggregate growth allowing us to reveal the mechanism of formation of such complex structures. The model is general and is able to predict aggregate morphologies occurring both in vivo and in vitro. Our findings provide a framework where the physical interactions between single proteins, the aggregate morphology, and the growth kinetics are connected into a single model in agreement with the experimental data.

  17. 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.

  18. 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.

  19. Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates.

    PubMed

    Gorovits, Rena; Fridman, Lilia; Kolot, Mikhail; Rotem, Or; Ghanim, Murad; Shriki, Oz; Czosnek, Henryk

    2016-02-01

    Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted by the whitefly Bemisia tabaci to tomato and other crops. TYLCV proteins are endangered by the host defenses. We have analyzed the capacity of the tomato plant and of the whitefly insect vector to degrade the six proteins encoded by the TYLCV genome. Tomato and whitefly demonstrated the highest proteolytic activity in the fractions containing soluble proteins, less-in large protein aggregates; a significant decrease of TYLCV proteolysis was detected in the intermediate-sized aggregates. All the six TYLCV proteins were differently targeted by the cytoplasmic and nuclear degradation machineries (proteases, ubiquitin 26S proteasome, autophagy). TYLCV could confront host degradation by sheltering in small/midsized aggregates, where viral proteins are less exposed to proteolysis. Indeed, TYLCV proteins were localized in aggregates of various sizes in both host organisms. This is the first study comparing degradation machinery in plant and insect hosts targeting all TYLCV proteins. PMID:26654789

  20. Serum proteins prevent aggregation of Fe2O3 and ZnO nanoparticles

    PubMed Central

    Wells, Mark A.; Abid, Aamir; Kennedy, Ian M.; Barakat, Abdul I.

    2014-01-01

    Aggregation of metal oxide nanoparticles in aqueous media complicates interpretation of in vitro studies of nanoparticle–cell interactions. We used dynamic light scattering to investigate the aggregation dynamics of iron oxide and zinc oxide nanoparticles. Our results show that iron oxide particles aggregate more readily than zinc oxide particles. Pretreatment with serum stabilises iron oxide and zinc oxide nanoparticles against aggregation. Serum-treated iron oxide is stable only in pure water, while zinc oxide is stable in water or cell culture media. These findings, combined with zeta potential measurements and quantification of proteins adsorbed on particle surface, suggest that serum stabilisation of iron oxide particles occurs primarily through protein adsorption and resulting net surface charge. Zinc oxide stabilisation, however, also involves steric hindrance of particle aggregation. Fluid shear at levels used in flow experiments breaks up iron oxide particle aggregates. These results enhance our understanding of nanoparticle aggregation and its consequences for research on the biological effects of nanomaterials. PMID:22149273

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2016-04-01

    Due to their colloidal nature, all protein aggregates scatter light in the visible wavelength region when formed in aqueous solution. This phenomenon makes solution turbidity, a quantity proportional to the relative loss in forward intensity of scattered light, a convenient method for monitoring protein aggregation in biochemical assays. Although turbidity is often taken to be a linear descriptor of the progress of aggregation reactions, this assumption is usually made without performing the necessary checks to provide it with a firm underlying basis. In this article, we outline utilitarian methods for simulating the turbidity generated by homogeneous and mixed-protein aggregation reactions containing fibrous, amorphous, and crystalline structures. The approach is based on a combination of Rayleigh-Gans-Debye theory and approximate forms of the Mie scattering equations. PMID:26763936

  3. Cell-to-cell propagation of infectious cytosolic protein aggregates

    PubMed Central

    Hofmann, Julia P.; Denner, Philip; Nussbaum-Krammer, Carmen; Kuhn, Peer-Hendrik; Suhre, Michael H.; Scheibel, Thomas; Lichtenthaler, Stefan F.; Schätzl, Hermann M.; Bano, Daniele; Vorberg, Ina M.

    2013-01-01

    Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals. PMID:23509289

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

    PubMed

    Kuipers, Bas J H; Alting, Arno C; Gruppen, Harry

    2007-01-01

    Soy-derived proteins (soy protein isolate, glycinin, and beta-conglycinin) and bovine whey-derived proteins (whey protein isolate, alpha-lactalbumin, beta-lactoglobulin) were hydrolyzed using subtilisin Carlsberg, chymotrypsin, trypsin, bromelain, and papain. The (in)solubility of the hydrolysates obtained was studied as a function of pH. At neutral pH, all soy-derived protein hydrolysates, particularly those from glycinin, obtained by hydrolysis with subtilisin Carlsberg, chymotrypsin, bromelain, and papain showed a stronger aggregation compared to the non-hydrolyzed ones. This increase in aggregation was not observed upon hydrolysis by trypsin. None of the whey-derived protein hydrolysates exhibited an increase in aggregation at neutral pH. The high abundance of theoretical cleavage sites in the hydrophobic regions of glycinin probably explains the stronger exposure of hydrophobic groups than for the other proteins, which is suggested to be the driving force in the aggregate formation.

  5. 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.

  6. 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

  7. Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

    PubMed Central

    El Ayadi, Amina; Stieren, Emily S.; Barral, José M.; Oberhauser, Andres F.; Boehning, Darren

    2012-01-01

    Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed α, β, and γ-secretases1. Sequential proteolytic cleavage of APP with β and γ-secretases leads to the production of a small proteolytic peptide, termed Aβ, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a

  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. 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

  10. Predicting protein functions from PPI networks using functional aggregation.

    PubMed

    Hou, Jingyu; Chi, Xiaoxiao

    2012-11-01

    Predicting protein functions computationally from massive protein-protein interaction (PPI) data generated by high-throughput technology is one of the challenges and fundamental problems in the post-genomic era. Although there have been many approaches developed for computationally predicting protein functions, the mutual correlations among proteins in terms of protein functions have not been thoroughly investigated and incorporated into existing prediction methods, especially in voting based prediction methods. In this paper, we propose an innovative method to predict protein functions from PPI data by aggregating the functional correlations among relevant proteins using the Choquet-Integral in fuzzy theory. This functional aggregation measures the real impact of each relevant protein function on the final prediction results, and reduces the impact of repeated functional information on the prediction. Accordingly, a new protein similarity and a new iterative prediction algorithm are proposed in this paper. The experimental evaluations on real PPI datasets demonstrate the effectiveness of our method.

  11. 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.

  12. 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.

  13. Sans study of asphaltene aggregation

    SciTech Connect

    Overfield, R.E.; Sheu, E.Y.; Sinha, S.K.; Liang, K.S. )

    1988-06-01

    The colloidal properties of asphaltenes have long been recognized from peculiarities in their solubility and colligative properties. A layered micellar model or asphaltenes was proposed by others in which a highly condensed alkyl aromatic formed the central part, and molecules of decreasingly aromatic character (resins) clustered around them. Numerous studies, based on a variety of techniques such as ultracentrifugation and electron microscopy indicated a particulate nature for asphaltenes with size 20-40 A diameter. Others have proposed a refined model based on x-ray diffraction and small angle scattering. In this model, interactions between flat sheets of condensed aromatic rings form the central ''crystallite'' part of a spherical particle with the outer part being comprised of the aliphatic positions of the same molecules. These particles are bunched together with some degree of entanglement into ''micelles''. Concentration and solvent dependent radii of gyration, ranging from 30-50 A were reported. The aggregation creates a good deal of uncertainty as to the true molecular size or weight of asphaltenes. Neutron scattering offers novel contrast relative to light scattering (refractive index) and x-ray scattering (electron density). This is because the scattering length of proton is negative, whereas that from deuterium and other nuclei such as C, S, O, and N are positive. Thus by replacing hydrogen with deuterium in either the solvent or the scatterer the contrast can be varied, and different parts of the molecule can be highlighted.

  14. Lubrication properties of protein aggregate dispersions in a soft contact.

    PubMed

    Chojnicka, Agnieszka; de Jong, Saskia; de Kruif, Cornelus G; Visschers, Ronald W

    2008-02-27

    The lubrication, rheological, and molecular properties of two different protein aggregate dispersions were compared: globular aggregates of whey protein isolate (WPI) and fibrillar aggregates of ovalbumin from egg white. These dispersions are models for the lubricating fluid that is present between the tongue and the palate when consuming liquid or gelled products. To simulate oral conditions, a commercial tribometer was modified so that soft rubber surfaces could be used. This allowed us to measure friction at low contact pressures similar to those present between the tongue and palate. Clear correlations were observed between the measured friction coefficients and specific properties of the lubricating fluid such as protein concentration and aggregate size and shape. Furthermore, surface properties like elasticity, surface-surface interactions, and surface roughness had a significant effect on the friction under conditions that are relevant for texture perception. We conclude that in vitro measurements at low contact pressure provide valuable information for understanding and controlling food properties that modulate oral friction.

  15. 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.

  16. 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.

  17. Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

    PubMed

    Jacobson, Therese; Navarrete, Clara; Sharma, Sandeep K; Sideri, Theodora C; Ibstedt, Sebastian; Priya, Smriti; Grant, Chris M; Christen, Philipp; Goloubinoff, Pierre; Tamás, Markus J

    2012-11-01

    Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

  18. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA.

    PubMed

    Woerner, Andreas C; Frottin, Frédéric; Hornburg, Daniel; Feng, Li R; Meissner, Felix; Patra, Maria; Tatzelt, Jörg; Mann, Matthias; Winklhofer, Konstanze F; Hartl, F Ulrich; Hipp, Mark S

    2016-01-01

    Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial β-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein-43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases. PMID:26634439

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. Increased levels of hyper-stable protein aggregates in plasma of older adults.

    PubMed

    Xia, Ke; Trasatti, Hannah; Wymer, James P; Colón, Wilfredo

    2016-06-01

    Proteins that misfold into hyper-stable/degradation-resistant species during aging may accumulate and disrupt protein homeostasis (i.e., proteostasis), thereby posing a survival risk to any organism. Using the method diagonal two-dimensional (D2D) SDS-PAGE, which separates hyper-stable SDS-resistant proteins at a proteomics level, we analyzed the plasma of healthy young (<30 years) and older (60-80 years) adults. We discovered the presence of soluble SDS-resistant protein aggregates in the plasma of older adults, but found significantly lower levels in the plasma of young adults. We identified the inflammation-related chaperone protein haptoglobin as the main component of the hyper-stable aggregates. This observation is consistent with the growing link between accumulations of protein aggregates and aging across many organisms. It is plausible higher amounts of SDS-resistant protein aggregates in the plasma of older adults may reflect a compromise in proteostasis that may potentially indicate cellular aging and/or disease risk. The results of this study have implications for further understanding the link between aging and the accumulation of protein aggregates, as well as potential for the development of aging-related biomarkers. More broadly, this novel application of D2D SDS-PAGE may be used to identify, quantify, and characterize the degradation-resistant protein aggregates in human plasma or any biological system. PMID:27179971

  1. 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

  2. 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.

  3. 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

  4. 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. PMID:12783846

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

    PubMed

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

    2016-07-29

    Antibody aggregate is a common issue in therapeutic antibodies, which may compromise product efficacy and cause adverse effects. Antibody aggregate level is normally controlled in bioprocessing by polishing steps after Protein A capture. This paper studied the Higher Order Structures (HOS) of antibody aggregates (dimer H1 and H2) and their adsorption on Protein A resin and thus elucidated the mechanism using Protein A capture for enhanced aggregate removal. The HOS of antibody aggregates and their complex with Protein A were characterized using HDX-MS combined with SEC-MALS, Protein Conformational Array (PCA), and molecular modeling. The aggregate size and Protein A binding ratio suggested that H2 has much more compact structure than H1. HDX-MS and PCA further revealed that H1 was formed by single Fab-Fab interaction while H2 formed by Fab-Fab and likely Fc-Fc interaction. On Protein A resin, both the molar binding ratio and the correlation between protein size and ligand distance support that each monomer can only bind one Protein A ligand, while each dimer can bind two ligands, thus resulting in stronger resin binding. Furthermore, dimer H2 binds stronger than dimer H1 due to its compact structure. By integrating biophysical analysis and molecular modeling with process development, this study revealed the antibody aggregate structures and the mechanism of aggregate removal using Protein A chromatography. It also provided a general strategy for in-depth product and process understanding in antibody and other biologics development. PMID:27344283

  6. 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

  7. 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

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

    PubMed

    Saunders, Janet C; Young, Lydia M; Mahood, Rachel A; Jackson, Matthew P; Revill, Charlotte H; Foster, Richard J; Smith, D Alastair; Ashcroft, Alison E; Brockwell, David J; Radford, Sheena E

    2016-02-01

    Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, that is 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 islet amyloid polypeptide 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

  9. [Aggregating ability of seed storage proteins from cereals differing in gluten quality].

    PubMed

    Trufanov, V A; Permiakova, M D; Berezovskaia, E V

    2003-01-01

    The effects of pH, ionic strength, and medium composition on formation of macrocomplexes of seed storage proteins from wheat, rye, and barley have been studied. It has been found that various noncovalent interactions (electrostatic and hydrophobic interactions and hydrogen bonds) are involved in protein aggregation. Their combined action depends significantly on the biochemical nature of storage proteins and on the medium.

  10. 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.

  11. A plant cell model of polyglutamine aggregation: Identification and characterisation of macromolecular and small-molecule anti-protein aggregation activity in vivo.

    PubMed

    Liu, Guobao; Hu, Yueming; Tunnacliffe, Alan; Zheng, Yizhi

    2015-08-10

    In vitro studies have shown that LEA proteins from plants and invertebrates protect and stabilise other proteins under conditions of water stress, suggesting a role in stress tolerance. However, there is little information on LEA protein function in whole plants or plant cells, particularly with respect to their anti-aggregation activity. To address this, we expressed in tobacco BY-2 suspension cells an aggregation-prone protein based on that responsible for Huntington's disease (HD). In HD, abnormally long stretches of polyglutamine (polyQ) in huntingtin (Htt) protein cause aggregation of Htt fragments within cells. We constructed stably transformed BY-2 cell lines expressing enhanced green fluorescent protein (EGFP)-HttQ23 or EGFP-HttQ52 fusion proteins (encoding 23 or 52 glutamine residues, pertaining to the normal and disease states, respectively), as well as an EGFP control. EGFP-HttQ52 protein aggregated in the cytoplasm of transformed tobacco cells, which showed slow growth kinetics; in contrast, EGFP-HttQ23 or EGFP did not form aggregates and cells expressing these constructs grew normally. To test the effect of LEA proteins on protein aggregation in plant cells, we constructed cell lines expressing both EGFP-HttQ52 and LEA proteins (PM1, PM18, ZLDE-2 or AavLEA1) or a sHSP (PM31). Of these, AavLEA1 and PM31 reduced intracellular EGFP-HttQ52 aggregation and alleviated the associated growth inhibition, while PM18 and ZLDE-2 partially restored growth rates. Treatment of EGFP-HttQ52-expressing BY2 cells with the polyphenol epigallocatechin-3-gallate (EGCG) also reduced EGFP-HttQ52 aggregation and improved cell growth rate. The EGFP-HttQ52 cell line therefore has potential for characterising both macromolecular and small molecule inhibitors of protein aggregation in plant cells. PMID:26003885

  12. A plant cell model of polyglutamine aggregation: Identification and characterisation of macromolecular and small-molecule anti-protein aggregation activity in vivo.

    PubMed

    Liu, Guobao; Hu, Yueming; Tunnacliffe, Alan; Zheng, Yizhi

    2015-08-10

    In vitro studies have shown that LEA proteins from plants and invertebrates protect and stabilise other proteins under conditions of water stress, suggesting a role in stress tolerance. However, there is little information on LEA protein function in whole plants or plant cells, particularly with respect to their anti-aggregation activity. To address this, we expressed in tobacco BY-2 suspension cells an aggregation-prone protein based on that responsible for Huntington's disease (HD). In HD, abnormally long stretches of polyglutamine (polyQ) in huntingtin (Htt) protein cause aggregation of Htt fragments within cells. We constructed stably transformed BY-2 cell lines expressing enhanced green fluorescent protein (EGFP)-HttQ23 or EGFP-HttQ52 fusion proteins (encoding 23 or 52 glutamine residues, pertaining to the normal and disease states, respectively), as well as an EGFP control. EGFP-HttQ52 protein aggregated in the cytoplasm of transformed tobacco cells, which showed slow growth kinetics; in contrast, EGFP-HttQ23 or EGFP did not form aggregates and cells expressing these constructs grew normally. To test the effect of LEA proteins on protein aggregation in plant cells, we constructed cell lines expressing both EGFP-HttQ52 and LEA proteins (PM1, PM18, ZLDE-2 or AavLEA1) or a sHSP (PM31). Of these, AavLEA1 and PM31 reduced intracellular EGFP-HttQ52 aggregation and alleviated the associated growth inhibition, while PM18 and ZLDE-2 partially restored growth rates. Treatment of EGFP-HttQ52-expressing BY2 cells with the polyphenol epigallocatechin-3-gallate (EGCG) also reduced EGFP-HttQ52 aggregation and improved cell growth rate. The EGFP-HttQ52 cell line therefore has potential for characterising both macromolecular and small molecule inhibitors of protein aggregation in plant cells.

  13. Glycation by Ascorbic Acid Oxidation Products Leads to the Aggregation of Lens Proteins

    PubMed Central

    Linetsky, Mikhail; Shipova, Ekaterina; Cheng, Rongzhu; Ortwerth, Beryl J.

    2008-01-01

    Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 × 106 up to 3.0 × 108 Da. This compared to proteins ranging from 1.8 × 106 up to 3.6 × 108 Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-14C] ascorbate was incorporated into the aggregate fraction and because CNBH3, which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of α-crystallin, significant aggregation of βH-crystallin, but rapid precipitation of purified βL- and γ-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, α-crystallin prevented the precipitation of the purified βL- and γ-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with βL-, or γ-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from

  14. 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

  15. Effect of Excipients on Liquid-Liquid Phase Separation and Aggregation in Dual Variable Domain Immunoglobulin Protein Solutions.

    PubMed

    Raut, Ashlesha S; Kalonia, Devendra S

    2016-03-01

    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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

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

    SciTech Connect

    Emadi, Saeed Behzadi, Maliheh

    2014-08-08

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

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

    PubMed

    Zeineddine, Rafaa; Yerbury, Justin J

    2015-01-01

    With the onset of the rapidly aging population, the impact of age related neurodegenerative diseases is becoming a predominant health and economic concern. Neurodegenerative diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease (CJD), Parkinson's disease, Huntington's disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) result from the loss of a specific subsets of neurons, which is closely associated with accumulation and deposition of specific protein aggregates. Protein aggregation, or fibril formation, is a well-studied phenomenon that occurs in a nucleation-dependent growth reaction. Recently, there has been a swell of literature implicating protein aggregation and its ability to propagate cell-to-cell in the rapid progression of these diseases. In order for protein aggregation to be kindled in recipient cells it is a requisite that aggregates must be able to be released from one cell and then taken up by others. In this article we will explore the relationship between protein aggregates, their propagation and the role of macropinocytosis in their uptake. We highlight the ability of neurons to undergo stimulated macropinocytosis and identify potential therapeutic targets. PMID:26528186

  4. A desolvation model for trifluoroethanol-induced aggregation of enhanced green fluorescent protein.

    PubMed

    Anderson, Valerie L; Webb, Watt W

    2012-02-22

    Studies of amyloid disease-associated proteins in aqueous solutions containing 2,2,2-trifluoroethanol (TFE) have shown that the formation of structural intermediates is often correlated with enhanced protein aggregation. Here, enhanced green fluorescent protein (EGFP) is used as a model protein system to investigate the causal relationship between TFE-induced structural transitions and aggregation. Using circular dichroism spectroscopy, light scattering measurements, and transmission electron microscopy imaging, we demonstrate that population of a partially α-helical, monomeric intermediate is roughly correlated with the growth of β-sheet-rich, flexible fibrils for acid-denatured EGFP. By fitting our circular dichroism data to a model in which TFE-water mixtures are assumed to be ideal solutions, we show that increasing entropic costs of protein solvation in TFE-water mixtures may both cause the population of the intermediate state and increase aggregate production. Tertiary structure and electrostatic repulsion also impede aggregation. We conclude that initiation of EGFP aggregation in TFE likely involves overcoming of multiple protective factors, rather than stabilization of aggregation-prone structural elements.

  5. Formation of assemblies on cell membranes by secreted proteins: molecular studies of free λ light chain aggregates found on the surface of myeloma cells.

    PubMed

    Hutchinson, Andrew T; Malik, Ansha; Berkahn, Mark B; Agostino, Mark; To, Joyce; Tacchi, Jessica L; Djordjevic, Steven P; Turnbull, Lynne; Whitchurch, Cynthia B; Edmundson, Allen B; Jones, Darren R; Raison, Robert L; Ramsland, Paul A

    2013-09-15

    We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.

  6. 4D imaging of protein aggregation in live cells.

    PubMed

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

  7. Proteome response at the edge of protein aggregation

    PubMed Central

    Sanchez de Groot, Natalia; Gomes, Ricardo A.; Villar-Pique, Anna; Babu, M. Madan; Coelho, Ana Varela; Ventura, Salvador

    2015-01-01

    Proteins adopt defined structures and are crucial to most cellular functions. Their misfolding and aggregation is associated with numerous degenerative human disorders such as type II diabetes, Huntington's or Alzheimer's diseases. Here, we aim to understand why cells promote the formation of protein foci. Comparison of two amyloid-β-peptide variants, mostly insoluble but differently recruited by the cell (inclusion body versus diffused), reveals small differences in cell fitness and proteome response. We suggest that the levels of oxidative stress act as a sensor to trigger protein recruitment into foci. Our data support a common cytoplasmic response being able to discern and react to the specific properties of polypeptides. PMID:25673330

  8. Proteome response at the edge of protein aggregation.

    PubMed

    Sanchez de Groot, Natalia; Gomes, Ricardo A; Villar-Pique, Anna; Babu, M Madan; Coelho, Ana Varela; Ventura, Salvador

    2015-02-01

    Proteins adopt defined structures and are crucial to most cellular functions. Their misfolding and aggregation is associated with numerous degenerative human disorders such as type II diabetes, Huntington's or Alzheimer's diseases. Here, we aim to understand why cells promote the formation of protein foci. Comparison of two amyloid-β-peptide variants, mostly insoluble but differently recruited by the cell (inclusion body versus diffused), reveals small differences in cell fitness and proteome response. We suggest that the levels of oxidative stress act as a sensor to trigger protein recruitment into foci. Our data support a common cytoplasmic response being able to discern and react to the specific properties of polypeptides. PMID:25673330

  9. Self-propagation of pathogenic protein aggregates in neurodegenerative diseases.

    PubMed

    Jucker, Mathias; Walker, Lary C

    2013-09-01

    For several decades scientists have speculated that the key to understanding age-related neurodegenerative disorders may be found in the unusual biology of the prion diseases. Recently, owing largely to the advent of new disease models, this hypothesis has gained experimental momentum. In a remarkable variety of diseases, specific proteins have been found to misfold and aggregate into seeds that structurally corrupt like proteins, causing them to aggregate and form pathogenic assemblies ranging from small oligomers to large masses of amyloid. Proteinaceous seeds can therefore serve as self-propagating agents for the instigation and progression of disease. Alzheimer's disease and other cerebral proteopathies seem to arise from the de novo misfolding and sustained corruption of endogenous proteins, whereas prion diseases can also be infectious in origin. However, the outcome in all cases is the functional compromise of the nervous system, because the aggregated proteins gain a toxic function and/or lose their normal function. As a unifying pathogenic principle, the prion paradigm suggests broadly relevant therapeutic directions for a large class of currently intractable diseases.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. The Interplay between PolyQ and Protein Context Delays Aggregation by Forming a Reservoir of Protofibrils

    PubMed Central

    Thomas, David J.; San Biagio, Pier Luigi; Pastore, Annalisa

    2006-01-01

    Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway. PMID:17205115

  15. Impact of asymmetrical flow field-flow fractionation on protein aggregates stability.

    PubMed

    Bria, Carmen R M; Williams, S Kim Ratanathanawongs

    2016-09-23

    The impact of asymmetrical flow field-flow fractionation (AF4) on protein aggregate species is investigated with the aid of multiangle light scattering (MALS) and dynamic light scattering (DLS). The experimental parameters probed in this study include aggregate stability in different carrier liquids, shear stress (related to sample injection), sample concentration (during AF4 focusing), and sample dilution (during separation). Two anti-streptavidin (anti-SA) IgG1 samples composed of low and high molar mass (M) aggregates are subjected to different AF4 conditions. Aggregates suspended and separated in phosphate buffer are observed to dissociate almost entirely to monomer. However, aggregates in citric acid buffer are partially stable with dissociation to 25% and 5% monomer for the low and high M samples, respectively. These results demonstrate that different carrier liquids change the aggregate stability and low M aggregates can behave differently than their larger counterparts. Increasing the duration of the AF4 focusing step showed no significant changes in the percent monomer, percent aggregates, or the average Ms in either sample. Syringe-induced shear related to sample injection resulted in an increase in hydrodynamic diameter (dh) as measured by batch mode DLS. Finally, calculations showed that dilution during AF4 separation is significantly lower than in size exclusion chromatography with dilution occurring mainly at the AF4 channel outlet and not during the separation. This has important ramifications when analyzing aggregates that rapidly dissociate (<∼2s) upon dilution as the size calculated by AF4 theory may be more accurate than that measured by online DLS. Experimentally, the dhs determined by online DLS generally agreed with AF4 theory except for the more well retained larger aggregates for which DLS showed smaller sizes. These results highlight the importance of using AF4 retention theory to understand the impacts of dilution on analytes. PMID

  16. Chaperone potential of Pulicaria undulata extract in preventing aggregation of stressed proteins.

    PubMed

    Ghahghaei, Arezou; Valizadeh, Jafar; Nazari, Shahrzad; Ravandeh, Mehdi

    2014-06-01

    This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.

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

    NASA Astrophysics Data System (ADS)

    Taniguchi, Y.; Okuno, A.; Kato, M.

    2010-03-01

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

  18. Using intramolecular disulfide bonds in tau protein to deduce structural features of aggregation-resistant conformations.

    PubMed

    Walker, Sophie; Ullman, Orly; Stultz, Collin M

    2012-03-16

    Because tau aggregation likely plays a role in a number of neurodegenerative diseases, understanding the processes that affect tau aggregation is of considerable importance. One factor that has been shown to influence the aggregation propensity is the oxidation state of the protein itself. Tau protein, which contains two naturally occurring cysteine residues, can form both intermolecular disulfide bonds and intramolecular disulfide bonds. Several studies suggest that intermolecular disulfide bonds can promote tau aggregation in vitro. By contrast, although there are data to suggest that intramolecular disulfide bond formation retards tau aggregation in vitro, the precise mechanism underlying this observation remains unclear. While it has been hypothesized that a single intramolecular disulfide bond in tau leads to compact conformations that cannot form extended structure consistent with tau fibrils, there are few data to support this conjecture. In the present study we generate oxidized forms of the truncation mutant, K18, which contains all four microtubule binding repeats, and isolate the monomeric fraction, which corresponds to K18 monomers that have a single intramolecular disulfide bond. We study the aggregation propensity of the oxidized monomeric fraction and relate these data to an atomistic model of the K18 unfolded ensemble. Our results argue that the main effect of intramolecular disulfide bond formation is to preferentially stabilize conformers within the unfolded ensemble that place the aggregation-prone tau subsequences, PHF6* and PHF6, in conformations that are inconsistent with the formation of cross-β-structure. These data further our understanding of the precise structural features that retard tau aggregation.

  19. Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering.

    PubMed

    Barnett, Gregory V; Drenski, Michael; Razinkov, Vladimir; Reed, Wayne F; Roberts, Christopher J

    2016-10-15

    Parallel temperature initial rates (PTIR) from chromatographic separation of aggregating protein solutions are combined with continuous simultaneous multiple sample light scattering (SMSLS) to make quantitative deductions about protein aggregation kinetics and mechanisms. PTIR determines the rates at which initially monomeric proteins are converted to aggregates over a range of temperatures, under initial-rate conditions. Using SMSLS for the same set of conditions provides time courses of the absolute Rayleigh scattering ratio, IR(t), from which a potentially different measure of aggregation rates can be quantified. The present report compares these measures of aggregation rates across a range of solution conditions that result in different aggregation mechanisms for anti-streptavidin (AS) immunoglobulin gamma-1 (IgG1). The results illustrate how the two methods provide complementary information when deducing aggregation mechanisms, as well as cases where they provide new mechanistic details that were not possible to deduce in previous work. Criteria are presented for when the two techniques are expected to give equivalent results for quantitative rates, the potential limitations when solution non-idealities are large, as well as a comparison of the temperature dependence of AS-IgG1 aggregation rates with published data for other antibodies. PMID:27510552

  20. Manipulation of unfolding-induced protein aggregation by peptides selected for aggregate-binding ability through phage display library screening.

    PubMed

    Kundu, Bishwajit; Shukla, Anshuman; Guptasarma, Purnananda

    2002-03-01

    A phage-displayed library of peptides (12-mer) was screened for the ability to bind to thermally aggregated bovine carbonic anhydrase (BCA), with a view toward examining whether peptides possessing this ability might bind to partially structured intermediates on the protein's unfolding pathway and, therefore, constitute useful tools for manipulation of the kinetic partitioning of molecules between the unfolded and aggregated states. Two peptides [N-HPSTMGLRTMHP-C and N-TPSAWKTALVKA-C] were identified and tested. While neither showed thermal aggregation autonomously, both peptides individually elicited remarkable increases in the levels of thermal aggregation of BCA. A possible explanation is that both peptides bind to surfaces on molten BCA that are not directly involved in aggregation. Such binding could slow down interconversions between folded and unfolded states and stabilize aggregation-prone intermediate(s) to make them more prone to aggregation, while failing to achieve any steric prevention of aggregation. The approach has the potential of yielding useful aggregation-aiding/inhibiting agents, and may provide clues to whether amorphous aggregates are "immobilized" forms of folding intermediates. PMID:11866450

  1. 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.

  2. [Seed, aggregation and propagation of abnormal proteins could explain neurodegeneration?].

    PubMed

    Murayama, Shigeo

    2011-11-01

    Braak proposed propagation staging paradigm of Lewy- related alpha-synucleinopathy, which starts from medulla oblongata and extends rostrally to neocortex. Since this propagation shares that of bovine spongiformic encephalopathy, alpha- synuclein- prionopathy hypothesis was presented and augumented by pathological reports of Lewy body pathology in fetal tansplants of midbrain to patients with Parkinson disease (PD). The prionopathy hypothesis expanded to include tau and TDP- 43, is now receiving considerable attention world wide. Laterality of clinical symptoms can be explained with this hypothesis in PD, amyotrophic lateral sclerosis- TDP43, frontotemoral lobar degeneration- semantic dementia- TDP43 and tauopathy including corticobasal degeneration and argyrophilic grain dementia. Major cons of prionopathy hypothesis is how to explain cell to cell transmission of intracellular amyloid- like proteins. Several clinical and experimental data are now accumulated to answer this question. The difference in speed of spread between prion disease and neurodegenerative disease could be explained by aggregation size of abnormal proteins. The hypothesis could also explain glinoneuronal interaction, which is receiving another hot topic of neurodeneration. We propose that seed, aggregation propagation of abnormal protein should form one factor of clinical progression of neurodegenerative diseases and can be a therapeutic targets in future research.

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

    PubMed

    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. PMID:23224114

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

    PubMed

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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.

  7. Temporal changes in aggregation: a laboratory study.

    PubMed

    Scott, M E

    1987-06-01

    Changes in the variance to mean ratio and the parameter k of the negative binomial distribution were used to study temporal changes in the degree of aggregation of the monogenean parasite, Gyrodactylus turnbulli in free-running laboratory populations of the guppy, Poecilia reticulata. The parasite undergoes recurrent epidemic cycles in the host population under conditions of regular immigration of uninfected guppies. During the early phase of the epidemic, heterogeneity among fish together with direct reproduction are thought to contribute to the increasing degree of aggregation. During the increasing phase of the epidemic cycle, parasites become increasingly aggregated in the host population, presumably because of the direct reproduction of the parasite on the surface of a single host. As the peak prevalance and abundance are approached, the parasites become less aggregated with lowest clumping occurring during the declining phase of the cycle. This is thought to be a function of density-dependent death of infected hosts, and density-dependent reduction in parasite survival and reproduction on hosts that recover from infection. This study clearly indicates that the variance to mean ratio and the parameter k of the negative binomial distribution do not quantify the same aspect of the frequency distribution. It is suggested that the variance to mean ratio is a better measure when the prevalence and/or mean burden are changing and when the tail of the distribution is of particular interest, and that k may be a preferred parameter when the zero class or the lightly infected hosts are of primary interest. PMID:3614993

  8. An experimental study of dense aerosol aggregations

    NASA Astrophysics Data System (ADS)

    Dhaubhadel, Rajan

    We demonstrated that an aerosol can gel. This gelation was then used for a one-step method to produce an ultralow density porous carbon or silica material. This material was named an aerosol gel because it was made via gelation of particles in the aerosol phase. The carbon and silica aerosol gels had high specific surface area (200--350 sq m2/g for carbon and 300--500 sq m2/g for silica) and an extremely low density (2.5--6.0 mg/cm3), properties similar to conventional aerogels. Key aspects to form a gel from an aerosol are large volume fraction, ca. 10-4 or greater, and small primary particle size, 50 nm or smaller, so that the gel time is fast compared to other characteristic times. Next we report the results of a study of the cluster morphology and kinetics of a dense aggregating aerosol system using the small angle light scattering technique. The soot particles started as individual monomers, ca. 38 nm radius, grew to bigger clusters with time and finally stopped evolving after spanning a network across the whole system volume. This spanning is aerosol gelation. The gelled system showed a hybrid morphology with a lower fractal dimension at length scales of a micron or smaller and a higher fractal dimension at length scales greater than a micron. The study of the kinetics of the aggregating system showed that when the system gelled, the aggregation kernel homogeneity lambda attained a value 0.4 or higher. The magnitude of the aggregation kernel showed an increase with increasing volume fraction. We also used image analysis technique to study the cluster morphology. From the digitized pictures of soot clusters the cluster morphology was determined by two different methods: structure factor and perimeter analysis. We find a hybrid, superaggregate morphology characterized by a fractal dimension of Df ≈ to 1.8 between the monomer size, ca. 50 nm, and 1 mum micron and Df ≈ to 2.6 at larger length scales up to ˜ 10 mum. The superaggregate morphology is a

  9. 'Insulator bodies' are aggregates of proteins but not of insulators.

    PubMed

    Golovnin, Anton; Melnikova, Larisa; Volkov, Ilya; Kostuchenko, Margarita; Galkin, Alexander V; Georgiev, Pavel

    2008-05-01

    Chromatin insulators are thought to restrict the action of enhancers and silencers. The best-known insulators in Drosophila require proteins such as Suppressor of Hairy wing (Su(Hw)) and Modifier of mdg4 (Mod(mdg4)) to be functional. The insulator-related proteins apparently colocalize as nuclear speckles in immunostained cells. It has been asserted that these speckles are 'insulator bodies' of many Su(Hw)-insulator DNA sites held together by associated proteins, including Mod(mdg4). As we show here using flies, larvae and S2 cells, a mutant Mod(mdg4) protein devoid of the Q-rich domain supports the function of Su(Hw)-dependent insulators and efficiently binds to correct insulator sites on the chromosome, but does not form or enter the Su(Hw)-marked nuclear speckles; conversely, the latter accumulate another (C-truncated) Mod(mdg4) mutant that cannot interact with Su(Hw) or with the genuine insulators. Hence, it is not the functional genomic insulators but rather aggregated proteins that make the so-called 'insulator bodies'.

  10. The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation*

    PubMed Central

    Entzminger, Kevin C.; Chang, Christine; Myhre, Ryan O.; McCallum, Katie C.; Maynard, Jennifer A.

    2013-01-01

    The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp co-expression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression, and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp co-expression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence. PMID:22650963

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

    PubMed Central

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

    2016-01-01

    Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases. PMID:27230476

  12. 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

  13. Lumenal peroxisomal protein aggregates are removed by concerted fission and autophagy events.

    PubMed

    Manivannan, Selvambigai; de Boer, Rinse; Veenhuis, Marten; van der Klei, Ida J

    2013-07-01

    We demonstrated that in the yeast Hansenula polymorpha peroxisome fission and degradation are coupled processes that are important to remove intra-organellar protein aggregates. Protein aggregates were formed in peroxisomes upon synthesis of a mutant catalase variant. We showed that the introduction of these aggregates in the peroxisomal lumen had physiological disadvantages as it affected growth and caused enhanced levels of reactive oxygen species. Formation of the protein aggregates was followed by asymmetric peroxisome fission to separate the aggregate from the mother organelle. Subsequently, these small, protein aggregate-containing organelles were degraded by autophagy. In line with this observation we showed that the degradation of the protein aggregates was strongly reduced in dnm1 and pex11 cells in which peroxisome fission is reduced. Moreover, this process was dependent on Atg1 and Atg11. PMID:23614977

  14. Expression of Cardamom mosaic virus coat protein in Escherichia coli and its assembly into filamentous aggregates.

    PubMed

    Jacob, Thomas; Usha, R

    2002-06-01

    Cardamom mosaic virus (CdMV), a member of the genus Macluravirus of Potyviridae, causes a mosaic disease in cardamom. A polyclonal antiserum was raised against the purified virus and IgG was prepared. Electron microscopic studies on the purified virus showed flexuous filamentous particles of approximately 800 nm in length, typical of members of Potyviridae. The coat protein (CP) encoding sequence of the virus was expressed in Escherichia coli and the protein purified by affinity chromatography under denaturing conditions. The viral nature of the expressed CP was confirmed by positive reaction with anti CdMV IgG in a Western blot. The expressed CP aggregated irreversibly upon renaturation at concentrations above 0.07 mg/ml. The expression of the CP led to the formation of filamentous aggregates in E. coli as observed by immuno-gold electron microscopy. The filamentous aggregates were of 100-150 nm in length. Immuno-capture RT-PCR confirmed the absence of coat protein mRNA in the filamentous aggregates. Deletion mutations, which were expected to inhibit virus assembly, were introduced in the core region of the coat protein. However, these mutations did not improve the solubility of the CP in non-denaturing buffers.

  15. Predictive response surface model for heat-induced rheological changes and aggregation of whey protein concentrate.

    PubMed

    Alvarez, Pedro A; Emond, Charles; Gomaa, Ahmed; Remondetto, Gabriel E; Subirade, Muriel

    2015-02-01

    Whey proteins are now far more than a by-product of cheese processing. In the last 2 decades, food manufacturers have developed them as ingredients, with the dairy industry remaining as a major user. For many applications, whey proteins are modified (denatured) to alter their structure and functional properties. The objective of this research was to study the influence of 85 to 100 °C, with protein concentration of 8% to 12%, and treatment times of 5 to 30 min, while measuring rheological properties (storage modulus, loss modulus, and complex viscosity) and aggregation (intermolecular beta-sheet formation) in dispersions of whey protein concentrate (WPC). A Box-Behnken Response Surface Methodology modeled the heat denaturation of liquid sweet WPC at 3 variables and 3 levels. The model revealed a very significant fit for viscoelastic properties, and a lesser fit for protein aggregation, at temperatures not previously studied. An exponential increase of rheological parameters was governed by protein concentration and temperature, while a modest linear relationship of aggregation was governed by temperature. Models such as these can serve as valuable guides to the ingredient and dairy industries to develop target products, as whey is a major ingredient in many functional foods.

  16. Effect of Electrostatics on Aggregation of Prion Protein Sup35 Peptide

    PubMed Central

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

    2012-01-01

    Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. 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 the 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 hours, whereas the lag time decreases ~5 times when ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 hours under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. 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 on dissociation of transient peptide dimers. PMID:22466073

  17. 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.

  18. Simple off-lattice model to study the folding and aggregation of peptides

    NASA Astrophysics Data System (ADS)

    Combe, Nicolas; Frenkel, Daan

    We present a numerical study of a new protein model. This off-lattice model takes into account both the hydrogen bonds and the amino-acid interactions. It reproduces the folding of a small protein (peptide): morphological analysis of the conformations at low temperature shows two well-known substructures α-helix and β-sheet depending on the chosen sequence. The folding pathway in the scope of this model is studied through a free-energy analysis. We then study the aggregation of proteins. Proteins in the aggregate are mainly bound via hydrogen bonds. Performing a free-energy analysis we show that the addition of a peptide to such an aggregate is not favourable. We qualitatively reproduce the abnormal aggregation of proteins in prion diseases.

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

    SciTech Connect

    Takanashi, Keisuke; Yamaguchi, Atsushi

    2014-09-26

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

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

    PubMed

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

    2016-08-19

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

  2. Synthesis of several membrane proteins during developmental aggregation in Myxococcus xanthus.

    PubMed

    Orndorff, P E; Dworkin, M

    1982-01-01

    We have examined the pattern of synthesis of several membrane proteins during the aggregation phase of development in Myxococcus xanthus. Development was initiated by plating vegetative cells on polycarbonate filters placed on top of an agar medium that supported fruiting body formation. At various times during aggregation a filter was removed, the cells were pulse-labeled with [35S]methionine, and the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rate of synthesis of numerous individual proteins changed during aggregation; we concentrated on six whose pattern of synthesis was greatly altered during aggregation. The rate of synthesis of five of the six proteins increased considerably during aggregation; that of the remaining protein was curtailed and appeared to be regulated by nutrient conditions. Three of the five major membrane proteins that increased during aggregation had a unique pattern of synthesis that was displayed only under conditions that are are required for development - high cell density, nutrient depletion, and a solid (agar) surface. The remaining two proteins were not unique to development; the appearance of one protein could be induced under conditions of high cell density, whereas the other could be induced by placing the cells on a solid agar surface. All of the five major proteins that appeared during development did so during the preaggregation stage, and the synthesis of four of the five proteins appeared to be curtailed late in aggregation. The synthesis of the remaining protein continued throughout aggregation. PMID:6798022

  3. 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

  4. 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.

  5. 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

  6. 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. PMID:26492083

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

    PubMed Central

    Stefani, Massimo; Rigacci, Stefania

    2013-01-01

    Amyloid aggregation is a hallmark of several degenerative diseases affecting the brain or peripheral tissues, whose intermediates (oligomers, protofibrils) and final mature fibrils display different toxicity. Consequently, compounds counteracting amyloid aggregation have been investigated for their ability (i) to stabilize toxic amyloid precursors; (ii) to prevent the growth of toxic oligomers or speed that of fibrils; (iii) to inhibit fibril growth and deposition; (iv) to disassemble preformed fibrils; and (v) to favor amyloid clearance. Natural phenols, a wide panel of plant molecules, are one of the most actively investigated categories of potential amyloid inhibitors. They are considered responsible for the beneficial effects of several traditional diets being present in green tea, extra virgin olive oil, red wine, spices, berries and aromatic herbs. Accordingly, it has been proposed that some natural phenols could be exploited to prevent and to treat amyloid diseases, and recent studies have provided significant information on their ability to inhibit peptide/protein aggregation in various ways and to stimulate cell defenses, leading to identify shared or specific mechanisms. In the first part of this review, we will overview the significance and mechanisms of amyloid aggregation and aggregate toxicity; then, we will summarize the recent achievements on protection against amyloid diseases by many natural phenols. PMID:23765219

  8. The yeast peroxiredoxin Tsa1 protects against protein-aggregate-induced oxidative stress

    PubMed Central

    Weids, Alan J.; Grant, Chris M.

    2014-01-01

    ABSTRACT Peroxiredoxins are ubiquitous thiol-specific proteins that have multiple functions in stress protection, including protection against oxidative stress. Tsa1 is the major yeast peroxiredoxin and we show that it functions as a specific antioxidant to protect the cell against the oxidative stress caused by nascent-protein misfolding and aggregation. Yeast mutants lacking TSA1 are sensitive to misfolding caused by exposure to the proline analogue azetidine-2-carboxylic acid (AZC). AZC promotes protein aggregation, and its toxicity to a tsa1 mutant is caused by the production of reactive oxygen species (ROS). The generation of [rho0] cells, which lack mitochondrial DNA, rescues the tsa1 mutant AZC sensitivity, indicating that mitochondria are the source of ROS. Inhibition of nascent-protein synthesis with cycloheximide prevents AZC-induced protein aggregation and abrogates ROS generation, confirming that the formation of aggregates causes ROS production. Protein aggregation is accompanied by mitochondrial fragmentation, and we show that Tsa1 localises to the sites of protein aggregation. Protein aggregates are formed adjacent to mitochondria, and our data indicate that active mitochondria generate ROS. These data indicate a new role for peroxiredoxins in protecting against ROS that are generated as a result of protein misfolding and aggregate formation. PMID:24424024

  9. Structure and flow of dense suspensions of protein fractal aggregates in comparison with microgels.

    PubMed

    Inthavong, Walailuk; Kharlamova, Anna; Chassenieux, Christophe; Nicolai, Taco

    2016-03-14

    Solutions of the globular whey protein β-lactoglobulin (β-lg) were heated at different protein concentrations leading to the formation of polydisperse fractal aggregates with different average sizes. The structure of the solutions was analyzed with light scattering as a function of the protein concentration. The osmotic compressibility and the dynamic correlation length decreased with increasing concentration and became independent of the aggregate size in dense suspensions. The results obtained for different aggregate sizes could be superimposed after normalizing the concentration with the overlap concentration. Dense suspensions of fractal protein aggregates are strongly interpenetrated and can be visualized as an ensemble of fractal 'blobs'. The viscosity of the heated β-lg solutions increased extremely sharply above 80 g L(-1) and diverged at 98 g L(-1), mainly due to the sharply increasing aggregate size. At a fixed aggregate size, the viscosity increased initially exponentially with increasing concentration and then diverged. The increase was stronger when the aggregates were larger, but the dependence of the viscosity on the aggregate size was weaker than that of the osmotic compressibility and the dynamic correlation length. The concentration dependence of the viscosity of solutions of fractal β-lg aggregates is much stronger than that of homogeneous β-lg microgels. The behavior of fractal aggregates formed by whey protein isolates was similar. PMID:26864954

  10. Structural insights into the aggregation behavior of Murraya koenigii miraculin-like protein below pH 7.5.

    PubMed

    Selvakumar, Purushotham; Sharma, Nidhi; Tomar, Prabhat Pratap Singh; Kumar, Pravindra; Sharma, Ashwani Kumar

    2014-05-01

    Murraya koenigii miraculin-like protein (MKMLP) gradually precipitates below pH 7.5. Here, we explore the basis for this aggregation by identifying the aggregation-prone regions via comparative analysis of crystal structures acquired at several pH values. The prediction of aggregation-prone regions showed the presence of four short peptides either in beta sheets or loops on surface of the protein. These peptides were distributed in two patches far apart on the surface. Comparison of crystal structures of MKMLP, determined at 2.2 Å resolution in pH 7.0 and 4.6 in the present study and determined at 2.9 Å in pH 8.0 in an earlier reported study, reveal subtle conformational differences resulting in gradual exposure of aggregation-prone regions. As the pH is lowered, there are alterations in ionic interactions within the protein interactions of the chain with water molecules and exposure of hydrophobic residues. The analysis of symmetry-related molecular interfaces involving one patch revealed shortening of nonpolar intermolecular contacts as the pH decreased. In particular, a decrease in the intermolecular distance between Trp103 of the aggregation-prone peptide WFITTG (103-108) unique to MLPs was observed. These results demonstrated that aggregation occurs due to the cumulative effect of the changes in interactions in two aggregation-prone defined regions. PMID:24265134

  11. 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

  12. 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

  13. The Copper Metabolism MURR1 Domain Protein 1 (COMMD1) Modulates the Aggregation of Misfolded Protein Species in a Client-Specific Manner

    PubMed Central

    Vonk, Willianne I. M.; Kakkar, Vaishali; Bartuzi, Paulina; Jaarsma, Dick; Berger, Ruud; Hofker, Marten H.; Klomp, Leo W. J.; Wijmenga, Cisca; Kampinga, Harm H.; van de Sluis, Bart

    2014-01-01

    The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson’s disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington’s disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies. PMID:24691167

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

    PubMed

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

    2015-05-01

    Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the relationship(s) among protein-protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate. Aggregate size and shape were determined using a combination of laser light scattering and small-angle neutron or X-ray scattering. Protein-protein interactions were quantified in terms of the protein-protein Kirkwood-Buff integral (G22) determined from static light scattering and in terms of the protein effective charge (Zeff) measured using electrophoretic light scattering. Changing from citrate to acetate resulted in significantly different protein-protein interactions as a function of pH for low NaCl concentrations when the protein displayed positive Zeff. Overall, the results suggest that electrostatic repulsions between proteins were lessened because of preferential accumulation of citrate anions, compared to acetate anions, at the protein surface. The predominant aggregation mechanisms correlated well with G22, indicating that ion-specific effects beyond traditional mean-field descriptions of electrostatic protein-protein interactions are important for predicting qualitative shifts in protein aggregation state diagrams. Interestingly, while solution conditions dictated which mechanisms predominated, aggregate average molecular weight and size displayed a common scaling behavior across both citrate- and acetate-based systems. PMID:25885209

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

    NASA Astrophysics Data System (ADS)

    Mezzenga, Raffaele; Fischer, Peter

    2013-04-01

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

  16. Protein aggregation in E. coli : short term and long term effects of nutrient density.

    PubMed

    Baig, Ulfat I; Bhadbhade, Bharati J; Mariyam, Dincy; Watve, Milind G

    2014-01-01

    During exponential growth some cells of E. coli undergo senescence mediated by asymmetric segregation of damaged components, particularly protein aggregates. We showed previously that functional cell division asymmetry in E. coli was responsive to the nutritional environment. Short term exposure as well as long term selection in low calorie environments led to greater cell division symmetry and decreased frequency of senescent cells as compared to high calorie environments. We show here that long term selection in low nutrient environment decreased protein aggregation as revealed by fluorescence microscopy and proportion of insoluble proteins. Across selection lines protein aggregation was correlated significantly positively with the RNA content, presumably indicating metabolic rate. This suggests that the effects of caloric restriction on cell division symmetry and aging in E. coli may work via altered protein handling mechanisms. The demonstrable effects of long term selection on protein aggregation suggest that protein aggregation is an evolvable phenomenon rather than being a passive inevitable process. The aggregated proteins progressively disappeared on facing starvation indicating degradation and recycling demonstrating that protein aggregation is a reversible process in E. coli. PMID:25210787

  17. Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons

    PubMed Central

    Trushina, Eugenia; Rana, Sandeep; McMurray, Cynthia T; Hua, Duy H

    2009-01-01

    Background Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-β (Aβ) aggregates in cell and mouse models representing Alzheimer's Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD. Results TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons. Conclusion We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD. PMID:19586540

  18. Milk protein suspensions enriched with three essential minerals: Physicochemical characterization and aggregation induced by a novel enzymatic pool.

    PubMed

    Lombardi, Julia; Spelzini, Darío; Corrêa, Ana Paula Folmer; Brandelli, Adriano; Risso, Patricia; Boeris, Valeria

    2016-04-01

    Structural changes of casein micelles and their aggregation induced by a novel enzymatic pool isolated from Bacillus spp. in the presence of calcium, magnesium or zinc were investigated. The effect of cations on milk protein structure was studied using fluorescence and dynamic light scattering. In the presence of cations, milk protein structure rearrangements and larger casein micelle size were observed. The interaction of milk proteins with zinc appears to be of a different nature than that with calcium or magnesium. Under the experimental conditions assayed, the affinity of each cation for some groups present in milk proteins seems to play an important role, besides electrostatic interaction. On the other hand, the lowest aggregation times were achieved at the highest calcium and zinc concentrations (15 mM and 0.25 mM, respectively). The study found that the faster the aggregation of casein micelles, the less compact the gel matrix obtained. Cation concentrations affected milk protein aggregation kinetics and the structure of the aggregates formed.

  19. Hybrid molecules synergistically acting against protein aggregation diseases.

    PubMed

    Korth, Carsten; Klingenstein, Ralf; Müller-Schiffmann, Andreas

    2013-01-01

    An emerging common feature of the age-associated neurodegenerative disorders like Alzheimer's disease (AD) and Creutzfeldt-Jakob disease (CJD) is the ability of many disease-associated protein aggregates to induce conversion of a normal counterpart conformer leading to an acceleration of disease progression. Curative pharmacotherapy has not been achieved so far despite successes in elucidating pathomechanisms. Here, we review the pharmaceutical strategy of generating hybrid compounds, i.e. compounds consisting of several independently acting moieties with synergistic effects, on key molecular players in AD and CJD. For prion diseases, we review hybrid compounds consisting of two different heterocyclic compounds, their synergistic effects on prion replication in a cell culture model and their ability to prolong survival of experimentally prion-infected mice in vivo. While a combination therapy of several antiprion compounds including quinacrine, clomipramine, simvastatin and tocopherol prolonged survival time to 10-25%, administration of hybrid compound quinpramine alone, a chimera of acridine and iminodibenzyl scaffolds, led to 10% survival time extension. For AD, we review a hybrid compound consisting of an Aβ recognizing D-peptide fused to a small molecule β-sheet breaker, an aminopyrazole. This molecule was able to diminish Aβ oligomers in cell culture and significantly decrease synaptotoxicity as measured by miniature excitatory postsynaptic responses in vitro. Hybrid compounds can dramatically increase potency of their single moieties and lead to novel functions when they act in a simultaneous or sequential manner thereby revealing synergistic properties. Their systematic generation combining different classes of compounds from peptides to small molecules has the potential to significantly accelerate drug discovery. PMID:24059335

  20. DO NOT DROP: MECHANICAL SHOCK IN VIALS CAUSES CAVITATION, PROTEIN AGGREGATION AND PARTICLE FORMATION

    PubMed Central

    Randolph, Theodore W.; Schiltz, Elise; Sederstrom, Donn; Steinmann, Daniel; Mozziconacci, Olivier; Schöneich, Christian; Freund, Erwin; Ricci, Margaret S.; Carpenter, John F.; Lengsfeld, Corrine S.

    2014-01-01

    Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 μs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond timecourse. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues. PMID:25418950

  1. NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    PubMed Central

    Nivon, Mathieu; Fort, Loïc; Muller, Pascale; Richet, Emma; Simon, Stéphanie; Guey, Baptiste; Fournier, Maëlenn; Arrigo, André-Patrick; Hetz, Claudio; Atkin, Julie D.; Kretz-Remy, Carole

    2016-01-01

    During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called “protein conformational diseases,” such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases. PMID:27075172

  2. 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

    2014-01-01

    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.

  3. Interaction of polyanions with basic proteins, 2(a) : influence of complexing polyanions on the thermo-aggregation of oligomeric enzymes.

    PubMed

    Shalova, Irina N; Asryants, Regina A; Sholukh, Mikhail V; Saso, Luciano; Kurganov, Boris I; Muronetz, Vladimir I; Izumrudov, Vladimir A

    2005-12-15

    The ability of synthetic polyanions to suppress thermo-aggregation of the oligomeric enzymes (glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and aspartate aminotransferase) has been established. The ability of the polyanions to reduce the thermo-aggregation increased in the order poly(methacrylic acid) < poly(acrylic acid) < sodium poly(styrene sulphonate), which agreed well with the increase, in the same order, of the charge density of the chains. The lengthening of the chains, as well as the rise in their relative content, resulted in an increase of the ability to reduce thermo-aggregation, mentioned above. Complete prevention of the enzyme aggregation was achieved when highly charged polyanions of a relatively high degree of polymerization were used in a concentration sufficient to solubilize the protein. Complexing with the polyanions prevented thermo-aggregation of the enzymes, but not their thermo-denaturation. The adverse effect of the complexing polyanions on the catalytic activity was reduced by the addition of a synthetic polycation, which resulted in a significant reactivation (up to 40%) of the enzyme. The possibility of preventing the thermo-aggregation of enzyme molecules and then partly restoring the enzyme activity, appears to be of particular interest when studying the aggregation mechanism of proteins that are prone to form the amyloid structures responsible for the development of neurodegenerative diseases like Alzheimer's disease, bovine spongiform encephalopathy and Huntington disease. This finding can also be considered as an important step in the creation of artificial chaperones.

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

    PubMed

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

    2015-01-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm; 2000-250 μm; 250-53 μm; and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000-250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture. PMID:26423355

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm 2000-250 μm 250-53 μm and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000-250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture.

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

    PubMed Central

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

    2015-01-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm; 2000–250 μm; 250–53 μm; and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000–250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture. PMID:26423355

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

    PubMed Central

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

    2010-01-01

    How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under 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 non-aggregated 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° 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 knock-out, LH1 knock-out, 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 non-aggregated LH2 proteins in the photosynthetic membranes. PMID:19572507

  8. The Involvement of Microbially Derived Extracellular Proteins in Nanoparticle Formation and Aggregation

    NASA Astrophysics Data System (ADS)

    Pearce, C. I.; Moreau, J. W.

    2007-12-01

    While humans are newcomers to the field of nanoscience, microbes have been synthesizing functional nanoscale structures for billions of years. Bacteria have evolved the capability to produce proteins that can unite cellular processes with inorganic substrates, transfer electrons, template biomineralization, and facilitate adhesion. Biominerals are commonly nano-composite materials in which biomolecules such as proteins and/or polysaccharides act as a template to direct nanoparticle nucleation and growth. Understanding the capability of microbes to form nanoparticles and influence their reactive transport properties offers potential for bioremediation and materials synthesis applications. The identification of biomolecules and functional groups associated with biogenic nanoparticle formation in both environmental and laboratory systems is the objective of our research. Two such systems in which protein-nanoparticle interactions were studied are discussed. First, the biogenic reduction of selenium oxyanions to Se0 was studied in pure cultures of Veillonella atypica, Bacillus selenitireducens and Geobacter sulfurreducens. Biogenic Se0 nanostructures were observed as spherical, fibrillar, granular or amorphous aggregates, both in the cytoplasm or periplasmic space and extracellularly. These nanoparticles formed as protein-nanoparticle complexes that could be separated from the cells on the basis of density. A protein of ~39 kDa associated with biogenic nano-Se0 was recovered via polyacrylamide gel electrophoresis for characterization by MALDI-TOF mass spectrometry. Initial results suggest that this protein plays an integral, structural role in Se0 nanosphere formation. Second, the nanoparticulate products of bacterial sulfate reduction in a biofilm growing in minewater were investigated with multiple high- spatial resolution microanalyses. Biogenic zinc-sulfide nanoparticles exhibited evidence for rapid, highly efficient aggregation to form orders-of-magnitude larger

  9. Mechanisms of heat-mediated aggregation of wheat gluten protein upon pasta processing.

    PubMed

    Wagner, Magali; Morel, Marie-Helene; Bonicel, Joelle; Cuq, Bernard

    2011-04-13

    During pasta processing, structural changes of protein occur, due to changes in water content, mechanical energy input, and high temperature treatments. The present paper investigates the impact of successive and intense thermal treatments (high temperature drying, cooking, and overcooking) on aggregation of gluten protein in pasta. Protein aggregation was evaluated by the measurement of sensitivity of disulfide bonds toward reduction with dithioerythritol (DTE), at different reactions times. In addition to the loss in protein extractability in sodium dodecyl sulfate buffer, heat treatments induced a drastic change in disulfide bonds sensitivity toward DTE reduction and in size-exclusion high-performance liquid chromatography profiles of fully reduced protein. The protein solubility loss was assumed to derive from the increasing connectivity of protein upon heat treatments. The increasing degree of protein upon aggregation would be due to the formation of additional interchain disulfide bonds. PMID:21370874

  10. 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

  11. 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.

  12. 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.

  13. Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation.

    PubMed

    Rigg, Rachel A; Healy, Laura D; Nowak, Marie S; Mallet, Jérémy; Thierheimer, Marisa L D; Pang, Jiaqing; McCarty, Owen J T; Aslan, Joseph E

    2016-04-01

    Molecular chaperones that support protein quality control, including heat shock protein 70 (Hsp70), participate in diverse aspects of cellular and physiological function. Recent studies have reported roles for specific chaperone activities in blood platelets in maintaining hemostasis; however, the functions of Hsp70 in platelet physiology remain uninvestigated. Here we characterize roles for Hsp70 activity in platelet activation and function. In vitro biochemical, microscopy, flow cytometry, and aggregometry assays of platelet function, as well as ex vivo analyses of platelet aggregate formation in whole blood under shear, were carried out under Hsp70-inhibited conditions. Inhibition of platelet Hsp70 blocked platelet aggregation and granule secretion in response to collagen-related peptide (CRP), which engages the immunoreceptor tyrosine-based activation motif-bearing collagen receptor glycoprotein VI (GPVI)-Fc receptor-γ chain complex. Hsp70 inhibition also reduced platelet integrin-αIIbβ3 activation downstream of GPVI, as Hsp70-inhibited platelets showed reduced PAC-1 and fibrinogen binding. Ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear. Biochemical studies supported a role for Hsp70 in maintaining the assembly of the linker for activation of T cells signalosome, which couples GPVI-initiated signaling to integrin activation, secretion, and platelet function. Together, our results suggest that Hsp70 regulates platelet activation and function by supporting linker for activation of T cells-associated signaling events downstream of platelet GPVI engagement, suggesting a role for Hsp70 in the intracellular organization of signaling systems that mediate platelet secretion, "inside-out" activation of platelet integrin-αIIbβ3, platelet-platelet aggregation, and, ultimately, hemostatic plug and thrombus formation.

  14. 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.

  15. Early-Aggregation Studies of Polyglutamine in Solution

    NASA Astrophysics Data System (ADS)

    Fluitt, Aaron; de Pablo, Juan

    2012-02-01

    Several neurodegenerative diseases, notably Huntington's disease, are associated with certain proteins containing extended polyglutamine tracts. In all polyglutamine diseases, the age of onset is inversely correlated with the length of the polyglutamine domain beyond some pathological threshold. Diseased cells are characterized by intranuclear inclusions rich in aggregated polyglutamine. Experimental evidence suggests that oligomeric aggregate species, not mature amyloid fibrils, are the species most toxic to the cell. Little is known about the structures and aggregation dynamics of polyglutamine oligomers due to their short lifetimes. A better understanding of the pathway through which polyglutamine peptides form oligomeric aggregates will aid the design of therapies to inhibit their toxic activity. In this work, we report structural characterization of polyglutamine monomers and dimers from atomistic molecular dynamics simulations in explicit water. Umbrella sampling simulations reveal that the stability of the dimer species with respect to the disassociated monomers is an increasing function of the chain length.

  16. Thermal protein denaturation and protein aggregation in cells made thermotolerant by various chemicals: role of heat shock proteins.

    PubMed

    Kampinga, H H; Brunsting, J F; Stege, G J; Burgman, P W; Konings, A W

    1995-08-01

    Thermotolerance (TT) induced by sodium arsenite (A-TT: 100 microM, 1 h, 37 degrees C) was compared to heat-induced thermotolerance (H-TT: 15 min, 44 degrees C) using HeLa S3 cells. All four pretreatments led to comparable levels of thermotolerance and also induced resistance to arsenite-, ethanol-, and diamide-induced toxicity (clonogenic ability). Stress-induced expression of the major heat shock proteins (hsp27, hsc70(p73), hsp70(p72), and hsp90) was generally highest in H-TT cells and lowest in A-TT cells. Interestingly, the four types of TT cells showed distinct differences in certain aspects of resistance against thermal protein damage. Thermal protein denaturation and aggregation determined in isolated cellular membrane fractions was found to be attenuated when they were isolated from H-TT and A-TT cells but not when isolated from E-TT and D-TT cells. The heat resistance in the proteins of the membrane fraction corresponded with elevated levels of hsp70(p72) associated with the isolated membrane fractions. In the nuclear fraction, only marginal (not significant) attenuation of the formation of protein aggregates (as determined by TX-100 (in)solubility) was observed. However, the postheat recovery from heat-induced protein aggregation in the nucleus was faster in H-TT, E-TT, and D-TT cells, but not in A-TT cells. Despite the fact that elevated levels of hsp27, hsp70(p73), and hsp70(p72) were found in the TX-100 insoluble nuclear fraction derived from all TT cells, no correlation was found with the degree of resistance in terms of the accelerated recovery from nuclear protein aggregation. The only correlation between accelerated recovery from nuclear protein aggregates was that with total cellular levels of hsp27. The data indicate that heat-induced loss of clonogenic ability may be a multitarget rather than a single target event. A threshold of damage may exist in cells after exposure to heat; multiple sets of proteins in (different compartments of) the cell

  17. 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.

  18. 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.

  19. Comparison of three optical methods to study erythrocyte aggregation.

    PubMed

    Zhao, H; Wang, X; Stoltz, J F

    1999-01-01

    The aim of this work was to evaluate three optical methods designed to determine erythrocyte aggregation: Erythroaggregometer (EA; Regulest, France), Laser-assisted Optical Rotational Cell Analyzer (LORCA; Mechatronics, Netherlands) and Fully Automatic Erythrocyte Aggregometer (FAEA; Myrenne, GmbH, Germany). Blood samples were taken from fifty donors (26 males and 24 females). The aggregation of normal red blood cell (RBC) and RBCs suspended in three normo- and hyperaggregating suspending media was studied. The results revealed some significant correlations between parameters measured by these instruments, in particular, between the indexes of aggregation of EA and LORCA. Further, RBC aggregation of multiple myeloma patients was also studied and a hyper erythrocyte aggregation state was found by EA and LORCA.

  20. Bayesian model aggregation for ensemble-based estimates of protein pKa values

    SciTech Connect

    Gosink, Luke J.; Hogan, Emilie A.; Pulsipher, Trenton C.; Baker, Nathan A.

    2014-03-01

    This paper investigates an ensemble-based technique called Bayesian Model Averaging (BMA) to improve the performance of protein amino acid p$K_a$ predictions. Structure-based p$K_a$ calculations play an important role in the mechanistic interpretation of protein structure and are also used to determine a wide range of protein properties. A diverse set of methods currently exist for p$K_a$ prediction, ranging from empirical statistical models to {\\it ab initio} quantum mechanical approaches. However, each of these methods are based on a set of assumptions that have inherent bias and sensitivities that can effect a model's accuracy and generalizability for p$K_a$ prediction in complicated biomolecular systems. We use BMA to combine eleven diverse prediction methods that each estimate pKa values of amino acids in staphylococcal nuclease. These methods are based on work conducted for the pKa Cooperative and the pKa measurements are based on experimental work conducted by the Garc{\\'i}a-Moreno lab. Our study demonstrates that the aggregated estimate obtained from BMA outperforms all individual prediction methods in our cross-validation study with improvements from 40-70\\% over other method classes. This work illustrates a new possible mechanism for improving the accuracy of p$K_a$ prediction and lays the foundation for future work on aggregate models that balance computational cost with prediction accuracy.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Effects of oxidative modification on thermal aggregation and gel properties of soy protein by malondialdehyde.

    PubMed

    Wu, Wei; Hua, Yufei; Lin, Qinlu

    2014-03-01

    Malondialdehyde (MDA) was selected as a representative of lipid peroxidation products to investigate the effects of oxidative modification on thermal aggregation and gel properties of soy protein by lipid peroxidation products. Incubation of soy protein with increasing concentration of MDA resulted in gradual decrease of particle size and content of thermal aggregates during heat denaturation. Oxidative modification by MDA resulted in a decrease in water holding capacity, gel hardness, and gel strength of soy protein gel. An increase in coarseness and interstice of MDA modified protein gel network was accompanied by uneven distribution of interstice as MDA concentration increased. The results showed that degree of thermal aggregation of MDA-modified soy protein gradually decreased as MDA concentration increased, which contributed to a decrease in water holding capacity, gel hardness, and gel strength of MDA-modified soy protein gel. PMID:24587523

  4. Diminishing returns to aggregate level studies.

    PubMed

    Gravelle, H

    1999-10-01

    The idea that the health of individuals depends on the characteristics of the society in which they live and on their own characteristics is important. The aggregate level relation between income inequality and population mortality has been examined by empirical works. However, if the individual level relation between risk of mortality and income is curvilinear, at least part of any association between population mortality and income inequality is artifactual in the sense that it could arise even if individual risk was due only to individual income and not to its distribution. Wolfson et al attempted to estimate how much of the variation in cross-sectional US state-level mortality could be due to the curvature of the relation between individual level mortality and income interacting with differences in the distribution of income within states. They concluded that the artifact is not the main reason for the frequently documented correlations between population mortality and income distribution. However, in the absence of any detailed information on the regressions, it is difficult to determine if the difference between actual and hypothetical mortality is significantly related to income equality. The individual risk of mortality is also affected by other individual characteristics, like climate or public health infrastructure. The authors suggest that investigations of the determinants of individual health test the effect of societal factors and that such testing requires both individual level and aggregate data.

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

    PubMed Central

    Nieuwkoop, Andrew J.; Berthold, Deborah A.; Comellas, Gemma; Sperling, Lindsay J.; Tang, Ming; Shah, Gautam J.; Brea, Elliott J.; Lemkau, Luisel R.

    2012-01-01

    Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution. PMID:22986689

  6. 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

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

    PubMed

    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

  8. Early (pre-DLVO) studies of particle aggregation.

    PubMed

    Vincent, Brian

    2012-01-15

    The history of colloid science, from its modern foundations in the mid-nineteenth century, has been strongly concerned with studies of the aggregation of colloidal particles. It was Thomas Graham (1861) who defined the word "colloid" (from the Greek word for glue) for those materials which could not pass through membranes, unlike smaller, truly-dissolved materials. Subsequently, Graham (1864), following earlier studies, principally by Selmi and Faraday, described "the power possessed by salts for destroying colloidal solutions". Although numerous, quantitative studies of particle aggregation were made in the years that followed, in particular, the determination of minimum electrolyte concentrations for the onset of particle aggregation and aggregation rates, no general theoretical framework emerged to account for these quantitative findings until the middle of the twentieth century. It was during and immediately following the Second World War that two sets of authors, Derjaguin and Landau, in Russia, and Verwey and Overbeek, in the Netherlands, independently came up with the theory that is now universally referred to as the DLVO theory of particle interactions and aggregation. All modern developments of the theory of particle aggregation use the DLVO theory as the keystone. However, the DLVO theory itself was based on a large body of experimental data in regard to particle aggregation obtained over the previous hundred years or so. This article is an attempt to review that body of experimental data and to show how this guided the DLVO authors in their thinking.

  9. Covalent modifications of ribosomal proteins in growing and aggregation-competent dictyostelium discoideum: phosphorylation and methylation.

    PubMed

    Ramagopal, S

    1991-04-01

    Phosphorylated and methylated ribosomal proteins were identified in vegetatively growing amoebae and in the starvation-induced, aggregation-competent cells of Dictyostelium discoideum. Of the 15 developmentally regulated cell-specific ribosomal proteins reported earlier, protein A and the acidic proteins A1, A2, and A3 were identified as phosphoproteins, and S5, S6, S10, and D were identified as methylated proteins. Three other ribosomal proteins were phosphorylated and 19 others methylated. S19, L13, A1, A2, and A3 were the predominant phosphoproteins in growing amoebae, whereas S20 and A were the predominant ones in the aggregation-competent cells. Among the methylated proteins, eight (S6, S10, S13, S30, D, L1, L2, and L31) were modified only during growth phase, six (S5, S7, S8, S24, S31, and L36) were altered only during aggregation-competent phase, and nine (S9, S27, S28, S29, S34, L7, L35, L41, and L42) were modified under both phases. Five proteins (S6, S24, L7, L41, and L42) were heavily methylated and of these, the large subunit proteins were present in both growing amoebae and aggregation-competent cells. These findings demonstrate that covalent modification of specific ribosomal proteins is regulated during cell differentiation in D. discoideum.

  10. 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

  11. 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.

  12. Agitation of amyloid proteins to speed aggregation measured by ThT fluorescence: a call for standardization.

    PubMed

    Batzli, Kiersten M; Love, Brian J

    2015-03-01

    This retrospective study of protein aggregation measured by Thioflavin T (ThT) fluorescence assay in published literature has assessed protein sensitivity to denaturing conditions that include elevated temperatures, fluctuations in pH, and concentration and, in particular, agitation to induce amyloid structure formation. The dynamic tracking of fluorescence shows a sigmoidal evolution as aggregates form; the resulting kinetics of association have been analyzed to explore the range of aggregation behavior which occurs based on environmental parameters. Comparisons between the experimental results of different groups have been historically difficult due to subtleties of experimental procedures including denaturing temperature, protein type and concentration, formulation differences, and how agitation is achieved. While it is clear that agitation has a strong influence on the driving force for aggregation, the use of magnetic stirring bar or shaker table rotational speed is insufficient to characterize the degree of turbulence produced during shear. The pathway forward in resolving dependence of aggregate formation on shear may require alternative methodologies or better standardization of the experimental protocols.

  13. 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

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

    PubMed

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

    2016-01-01

    A mid-infrared free-electron laser (FEL) is a linearly polarized, high-peak powered pulse laser with tunable wavelength within the mid-infrared absorption region. It was recently found that pathogenic amyloid fibrils could be partially dissociated to the monomer form by the irradiation of the FEL targeting the amide I band (C=O stretching vibration), amide II band (N-H bending vibration) and amide III band (C-N stretching vibration). In this study, the irradiation effect of the FEL on keratin aggregate was tested as another model to demonstrate an applicability of the FEL for dissociation of protein aggregates. Synchrotron radiation infrared microscopy analysis showed that the α-helix content in the aggregate structure decreased to almost the same level as that in the monomer state after FEL irradiation tuned to 6.06 µm (amide I band). Both irradiations at 6.51 µm (amide II band) and 8.06 µm (amide III band) also decreased the content of the aggregate but to a lesser extent than for the irradiation at the amide I band. On the contrary, the irradiation tuned to 5.6 µm (non-absorbance region) changed little the secondary structure of the aggregate. Scanning-electron microscopy observation at the submicrometer order showed that the angular solid of the aggregate was converted to non-ordered fragments by the irradiation at each amide band, while the aggregate was hardly deformed by the irradiation at 5.6 µm. These results demonstrate that the amide-specific irradiation by the FEL was effective for dissociation of the protein aggregate to the monomer form. PMID:26698057

  15. 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

  16. Effect of protein thermo aggregation on the binding of BSA to gelatin type A.

    PubMed

    Antonov, Y A; Zhuravleva, I L

    2013-02-01

    We study the effect of limited heat-induced aggregation of BSA on structure development in the water-gelatin-thermally aggregated BSA (BSA(TA)) system. The pH is set at 5.4 and the temperature is higher than the conformation transition temperature of gelatin, but lower than the denaturation temperature of BSA. Dynamic light scattering, circular dichroism, and fluorescence measurements are used to monitor structure changes. Interaction of gelatin with BSA(TA) leads to formation of large complex particles with an average radius ∼1500 nm. BSA-gelatin complex formation accompanies partial destabilization of the secondary and tertiary structures of BSA and an additional exposure of hydrophobic tryptophan residues on the surface of the globule. It is shown that electrostatic interaction of the oppositely charged groups of BSA(TA) and gelatin is responsible for formation of such complex particles, whereas the secondary forces (hydrophobic interaction and hydrogen bonds) play an important role in stabilization of the complex particles. The zeta potentials of the native and the thermally aggregated BSA samples were determined, and the solvent quality has been quantified by determining the activity of the protein samples in their saturated solutions. It was shown that steric reasons (large size of the thermally aggregated BSA(TA) particles), and uncomplete charge compensation of the positively charged gelatin molecules by the negatively charged BSA(TA) particles are the main factors in determining structure formation, while the levels of the activity of the native BSA and BSA(TA) have a smaller effect on the structure of complex.

  17. Homolog of the maize beta-glucosidase aggregating factor from sorghum is a jacalin-related GalNAc-specific lectin but lacks protein aggregating activity.

    PubMed

    Kittur, Farooqahmed S; Yu, Hyun Young; Bevan, David R; Esen, Asim

    2009-03-01

    Recently, we identified the maize beta-glucosidase aggregating factor (BGAF) as a jacalin-related lectin (JRL) and showed that its lectin domain is responsible for beta-glucosidase aggregation. By searching for BGAF homologs in sorghum, we identified and obtained an EST clone and determined its complete sequence. The predicted protein had the same modular structure as maize BGAF, shared 67% sequence identity with it, and revealed the presence of two potential carbohydrate-binding sites (GG...ATYLQ, site I and GG...GVVLD, site II). Maize BGAF1 is the only lectin from a class of modular JRLs containing an N-terminal dirigent and a C-terminal JRL domain, whose sugar specificity and beta-glucosidase aggregating activity have been studied in detail. We purified to homogeneity a BGAF homolog designated as SL (Sorghum lectin) from sorghum and expressed its recombinant version in Escherichia coli. The native protein had a molecular mass of 32 kD and was monomeric. Both native and recombinant SL-agglutinated rabbit erythrocytes, and inhibition assays indicated that SL is a GalNAc-specific lectin. Exchanging the GG...GVVLD motif in SL with that of maize BGAF1 (GG...GIAVT) had no effect on GalNAc-binding, whereas binding to Man was abolished. Substitution of Thr(293) and Gln(296) in site I to corresponding residues (Val(294) and Asp(297)) of maize BGAF1 resulted in the loss of GalNAc-binding, indicating that site I is responsible for generating GalNAc specificity in SL. Gel-shift and pull-down assays after incubating SL with maize and sorghum beta-glucosidases showed no evidence of interaction nor were any SL-protein complexes detected in sorghum tissue extracts, suggesting that the sorghum homolog does not participate in protein-protein interactions. PMID:19056785

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

    PubMed Central

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

    2014-01-01

    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/. PMID:24848016

  19. 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

    2016-01-01

    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/. PMID:27581095

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

    PubMed Central

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

    2016-01-01

    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/. PMID:27581095

  1. Aggregation of concentrated monoclonal antibody solutions studied by rheology and neutron scattering

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

    Protein solutions are studied using rheology and scattering techniques to investigate aggregation. Here we present a monoclonal antibody (mAb) that aggregates after incubation at 40 °C (below its unfolding temperature), with a decrease in monomer purity of 6% in 10 days. The mAb solution contains surfactant and behaves as a Newtonian fluid when reconstituted into solution from the lyophilized form (before incubation at 40 °C). In contrast, mAb solutions incubated at 40 °C for 1 month exhibit shear yielding in torsional bulk rheometers. Interfacial rheology reveals that interfacial properties are controlled by the surfactant, producing a negligible surface contribution to the bulk yield stress. These results provide evidence that protein aggregates formed in the bulk are responsible for the yield stress. Small-angle neutron scattering (SANS) measurements show an increase in intensity at low wavevectors (q < 4*10-2 nm-1) that we attribute to protein aggregation, and is not observed in solutions stored at 4 °C for 3 days before the measurement. This work suggests a correlation between the aggregated state of the protein (stability) and the yield stress from rheology. Research funded by MedImmune

  2. Efficient Inhibition of Protein Aggregation, Disintegration of Aggregates, and Lowering of Cytotoxicity by Green Tea Polyphenol-Based Self-Assembled Polymer Nanoparticles.

    PubMed

    Debnath, Koushik; Shekhar, Shashi; Kumar, Vipendra; Jana, Nihar R; Jana, Nikhil R

    2016-08-10

    Green tea polyphenol epigallocatechin-3-gallate (EGCG) is known for its antiamyloidogenic property, and it is observed that molecular EGCG binds with amyloid structure, redirects fibrillation kinetics, remodels mature fibril, and lowers the amyloid-derived toxicity. However, this unique property of EGCG is difficult to utilize because of their poor chemical stability and substandard bioavailability. Here we report a nanoparticle form of EGCG of 25 nm size (nano-EGCG) which is 10-100 times more efficient than molecular EGCG in inhibiting protein aggregation, disintegrating mature protein aggregates, and lowering amyloidogenic cytotoxicity. The most attractive advantage of nano-EGCG is that it efficiently protects neuronal cells from the toxic effect of extracellular amyloid beta or intracellular mutant huntingtin protein aggregates by preventing their aggregation. We found that the better performance of nano-EGCG is due to the combined effect of increased chemical stability of EGCG against degradation, stronger binding with protein aggregates, and efficient entry into the cell for interaction with aggregated protein structure. This result indicates that the nanoparticle form of antiamyloidogenic molecules can be more powerful in prevention and curing of protein aggregation derived diseases. PMID:27427935

  3. Subvisible particle counting provides a sensitive method of detecting and quantifying aggregation of monoclonal antibody caused by freeze-thawing: insights into the roles of particles in the protein aggregation pathway.

    PubMed

    Barnard, James G; Singh, Satish; Randolph, Theodore W; Carpenter, John F

    2011-02-01

    The objective of this study was to evaluate microflow imaging (MFI) as a sensitive tool to detect and quantify subvisible particle formation during freeze-thawing of an IgG(2) monoclonal antibody (mAb). Solutions of the protein formulated in 20 mM of histidine buffer (pH 5.5) were subjected to three freeze-thaw cycles and analyzed by MFI and size-exclusion chromatography (SEC). MFI showed increased particle numbers after each freeze-thaw cycle, whereas aggregates were not detected by SEC. Estimates of the total mass of particles formed revealed that monitoring of particle formation allows for the detection of protein aggregates comprising only hundredths of a percent of the total protein mass. Furthermore, differences in protein aggregation levels due to different formulations or different freeze-thawing protocols were resolved, even though protein aggregation could not be detected by SEC. To examine whether SEC and MFI-based estimations of total aggregate mass were in quantitative agreement, mAb was freeze-thawed in phosphate-buffered saline. This process created sufficient level of insoluble aggregates to be detected by SEC as a reduction in the monomer peak area in the chromatogram. There was good agreement between the loss of monomer detected by SEC and the total mass of subvisible particles detected by MFI.

  4. Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation

    PubMed Central

    2016-01-01

    The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo. PMID:26751094

  5. Role of protein conformation and aggregation in pumping water in and out of a cell.

    PubMed

    Cameron, I L; Kanal, K M; Fullerton, G D

    2006-01-01

    Dialysis cassettes containing BSA solutions were used to simulate passive in vivo conditions to assess the effect of protein conformation and aggregation on cell water content. The cassettes were suspended in dextran solutions to provide a range of fixed osmotic stress values simulating blood plasma. The system was placed on a shaker for 24 h to attain equilibrium. Four manipulation methods; pH, cosolute salt concentration, e.g. NaCl, temperature annealing and urea concentration denaturant were varied to produce well-known manipulations of BSA conformation. It was observed that the cell water content varied from +14% to about -13% with changes in protein conformation and aggregation. The findings demonstrate that a change in protein conformation and aggregation, pumps water in and out of a cell to maintain equilibrium % water content matching the protein conformational hydration parameter. This concept supplements existing theories on cell volume regulation. PMID:16376581

  6. 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

  7. Role of protein conformation and aggregation in pumping water in and out of a cell.

    PubMed

    Cameron, I L; Kanal, K M; Fullerton, G D

    2006-01-01

    Dialysis cassettes containing BSA solutions were used to simulate passive in vivo conditions to assess the effect of protein conformation and aggregation on cell water content. The cassettes were suspended in dextran solutions to provide a range of fixed osmotic stress values simulating blood plasma. The system was placed on a shaker for 24 h to attain equilibrium. Four manipulation methods; pH, cosolute salt concentration, e.g. NaCl, temperature annealing and urea concentration denaturant were varied to produce well-known manipulations of BSA conformation. It was observed that the cell water content varied from +14% to about -13% with changes in protein conformation and aggregation. The findings demonstrate that a change in protein conformation and aggregation, pumps water in and out of a cell to maintain equilibrium % water content matching the protein conformational hydration parameter. This concept supplements existing theories on cell volume regulation.

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

    PubMed Central

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

    2012-01-01

    Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity. PMID:22955885

  9. Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping.

    PubMed

    Bergh, Johan; Zetterström, Per; Andersen, Peter M; Brännström, Thomas; Graffmo, Karin S; Jonsson, P Andreas; Lang, Lisa; Danielsson, Jens; Oliveberg, Mikael; Marklund, Stefan L

    2015-04-01

    Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease. PMID:25802384

  10. Caspase-1 causes truncation and aggregation of the Parkinson's disease-associated protein α-synuclein.

    PubMed

    Wang, Wei; Nguyen, Linh T T; Burlak, Christopher; Chegini, Fariba; Guo, Feng; Chataway, Tim; Ju, Shulin; Fisher, Oriana S; Miller, David W; Datta, Debajyoti; Wu, Fang; Wu, Chun-Xiang; Landeru, Anuradha; Wells, James A; Cookson, Mark R; Boxer, Matthew B; Thomas, Craig J; Gai, Wei Ping; Ringe, Dagmar; Petsko, Gregory A; Hoang, Quyen Q

    2016-08-23

    The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well. PMID:27482083

  11. Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation

    PubMed Central

    Ghosh, Dhiman; Singh, Pradeep K.; Sahay, Shruti; Jha, Narendra Nath; Jacob, Reeba S.; Sen, Shamik; Kumar, Ashutosh; Riek, Roland; Maji, Samir K.

    2015-01-01

    Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases. PMID:25784353

  12. TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins

    PubMed Central

    Prpar Mihevc, S.; Baralle, Marco; Buratti, Emanuele; Rogelj, Boris

    2016-01-01

    TDP-43 protein plays an important role in regulating transcriptional repression, RNA metabolism, and splicing. Typically it shuttles between the nucleus and the cytoplasm to perform its functions, while abnormal cytoplasmic aggregation of TDP-43 has been associated with neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). For the purpose of this study we selected a set of proteins that were misregulated following silencing of TDP-43 and analysed their expression in a TDP-43-aggregation model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Following TDP-43 sequestration in insoluble aggregates, we observed higher nuclear levels of EIF4A3, and POLDIP3β, whereas nuclear levels of DNMT3A, HNRNPA3, PABPC1 and POLDIP3α dropped, and cytoplasmic levels of RANBP1 dropped. In addition, immunofluorescence signal intensity quantifications showed increased nuclear expression of HNRNPL and YARS, and downregulation of cytoplasmic DPCD. Furthermore, cytoplasmic levels of predominantly nuclear protein ALYREF increased. In conclusion, by identifying a common set of proteins that are differentially expressed in a similar manner in these two different conditions, we show that TDP-43 aggregation has a comparable effect to TDP-43 knockdown. PMID:27665936

  13. Albinterferon α2b adsorption to silicone oil-water interfaces: effects on protein conformation, aggregation, and subvisible particle formation.

    PubMed

    Basu, Pinaki; Blake-Haskins, Angela W; O'Berry, Kristin B; Randolph, Theodore W; Carpenter, John F

    2014-02-01

    Silicone oil used as a lubricant in prefilled syringes has the potential to induce formation of particles in protein formulations. In the current study, we used a therapeutic fusion protein, albinterferon α2b , to evaluate protein aggregation and particle formation in the presence of silicone oil microdroplets or immobilized silicone interfaces. Tertiary structure of albinterferon α2b adsorbed on silicone oil microdroplets was perturbed in a formulation containing only buffer. In contrast, native-like tertiary structure was retained for albinterferon α2b adsorbed on silicone oil microdroplets in 300 mM sodium chloride or 300 mM sucrose formulations. Agitation of albinterferon α2b samples in the presence of silicone oil droplets or siliconized beads, respectively, caused albinterferon α2b aggregation and subvisible particle formation in formulations containing buffer or 300 mM sucrose. Adsorption of albinterferon α2b onto silicone oil was inhibited by addition of 0.01% (w/v) polysorbate 80, and this excipient prevented aggregation during agitation in the presence of silicone oil microdroplets. Aggregation was also reduced in the presence of 300 mM sodium chloride during agitation at least in part because of the increased conformational stability of the protein. PMID:24382812

  14. 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

  15. A Loss in Cellular Protein Partners Promotes α-Synuclein Aggregation in Cells Resulting from Oxidative Stress

    PubMed Central

    Guo, Yuanjian; Scarlata, Suzanne

    2015-01-01

    There is a consensus that oxidative stress promotes neurodegeneration and may be linked to plaque formation. α-Synuclein is the main component of neurodegenerative plaques. We have found that α-synuclein binds strongly to the enzyme phospholipase Cβ1 (PLCβ1) in vitro and in cells affecting both its G protein activation and its degradation. Because PLCβ1 binds to α-synuclein in cells, we tested whether decreasing its level would promote α-synuclein aggregation and whether overproducing PLCβ1 would inhibit aggregation. By imaging fluorescent α-synuclein in living HEK293, PC12, and SK-H-SH cells, we find that α-synuclein aggregation is directly related to the level of PLCβ1. Importantly, we found that oxidative stress does not affect the cellular levels of α-synuclein but results in the down-regulation of PLCβ1 thereby promoting α-synuclein aggregation. A peptide that mimics part of the α-synuclein binding site to PLCβ prevents aggregation. Our studies indicate that PLCβ1 can reduce cell damage under oxidative stress and offers a potential site that might be exploited to prevent α-synuclein aggregation. PMID:23659438

  16. Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens.

    PubMed Central

    Ward, J E; Dale, E M; Nester, E W; Binns, A N

    1990-01-01

    Products of the virB operon are proposed components of a membrane-associated T-DNA transport apparatus in Agrobacterium tumefaciens. Here we identified the virB10 gene product and raised specific antiserum to the protein. While the virB10 reading frame contains two potential ATG translation start sites located 32 codons apart, we found that only the downstream ATG was required for efficient VirB10 synthesis. Cellular localization studies and analysis of translational fusions with the Escherichia coli alkaline phosphatase gene (phoA) indicated that VirB10 was anchored in the inner membrane and contained a periplasmic domain. This work also demonstrated the utility of alkaline phosphatase as a reporter for secreted proteins in A. tumefaciens. Several high-molecular-weight forms of VirB10 were observed after treatment of A. tumefaciens whole cells or inner membranes with protein cross-linking agents, suggesting that VirB10 exists as a native oligomer or forms an aggregate with other membrane proteins. These results provide the first biochemical evidence that a VirB protein complex is membrane associated in A. tumefaciens. Images PMID:2394684

  17. Probing Alzheimer amyloid peptide aggregation using a cell-free fluorescent protein refolding method.

    PubMed

    Arslan, Pharhad Eli; Chakrabartty, Avijit

    2009-08-01

    Fibrillation of the Alzheimer beta-amyloid peptide (Abeta) and (or) formation of toxic oligomers are key pathological events in Alzheimer's disease. Several strategies have been introduced to identify small molecule aggregation inhibitors, and based on these methods, a number of aggregation inhibitors have been identified. However, most of these methods use chemically synthesized Abeta42 peptides, which are difficult to maintain in a monomeric state at neutral pH where anti-aggregation screening is usually carried out. We have developed a cell-free Abeta42 aggregation assay based on fluorescence protein refolding. This assay utilizes nanomolar concentrations of protein. We genetically fused Abeta42 to the N-terminus of vYFP, a variant of of GFP, and expressed and purified the folded fusion protein. The refolding efficiency of Abeta42-vYFP fusion was inversely correlated with the solubility of Abeta42. Using fluorescence to monitor refolding of Abeta42-vYFP, we confirmed that Zn2+ binds to Abeta42 and increases its aggregation. The IC50 value estimated for Zn binding is 3.03 +/- 0.65 micromol/L. We also show that this technique is capable of monitoring the aggregation of chemically synthesized Abeta42.

  18. Suppression of protein aggregation by chaperone modification of high molecular weight complexes.

    PubMed

    Labbadia, John; Novoselov, Sergey S; Bett, John S; Weiss, Andreas; Paganetti, Paolo; Bates, Gillian P; Cheetham, Michael E

    2012-04-01

    Protein misfolding and aggregation are associated with many neurodegenerative diseases, including Huntington's disease. The cellular machinery for maintaining proteostasis includes molecular chaperones that facilitate protein folding and reduce proteotoxicity. Increasing the protein folding capacity of cells through manipulation of DNAJ chaperones has been shown to suppress aggregation and ameliorate polyglutamine toxicity in cells and flies. However, to date these promising findings have not been translated to mammalian models of disease. To address this issue, we developed transgenic mice that over-express the neuronal chaperone HSJ1a (DNAJB2a) and crossed them with the R6/2 mouse model of Huntington's disease. Over-expression of HSJ1a significantly reduced mutant huntingtin aggregation and enhanced solubility. Surprisingly, this was mediated through specific association with K63 ubiquitylated, detergent insoluble, higher order mutant huntingtin assemblies that decreased their ability to nucleate further aggregation. This was dependent on HSJ1a client binding ability, ubiquitin interaction and functional co-operation with HSP70. Importantly, these changes in mutant huntingtin solubility and aggregation led to improved neurological performance in R6/2 mice. These data reveal that prevention of further aggregation of detergent insoluble mutant huntingtin is an additional level of quality control for late stage chaperone-mediated neuroprotection. Furthermore, our findings represent an important proof of principle that DNAJ manipulation is a valid therapeutic approach for intervention in Huntington's disease.

  19. A Consensus Method for the Prediction of ‘Aggregation-Prone’ Peptides in Globular Proteins

    PubMed Central

    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). PMID:23326595

  20. Proteasome dysfunction induces muscle growth defects and protein aggregation.

    PubMed

    Kitajima, Yasuo; Tashiro, Yoshitaka; Suzuki, Naoki; Warita, Hitoshi; Kato, Masaaki; Tateyama, Maki; Ando, Risa; Izumi, Rumiko; Yamazaki, Maya; Abe, Manabu; Sakimura, Kenji; Ito, Hidefumi; Urushitani, Makoto; Nagatomi, Ryoichi; Takahashi, Ryosuke; Aoki, Masashi

    2014-12-15

    The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes of protein and organelle clearance. The role of the proteasome pathway in mammalian muscle has not been examined in vivo. In this study, we report that the muscle-specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in profound muscle growth defects and a decrease in force production in mice. Specifically, developing muscles in conditional Rpt3-knockout animals showed dysregulated proteasomal activity. The autophagy pathway was upregulated, but the process of autophagosome formation was impaired. A microscopic analysis revealed the accumulation of basophilic inclusions and disorganization of the sarcomeres in young adult mice. Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways. The deletion of a component of the proteasome complex contributed to myofiber degeneration and weakness in muscle disorders that are characterized by the accumulation of abnormal inclusions.

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

    PubMed

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

    2014-06-01

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

  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. Kinetics of cold-set diffusion-limited aggregations of denatured whey protein isolate colloids.

    PubMed

    Wu, Hua; Xie, Jianjun; Morbidelli, Massimo

    2005-01-01

    The CaCl2-induced cold-set aggregation kinetics of the denatured whey protein isolate (WPI) colloids has been investigated under dilute diffusion-limited cluster aggregation (DLCA) conditions, using small-angle light scattering. In particular, the structure factor, the scattered intensity at zero angle and the average radius of gyration have been measured for the aggregating system as a function of time. It is found that the fractal dimension of the clusters is df= 1.85, in the range typical of clusters aggregated under DLCA conditions. The aggregation kinetics in this transition region can be described by a power law relation in the initial stage of the aggregation, but the exponent of the power law is equal to 0.7, i.e., significantly larger than 1/df= 0.54, which is the typical value of the DLCA kinetics. Since it is found that the average gyration radius of the clusters has reached a value of 80 microm, leading to a cumulative volume fraction of clusters equal to 0.25, it is legitimate to expect that the process is in the region of transition from aggregation to gelation. This confirmed by the fact that, at the later stage of the aggregation, the growth of the average cluster size further accelerates with time and eventually becomes explosive, leading to gelation. The observed aggregation kinetics has been compared with that reported in the literature from DLCA Monte Carlo simulations, and a good agreement has been found with the data corresponding to the transition region from aggregation to gelation. Numerical simulations using the Smoluchowski kinetic model have also been carried out in order to support the experimental findings.

  4. Effects of Protein Conformation, Apparent Solubility, and Protein-Protein Interactions on the Rates and Mechanisms of Aggregation for an IgG1Monoclonal Antibody.

    PubMed

    Kalonia, Cavan; Toprani, Vishal; Toth, Ronald; Wahome, Newton; Gabel, Ian; Middaugh, C Russell; Volkin, David B

    2016-07-28

    Non-native protein aggregation is a key degradation pathway of immunoglobulins. In this work, the aggregation kinetics of an immunoglobulin gamma-1 monoclonal antibody (IgG1 mAb) in different solution environments was monitored over a range of incubation temperatures for up to seven months using size exclusion chromatography. Histidine and citrate buffers with/without sodium chloride were employed to modulate the mAb's conformational stability, solubility (in the presence of polyethylene glycol, PEG), and protein-protein interactions as measured by differential scanning calorimetry, PEG precipitation, and static light scattering, respectively. The effect of these parameters on the mechanism(s) of mAb aggregation during storage at different temperatures was determined using kinetic models, which were used to fit aggregation data to determine rate constants for aggregate nucleation and growth processes. This approach was used to investigate the effects of colloidal protein-protein interactions and solubility values (in PEG solutions) on the mechanisms and rates of IgG1 mAb aggregation as a function of temperature-induced structural perturbations. Aggregate nucleation and growth pathways for this IgG1 mAb were sensitive to temperature and overall conformational stability. Aggregate growth, on the other hand, was also sensitive to conditions affecting the solubility of the mAb, particularly at elevated temperatures. PMID:27380437

  5. 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.

  6. Modeling the interplay between protein and lipid aggregation in supported membranes.

    PubMed

    de Prado Salas, Pablo González; Encinar, Mario; Alonso, Alvaro; Vélez, Marisela; Tarazona, Pedro

    2015-01-01

    We present a theoretical model that deals with the complex interplay between lipid segregation and the self-aggregation of lipid-attached proteins. The model, in contrast to previous ones that consider proteins only as passive elements affecting the lipid distribution, describes the system including three terms: the dynamic interactions between protein monomers, the interactions between lipid components, and a mixed term considering protein-lipid interactions. It is used to explain experimental results performed on a well-defined system in which a self-aggregating soluble bacterial cytoskeletal protein polymerizes on a lipid bilayer containing two lipid components. All the elements considered in a previously described protein model, including torsion of the monomers within the filament, are needed to account for the observed filament shapes. The model also points out that lipid segregation can affect the length and curvature of the filaments and that the dynamic behavior of the lipids and proteins can have different time scales, giving rise to memory effects. This simple model that considers a dynamic protein assembly on a fluid and active lipid surface can be easily extended to other biologically relevant situations in which the interplay between protein and lipid aggregation is needed to fully describe the system. PMID:24968242

  7. Modeling the interplay between protein and lipid aggregation in supported membranes.

    PubMed

    de Prado Salas, Pablo González; Encinar, Mario; Alonso, Alvaro; Vélez, Marisela; Tarazona, Pedro

    2015-01-01

    We present a theoretical model that deals with the complex interplay between lipid segregation and the self-aggregation of lipid-attached proteins. The model, in contrast to previous ones that consider proteins only as passive elements affecting the lipid distribution, describes the system including three terms: the dynamic interactions between protein monomers, the interactions between lipid components, and a mixed term considering protein-lipid interactions. It is used to explain experimental results performed on a well-defined system in which a self-aggregating soluble bacterial cytoskeletal protein polymerizes on a lipid bilayer containing two lipid components. All the elements considered in a previously described protein model, including torsion of the monomers within the filament, are needed to account for the observed filament shapes. The model also points out that lipid segregation can affect the length and curvature of the filaments and that the dynamic behavior of the lipids and proteins can have different time scales, giving rise to memory effects. This simple model that considers a dynamic protein assembly on a fluid and active lipid surface can be easily extended to other biologically relevant situations in which the interplay between protein and lipid aggregation is needed to fully describe the system.

  8. Protein binding-induced surfactant aggregation variation: a new strategy of developing fluorescent aqueous sensor for proteins.

    PubMed

    Hu, Wenting; Ding, Liping; Cao, Jianhua; Liu, Lili; Wei, Yuting; Fang, Yu

    2015-03-01

    Novel strategies of developing fluorescent sensors for proteins are highly demanded. In this work, we particularly synthesized a cholesterol-derivatized pyrene probe. Its fluorescence emission is effectively tuned by the aggregation state of a cationic surfactant dodecyltrimethylammonium bromide (DTAB). The used probe/DTAB assemblies exhibit highly sensitive ratiometric responses to pepsin and ovalbumin egg (o-egg) with detection limits of 4.8 and 18.9 nM, respectively. The fluorescence changes indicate the protein-surfactant interaction leads to further aggregation of DTAB assemblies. The results from Tyndall effect and dynamic light scattering verify this assumption. The responses to pepsin and o-egg are due to their strong electrostatic or hydrophobic interaction with DTAB assemblies at pH 7.4. The present noncovalent supramolecular sensor represents a novel and simple strategy for sensing proteins, which is based on the encapsulated fluorophore probing the aggregation variation of the surfactant assemblies.

  9. 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.

  10. Nitrosative stress mediated misfolded protein aggregation mitigated by Na-D-{beta}-hydroxybutyrate intervention

    SciTech Connect

    Kabiraj, Parijat; Pal, Rituraj; Varela-Ramirez, Armando; Miranda, Manuel; Narayan, Mahesh

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Rotenone is a model for inducing apoptosis and synphilin-1 accumulation in Parkinson Prime s studies. Black-Right-Pointing-Pointer The metabolite sodium betahydroxybutryate mitigates these effects in SHSY5Y cell lines. Black-Right-Pointing-Pointer Results reveal a novel and innate mechanism to prevent neurodegeneration/cell death. -- Abstract: Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species, is associated with the pathogenesis of neurodegenerative disorders. Rotenone, a mitochondrial stressor induces caspase-9 and caspase-3 activation leading proteolytic cleavage of substrate nuclear poly(ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1, as a rotenone-induced Parkinsonia-onset biomarker. We report that the innate ketone body, Na-D-{beta}-hydroxybutyrate (Na{beta}HB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, our data reveal that the metabolic byproduct also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. Together, these results suggest that Na{beta}HB is neuroprotective; it attenuates effects originating from mitochondrial insult and can serve as a scaffold for the design and development of sporadic neuropathies.

  11. Effects of crowders on the equilibrium and kinetic properties of protein aggregation

    NASA Astrophysics Data System (ADS)

    Bridstrup, John; Yuan, Jian-Min

    2016-08-01

    The equilibrium and kinetic properties of protein aggregation systems in the presence of crowders are investigated using simple, illuminating models based on mass-action laws. Our model yields analytic results for equilibrium properties of protein aggregates, which fit experimental data of actin and ApoC-II with crowders reasonably well. When the effects of crowders on rate constants are considered, our kinetic model is in good agreement with experimental results for actin with dextran as the crowder. Furthermore, the model shows that as crowder volume fraction increases, the length distribution of fibrils becomes narrower and shifts to shorter values due to volume exclusion.

  12. 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

  13. Induction of the neural cell adhesion molecule and neuronal aggregation by osteogenic protein 1.

    PubMed Central

    Perides, G; Safran, R M; Rueger, D C; Charness, M E

    1992-01-01

    The neural cell adhesion molecule (N-CAM) plays a fundamental role in nervous system development and regeneration, yet the regulation of the expression of N-CAM in different brain regions has remained poorly understood. Osteogenic protein 1 (OP-1) is a member of the transforming growth factor beta superfamily that is expressed in the nervous system. Treatment of the neuroblastoma-glioma hybrid cell line NG108-15 for 1-4 days with recombinant human OP-1 (hOP-1) induced alterations in cell shape, formation of epithelioid sheets, and aggregation of cells into multilayered clusters. Immunofluorescence studies and Western blots demonstrated a striking differential induction of the three N-CAM isoforms in hOP-1-treated cells. hOP-1 caused a 6-fold up-regulation of the 140-kDa N-CAM, the isoform showing the highest constitutive expression, and a 29-fold up-regulation of the 180-kDa isoform. The 120-kDa isoform was not detected in control NG108-15 cells but was readily identified in hOP-1-treated cells. Incubation of NG108-15 cells with an antisense N-CAM oligonucleotide reduced the induction of N-CAM by hOP-1 and decreased the formation of multilayered cell aggregates. Anti-N-CAM monoclonal antibodies also diminished the formation of multilayered cell aggregates by hOP-1 and decreased cell-cell adhesion when hOP-1-treated NG108-15 cells were dispersed and replated. Thus, hOP-1 produces morphologic changes in NG108-15 cells, at least in part, by inducing N-CAM. These observations suggest that OP-1 or a homologue may participate in the regulation of N-CAM during nervous system development and regeneration. Images PMID:1438217

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

    PubMed Central

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

    2016-01-01

    This study investigated the glucono-δ-lactone (GDL)-induced aggregation of isoflavones and soy proteins in soymilk. High-performance liquid chromatography (HPLC) analysis indicated that isoflavones mixed with β-conglycinin (7S) and glycinin (11S) proteins formed 7S-isoflavone and 11S-isoflavone complexes in soymilk supernatant fraction (SSF). Most of the soy protein-isoflavone complexes then precipitated into the soymilk pellet fraction (SPF) following the addition of 4 mM GDL, whereupon the pH value of the soymilk dropped from 6.6 to 5.9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis suggest that the addition of 4 mM GDL induced the aggregation of most 7S (α’, α and β subunits), 11S acidic and 11S basic proteins as well as isoflavones, including most aglycones, including daidzein, glycitein, genistein and a portion of glucosides, including daidzin, glycitin, genistin, malonyldaidzin and malonylgenistin. These results provide an important reference pertaining to the effects of GDL on the aggregation of soy protein-isoflavone complexes and could benefit future research regarding the production of tofu from soymilk. PMID:27760990

  15. Association Between Foldability and Aggregation Propensity in Small Disulfide-Rich Proteins

    PubMed Central

    Fraga, Hugo; Graña-Montes, Ricardo; Illa, Ricard; Covaleda, Giovanni

    2014-01-01

    Abstract Aims: Disulfide-rich domains (DRDs) are small proteins whose native structure is stabilized by the presence of covalent disulfide bonds. These domains are versatile and can perform a wide range of functions. Many of these domains readily unfold on disulfide bond reduction, suggesting that in the absence of covalent bonding they might display significant disorder. Results: Here, we analyzed the degree of disorder in 97 domains representative of the different DRDs families and demonstrate that, in terms of sequence, many of them can be classified as intrinsically disordered proteins (IDPs) or contain predicted disordered regions. The analysis of the aggregation propensity of these domains indicates that, similar to IDPs, their sequences are more soluble and have less aggregating regions than those of other globular domains, suggesting that they might have evolved to avoid aggregation after protein synthesis and before they can attain its compact and covalently linked native structure. Innovation and Conclusion: DRDs, which resemble IDPs in the reduced state and become globular when their disulfide bonds are formed, illustrate the link between protein folding and aggregation propensities and how these two properties cannot be easily dissociated, determining the main traits of the folding routes followed by these small proteins to attain their native oxidized states. Antioxid. Redox Signal. 21, 368–383. PMID:24635049

  16. 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

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

    PubMed

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

    2016-04-01

    The discovery that the alteration of aging by reducing the activity of the insulin/IGF-1 signaling (IIS) cascade protects nematodes and mice from neurodegeneration-linked, toxic protein aggregation (proteotoxicity) raises the prospect that IIS inhibitors bear therapeutic potential to counter neurodegenerative diseases. Recently, we reported that NT219, a highly efficient IGF-1 signaling inhibitor, protects model worms from the aggregation of amyloid β peptide and polyglutamine peptides that are linked to the manifestation of Alzheimer's and Huntington's diseases, respectively. Here, we employed cultured cell systems to investigate whether NT219 promotes protein homeostasis (proteostasis) in mammalian cells and to explore its underlying mechanisms. We found that NT219 enhances the aggregation of misfolded prion protein and promotes its deposition in quality control compartments known as "aggresomes." NT219 also elevates the levels of certain molecular chaperones but, surprisingly, reduces proteasome activity and impairs autophagy. Our findings show that IGF-1 signaling inhibitors in general and NT219 in particular can promote proteostasis in mammalian cells by hyperaggregating hazardous proteins, thereby bearing the potential to postpone the onset and slow the progression of neurodegenerative illnesses in the elderly.-Moll, L., Ben-Gedalya, T., Reuveni, H., Cohen, E. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition. PMID:26722006

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

    PubMed

    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. PMID:27435961

  19. 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.

  20. 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.

  1. Hydroxybutyrate prevents protein aggregation in the halotolerant bacterium Pseudomonas sp. CT13 under abiotic stress.

    PubMed

    Soto, Gabriela; Setten, Lorena; Lisi, Christian; Maurelis, Camila; Mozzicafreddo, Matteo; Cuccioloni, Massimiliano; Angeletti, Mauro; Ayub, Nicolás Daniel

    2012-05-01

    Polyhydroxybutyrate (PHB), a typical carbon and energy storage compound, is widely found in Bacteria and Archae domains. This polymer is produced in response to conditions of physiological stress. PHB is composed of repeating units of β-hydroxybutyrate (R-3HB). It has been previously shown that R-3HB functions as an osmolyte in extremophile strains. In this study, Pseudomonas sp. CT13, a halotolerant bacterium, and its PHB synthase-minus mutant (phaC) were used to analyze the chaperone role of R-3HB. The production of this compound was found to be essential to salt stress resistance and positively correlated with salt concentration, suggesting that PHB monomer acts as a compatible solute in Pseudomonas sp. CT13. R-3HB accumulation was also associated with the prevention of protein aggregation under combined salt and thermal stresses in Pseudomonas sp. CT13. Physiological concentrations of R-3HB efficiently reduced citrate synthase (CS) aggregation and stabilized the enzymatic activities of CS during thermal stress. Docking analysis of the CS/R-3HB interaction predicted the stability of this complex under physiological concentrations of R-3HB. Thus, in vivo, in vitro and in silico analyses suggest that R-3HB can act as a chemical chaperone. PMID:22527039

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

    PubMed

    Zhou, Jie; Wang, Jian; Cheng, Yuan; Chi, Ying-Jun; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2013-01-01

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

  3. NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses

    PubMed Central

    Cheng, Yuan; Chi, Ying-Jun; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2013-01-01

    Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions. PMID:23341779

  4. Self-assembly of protein amyloids: A competition between amorphous and ordered aggregation

    NASA Astrophysics Data System (ADS)

    Lee, Chiu Fan

    2009-09-01

    Protein aggregation in the form of amyloid fibrils has important biological and technological implications. Although the self-assembly process is highly efficient, aggregates not in the fibrillar form would also occur and it is important to include these disordered species when discussing the thermodynamic equilibrium behavior of the system. Here, we initiate such a task by considering a mixture of monomeric proteins and the corresponding aggregates in the disordered form (micelles) and in the fibrillar form (amyloid fibrils). Starting with a model on the respective binding free energies for these species, we calculate their concentrations at thermal equilibrium. We then discuss how the incorporation of the disordered structure furthers our understanding on the various amyloid promoting factors observed empirically, and on the kinetics of fibrilization.

  5. 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

  6. 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

  7. 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.

  8. 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. PMID:27019408

  9. Protective effects of caffeoylquinic acids on the aggregation and neurotoxicity of the 42-residue amyloid β-protein.

    PubMed

    Miyamae, Yusaku; Kurisu, Manami; Murakami, Kazuma; Han, Junkyu; Isoda, Hiroko; Irie, Kazuhiro; Shigemori, Hideyuki

    2012-10-01

    Alzheimer's disease (AD), a neurodegenerative disorder, is characterized by aggregation of 42-mer amyloid β-protein (Aβ42). Aβ42 aggregates through β-sheet formation and induces cytotoxicity against neuronal cells. Aβ42 oligomer, an intermediate of the aggregates, causes memory loss and synaptotoxicity in AD. Inhibition of Aβ42 aggregation by small molecules is thus a promising strategy for the treatment of AD. Caffeoylquinic acid (CQA), a phenylpropanoid found widely in natural sources including foods, shows various biological activities such as anti-oxidative ability. Previously, our group reported that 3,5-di-O-caffeoylquinic acid (3,5-di-CQA) rescued the cognitive impairment in senescence-accelerated-prone mice 8. However, structure-activity relationship of CQA derivatives on the aggregation and neurotoxicity of Aβ42 remains elusive. To evaluate the anti-amyloidogenic property of CQA-related compounds for AD therapy, we examined the effect of CQA and its derivatives on the aggregation and neurotoxicity of Aβ42. In particular, 4,5-di-O-caffeoylquinic acid (4,5-di-CQA) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-tri-CQA) strongly inhibited the aggregation of Aβ42 in a dose-dependent manner. Structure-activity relationship studies suggested that the caffeoyl group in CQA is essential for the inhibitory activity. These CQAs also suppressed the transformation into β-sheet and cytotoxicity against human neuroblastoma cells of Aβ42. Furthermore, 3,4,5-tri-CQA blocked the formation of Aβ42 oligomer. These results indicate that 3,4,5-tri-CQA could be a potential agent for the prevention of AD.

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

    PubMed

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

    2016-01-01

    As a major effective component in green tea, (-)-epigallocatechin-3-gallate (EGCG)'s potential benefits to human health have been widely investigated. Recent experimental evidences indicate that EGCG can induce the aggregation of HMGB1 protein, a late mediator of inflammation, which subsequently stimulates the autophagic degradation and thus provides protection from lethal endotoxemia and sepsis. In this study, we use molecular dynamics (MD) simulations to explore the underlying molecular mechanism of this aggregation of HMGB1 facilitated by EGCG. Our simulation results reveal that EGCG firmly binds to HMGB1 near Cys106, which supports previous preliminary experimental evidence. A large HMGB1 conformational change is observed, where Box A and Box B, two homogenous domains of HMGB1, are repositioned and packed together by EGCG. This new HMGB1 conformation has large molecular polarity and distinctive electrostatic potential surface. We suggest that the highly polarized charge distribution leads to the aggregation of HMGB1, which differs from the previous hypothesis that two HMGB1 monomers are linked by the dimer of EGCG. Possible aggregating modes have also been investigated with potential of mean force (PMF) calculations. Finally, we conclude that the conformation induced by EGCG is more aggregation-prone with higher binding free energies as compared to those without EGCG. PMID:26899177

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

    PubMed Central

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

    2016-01-01

    As a major effective component in green tea, (−)-epigallocatechin-3-gallate (EGCG)’s potential benefits to human health have been widely investigated. Recent experimental evidences indicate that EGCG can induce the aggregation of HMGB1 protein, a late mediator of inflammation, which subsequently stimulates the autophagic degradation and thus provides protection from lethal endotoxemia and sepsis. In this study, we use molecular dynamics (MD) simulations to explore the underlying molecular mechanism of this aggregation of HMGB1 facilitated by EGCG. Our simulation results reveal that EGCG firmly binds to HMGB1 near Cys106, which supports previous preliminary experimental evidence. A large HMGB1 conformational change is observed, where Box A and Box B, two homogenous domains of HMGB1, are repositioned and packed together by EGCG. This new HMGB1 conformation has large molecular polarity and distinctive electrostatic potential surface. We suggest that the highly polarized charge distribution leads to the aggregation of HMGB1, which differs from the previous hypothesis that two HMGB1 monomers are linked by the dimer of EGCG. Possible aggregating modes have also been investigated with potential of mean force (PMF) calculations. Finally, we conclude that the conformation induced by EGCG is more aggregation-prone with higher binding free energies as compared to those without EGCG. PMID:26899177

  12. Self-assembly of high molecular weight polypeptide copolymers studied via diffusion limited aggregation.

    PubMed

    Meier, Christoph; Wu, Yuzhou; Pramanik, Goutam; Weil, Tanja

    2014-01-13

    The assembly of high molecular weight polypeptides into complex architectures exhibiting structural complexity ranging from the nano- to the mesoscale is of fundamental importance for various protein-related diseases but also hold great promise for various nano- and biotechnological applications. Here, the aggregation of partially unfolded high molecular weight polypeptides into multiscale fractal structures is investigated by means of diffusion limited aggregation and atomic force microscopy. The zeta potential, the hydrodynamic radius, and the obtained fractal morphologies were correlated with the conformation of the polypeptide backbones as obtained from circular dichroism measurements. The polypeptides are modified with polyethylene oxide side chains to stabilize the polypeptides and to normalize intermolecular interactions. The modification with the hydrophobic thioctic acid alters the folding of the polypeptide backbone, resulting in a change in solution aggregation and fractal morphology. We found that a more compact folding results in dense and highly branched structures, whereas a less compact folded polypeptide chain yields a more directional assembly. Our results provide first evidence for the role of compactness of polypeptide folding on aggregation. Furthermore, the mesoscale-structured biofilms were used to achieve a hierarchical protein assembly, which is demonstrated by deposition of Rhodamine-labeled HSA with the preassembled fractal structures. These results contribute important insights to the fundamental understanding of the aggregation of high molecular weight polypeptides in general and provide opportunities to study nanostructure-related effects on biological systems such as adhesion, proliferation, and the development of, for example, neuronal cells. PMID:24354281

  13. Self-assembly of high molecular weight polypeptide copolymers studied via diffusion limited aggregation.

    PubMed

    Meier, Christoph; Wu, Yuzhou; Pramanik, Goutam; Weil, Tanja

    2014-01-13

    The assembly of high molecular weight polypeptides into complex architectures exhibiting structural complexity ranging from the nano- to the mesoscale is of fundamental importance for various protein-related diseases but also hold great promise for various nano- and biotechnological applications. Here, the aggregation of partially unfolded high molecular weight polypeptides into multiscale fractal structures is investigated by means of diffusion limited aggregation and atomic force microscopy. The zeta potential, the hydrodynamic radius, and the obtained fractal morphologies were correlated with the conformation of the polypeptide backbones as obtained from circular dichroism measurements. The polypeptides are modified with polyethylene oxide side chains to stabilize the polypeptides and to normalize intermolecular interactions. The modification with the hydrophobic thioctic acid alters the folding of the polypeptide backbone, resulting in a change in solution aggregation and fractal morphology. We found that a more compact folding results in dense and highly branched structures, whereas a less compact folded polypeptide chain yields a more directional assembly. Our results provide first evidence for the role of compactness of polypeptide folding on aggregation. Furthermore, the mesoscale-structured biofilms were used to achieve a hierarchical protein assembly, which is demonstrated by deposition of Rhodamine-labeled HSA with the preassembled fractal structures. These results contribute important insights to the fundamental understanding of the aggregation of high molecular weight polypeptides in general and provide opportunities to study nanostructure-related effects on biological systems such as adhesion, proliferation, and the development of, for example, neuronal cells.

  14. Protein adsorption, desorption, and aggregation mediated by solid-liquid interfaces.

    PubMed

    Perevozchikova, Tatiana; Nanda, Hirsh; Nesta, Douglas P; Roberts, Christopher J

    2015-06-01

    Adsorption of proteins to solid-fluid interfaces is often empirically found to promote formation of soluble aggregates and larger, subvisible, and visible particles, but key stages in this process are often difficult to probe directly. Aggregation mediated by adsorption to water-silicon oxide (SiOx) interfaces, akin to hydrated glass surfaces, was characterized as a function of pH and ionic strength for alpha-chymotrypsinogen (aCgn) and for a monoclonal antibody (IgG1). A flow cell permitted neutron reflectivity for protein layers adsorbed to clean SiOx surfaces, as well as after successive "rinse" steps. Aggregates recovered in solution after gently "rinsing" the surface were characterized by neutron scattering, microscopy, and fluorescence spectroscopy. IgG1 molecules oriented primarily "flat" against the SiOx surface, with the primary protein layer desorbed to a minimal extent, whereas a diffuse overlayer was easily rinsed off. aCgn molecules were resistant to desorption when they appeared to be unfolded at the interface, but were otherwise easily removed. For cases where strong binding occurred, protein that did desorb was a mixture of monomer and small amounts of HMW aggregates (for aCgn) or subvisible particles (for IgG1). Changes in adsorption and/or unfolding with pH indicated that electrostatic interactions were important in all cases.

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

    PubMed

    Chen, Min; von Mikecz, Anna

    2005-04-15

    Despite of their exponentially growing use, little is known about cell biological effects of nanoparticles. Here, we report uptake of silica (SiO(2)) nanoparticles to the cell nucleus where they induce aberrant clusters of topoisomerase I (topo I) in the nucleoplasm that additionally contain signature proteins of nuclear domains, and protein aggregation such as ubiquitin, proteasomes, cellular glutamine repeat (polyQ) proteins, and huntingtin. Formation of intranuclear protein aggregates (1) inhibits replication, transcription, and cell proliferation; (2) does not significantly alter proteasomal activity or cell viability; and (3) is reversible by Congo red and trehalose. Since SiO(2) nanoparticles trigger a subnuclear pathology resembling the one occurring in expanded polyglutamine neurodegenerative disorders, we suggest that integrity of the functional architecture of the cell nucleus should be used as a read out for cytotoxicity and considered in the development of safe nanotechnology. PMID:15777787

  16. Mathematical aspects of the kinetics of formation and degradation of linear peptide or protein aggregates.

    PubMed

    Zhdanov, Vladimir P

    2016-08-01

    In cells, peptides and proteins are sometimes prone to aggregation. In neurons, for example, amyloid β peptides form plaques related to Alzheimer's disease (AD). The corresponding kinetic models either ignore or do not pay attention to degradation of these species. Here, the author proposes a generic kinetic model describing formation and degradation of linear aggregates. The process is assumed to occur via reversible association of monomers and attachment of monomers to or detachment from terminal parts of aggregates. Degradation of monomers is described as a first-order process. Degradation of aggregates is considered to occur at their terminal and internal parts with different rates and these steps are described by first-order equations as well. Irrespective of the choice of the values of the rate constants, the model predicts that eventually the system reaches a stable steady state with the aggregate populations rapidly decreasing with increasing size at large sizes. The corresponding steady-state size distributions of aggregates are illustrated in detail. The transient kinetics are also shown. The observation of AD appears, however, to indicate that the peptide production becomes eventually unstable, i.e., the growth of the peptide population is not properly limited. This is expected to be related to the specifics of the genetic networks controlling the peptide production. Following this line, two likely general networks with, respectively, global negative and positive feedbacks in the peptide production are briefly discussed. PMID:27132946

  17. Pathological Propagation through Cell-to-Cell Transmission of Non-Prion Protein Aggregates in Neurodegenerative Disorders

    PubMed Central

    Lee, Seung-Jae; Desplats, Paula; Sigurdson, Christina; Tsigelny, Igor; Masliah, Eliezer

    2016-01-01

    Neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, fronto-temporal dementia, Huntington's Disease and Creutzfeldt-Jakob Disease (CJD) are characterized by progressive accumulation of protein aggregates in selected brain regions. Protein misfolding and templated assembly into aggregates might result from an imbalance between protein synthesis, aggregation and clearance. While protein misfolding and aggregation occur in most neurodegenerative disorders, the concept of spreading and infectivity of aggregates in the CNS has been reserved to prion diseases such as CJD and bovine spongiform encephalopathy. Emerging evidence suggests that prion-like spreading may occur in other neurodegenerative disorders, taking place with secreted proteins, such as amyloid-β,) and cytosolic proteins, such as tau, huntingtin and α-synuclein. Underlying molecular mechanisms and therapeutic implications are discussed. PMID:21045796

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

    PubMed Central

    2011-01-01

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

  19. Observation of precrystallization aggregation in protein solutions during centrifugation

    NASA Astrophysics Data System (ADS)

    Behlke, Joachim; Knespel, Andreas

    1996-01-01

    Sedimentation velocity experiments on supersaturated solutions of either lysozyme or papain were performed to analyse the nucleation process as a prerequisite for crystal growth. The method is based on the determination of oligomers from sedimentation patterns, the calculation of equilibrium constants for the association reactions and the estimation of the free energy involved in such processes. The transition from the state of unfavourable free energy that is necessary for the formation of smaller oligomers to that of free energy released in larger systems ensues in assemblies of fifteen to twenty molecules for both proteins. The observation of nuclei in less than one hour permits this method to be used for early recognition of crystal growth.

  20. Molecular-level insights of early-stage prion protein aggregation on mica and gold surface determined by AFM imaging and molecular simulation.

    PubMed

    Lou, Zhichao; Wang, Bin; Guo, Cunlan; Wang, Kun; Zhang, Haiqian; Xu, Bingqian

    2015-11-01

    By in situ time-lapse AFM, we investigated early-stage aggregates of PrP formed at low concentration (100 ng/mL) on mica and Au(111) surfaces in acetate buffer (pH 4.5). Remarkably different PrP assemblies were observed. Oligomeric structures of PrP aggregates were observed on mica surface, which was in sharp contrast to the multi-layer PrP aggregates yielding parallel linear patterns observed Au(111) surface. Combining molecular dynamics and docking simulations, PrP monomers, dimers and trimers were revealed as the basic units of the observed aggregates. Besides, the mechanisms of the observed PrP aggregations and the corresponding molecular-substrate and intermolecular interactions were suggested. These interactions involved gold-sulfur interaction, electrostatic interaction, hydrophobic interaction, and hydrogen binding interaction. In contrast, the PrP aggregates observed in pH 7.2 PBS buffer demonstrated similar large ball-like structures on both mica and Au(111) surfaces. The results indicate that the pH of a solution and the surface of the system can have strong effects on supramolecular assemblies of prion proteins. This study provides in-depth understanding on the structural and mechanistic nature of PrP aggregation, and can be used to study the aggregation mechanisms of other proteins with similar misfolding properties.

  1. Specification aggregate quarry expansion: a case study demonstrating sustainable management of natural aggregate resources

    USGS Publications Warehouse

    Langer, William H.; Tucker, M.L.

    2003-01-01

    Many countries, provinces, territories, or states in the European Union, Australia, Canada, the United States, and elsewhere have begun implementing sustainability programs, but most of those programs stop short of sustainable management of aggregate resources. Sustainable practices do not always have to be conducted under the title of sustainability. This case study describes how Lafarge, a large multinational construction materials supplier, implemented the principles of sustainability even though there was an absence of existing local government policies or procedures addressing sustainable resource management. Jefferson County, Colorado, USA, is one of three counties in the six-county Denver, Colorado, region that has potentially available sources of crushed stone. Crushed stone comprises 30 percent of the aggregate produced in the area and plays a major role in regional aggregate resource needs. Jefferson County is home to four of the five crushed stone operations in the Denver region. Lafarge operates one of those four quarries. Lafarge recently proposed to expand its reserves by exchanging company-owned land for existing dedicated open space land adjacent to their quarry but owned by Jefferson County. A similar proposal submitted about 10 years earlier had been denied. Contrary to the earlier proposal, which was predicated on public relations, the new proposal was predicated on public trust. Although not explicitly managed under the moniker of sustainability, Lafarge used basic management principles that embody the tenets of sustainability. To achieve the goals of sustainable aggregate management where no governmental policies existed, Lafarge not only assumed their role of being a responsible corporate and environmental member of the community, but also assumed the role of facilitator to encourage and enable other stakeholders to responsibly resolve legitimate concerns regarding the Lafarge quarry proposal. Lafarge successfully presented an enlightened

  2. Regulation of Protein Secretion Through Controlled Aggregation in the Endoplasmic Reticulum

    NASA Astrophysics Data System (ADS)

    Rivera, Victor M.; Wang, Xiurong; Wardwell, Scott; Courage, Nancy L.; Volchuk, Allen; Keenan, Terence; Holt, Dennis A.; Gilman, Michael; Orci, Lelio; Cerasoli, Frank; Rothman, James E.; Clackson, Tim

    2000-02-01

    A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

  3. Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum.

    PubMed

    Rivera, V M; Wang, X; Wardwell, S; Courage, N L; Volchuk, A; Keenan, T; Holt, D A; Gilman, M; Orci, L; Cerasoli, F; Rothman, J E; Clackson, T

    2000-02-01

    A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery. PMID:10657290

  4. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development.

    PubMed

    den Engelsman, John; Garidel, Patrick; Smulders, Ronald; Koll, Hans; Smith, Bryan; Bassarab, Stefan; Seidl, Andreas; Hainzl, Otmar; Jiskoot, Wim

    2011-04-01

    Within the European Immunogenicity Platform (EIP) ( http://www.e-i-p.eu ), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.

  5. Design of heat shock-resistant surfaces to prevent protein aggregation: Enhanced chaperone activity of immobilized α-Crystallin.

    PubMed

    Ray, Namrata; Roy, Sarita; Singha, Santiswarup; Chandra, Bappaditya; Dasgupta, Anjan Kr; Sarkar, Amitabha

    2014-05-21

    α-Crystallin is a multimeric protein belonging to the family of small heat shock proteins, which function as molecular chaperones by resisting heat and oxidative stress induced aggregation of other proteins. We immobilized α-Crystallin on a self-assembled monolayer on glass surface and studied its activity in terms of the prevention of aggregation of aldolase. We discovered that playing with grafted protein density led to interesting variations in the chaperone activity of immobilized α-Crystallin. This result is in accordance with the hypothesis that dynamicity of subunits plays a vital role in the functioning of α-Crystallin and might be able to throw light on the structure-activity relationship. We showed that the chaperone activity of a certain number of immobilized α-Crystallins was superior compared to a solution containing an equivalent number of the protein and 10 times the number of the protein at temperatures >60 °C. The α-Crystallin grafted surfaces retained activity on reuse. This could also lead to the design of potent heat-shock resistant surfaces that can find wide applications in storage and shipping of protein based biopharmaceuticals.

  6. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins

    PubMed Central

    Bezawork-Geleta, Ayenachew; Brodie, Erica J.; Dougan, David A.; Truscott, Kaye N.

    2015-01-01

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPRmt). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPRmt. Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPRmt, this pathway may preferentially act to promote chaperone mediated refolding of proteins. PMID:26627475

  7. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins.

    PubMed

    Bezawork-Geleta, Ayenachew; Brodie, Erica J; Dougan, David A; Truscott, Kaye N

    2015-12-02

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPR(mt)). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPR(mt). Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPR(mt), this pathway may preferentially act to promote chaperone mediated refolding of proteins.

  8. Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

    PubMed Central

    Wu, Haixia; Truncali, Kristopher; Ritchie, Julie; Kroe-Barrett, Rachel; Singh, Sanjaya; Robinson, Anne S; Roberts, Christopher J

    2015-01-01

    The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77°C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the CH2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the CH2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs. PMID:26267255

  9. Characterization of Detergent-Insoluble Proteins in ALS Indicates a Causal Link between Nitrative Stress and Aggregation in Pathogenesis

    PubMed Central

    Basso, Manuela; Samengo, Giuseppina; Nardo, Giovanni; Massignan, Tania; D'Alessandro, Giuseppina; Tartari, Silvia; Cantoni, Lavinia; Marino, Marianna; Cheroni, Cristina; De Biasi, Silvia; Giordana, Maria Teresa; Strong, Michael J.; Estevez, Alvaro G.; Salmona, Mario; Bendotti, Caterina; Bonetto, Valentina

    2009-01-01

    Background Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited. Methodology/Principal Findings We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced. Conclusion/Significance Analysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS. PMID:19956584

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

    PubMed

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

    2016-04-19

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

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

    PubMed

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

    2016-04-19

    The surface and foaming properties of native soy glycinin (11S) and its heat-induced fibrillar aggregates, in the presence of natural surfactant steviol glycoside (STE), were investigated and compared at pH 7.0 to determine the impact of protein structure modification on protein-surfactant interfacial interactions. The adsorption at, and nonlinear dilatational rheological behavior of, the air-water interface were studied by combining drop shape analysis tensiometry, ellipsometry, and large-amplitude oscillatory dilatational rheology. Lissajous plots of surface pressure versus deformation were used to analyze the surface rheological response in terms of interfacial microstructure. The heat treatment generates a mixture of long fibrils and unconverted peptides. The presence of small peptides in 11S fibril samples resulted in a faster adsorption kinetics than that of native 11S. The addition of STE affected the adsorption of 11S significantly, whereas no apparent effect on the adsorption of the 11S fibril-peptide system was observed. The rheological response of interfaces stabilized by 11S-STE mixtures also differed significantly from the response for 11S fibril-peptide-STE mixtures. For 11S, the STE reduces the degree of strain hardening in extension and increases strain hardening in compression, suggesting the interfacial structure may change from a surface gel to a mixed phase of protein patches and STE domains. The foams generated from the mixtures displayed comparable foam stability to that of pure 11S. For 11S fibril-peptide mixtures STE only significantly affects the response in extension, where the degree of strain softening is decreased compared to the pure fibril-peptide system. The foam stability of the fibril-peptide system was significantly reduced by STE. These findings indicate that fibrillization of globular proteins could be a potential strategy to modify the complex surface and foaming behaviors of protein-surfactant mixtures. PMID:27043221

  12. Automatic segmentation and 3D feature extraction of protein aggregates in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Rodrigues, Pedro L.; Moreira, António H. J.; Teixeira-Castro, Andreia; Oliveira, João; Dias, Nuno; Rodrigues, Nuno F.; Vilaça, João L.

    2012-03-01

    In the last years, it has become increasingly clear that neurodegenerative diseases involve protein aggregation, a process often used as disease progression readout and to develop therapeutic strategies. This work presents an image processing tool to automatic segment, classify and quantify these aggregates and the whole 3D body of the nematode Caenorhabditis Elegans. A total of 150 data set images, containing different slices, were captured with a confocal microscope from animals of distinct genetic conditions. Because of the animals' transparency, most of the slices pixels appeared dark, hampering their body volume direct reconstruction. Therefore, for each data set, all slices were stacked in one single 2D image in order to determine a volume approximation. The gradient of this image was input to an anisotropic diffusion algorithm that uses the Tukey's biweight as edge-stopping function. The image histogram median of this outcome was used to dynamically determine a thresholding level, which allows the determination of a smoothed exterior contour of the worm and the medial axis of the worm body from thinning its skeleton. Based on this exterior contour diameter and the medial animal axis, random 3D points were then calculated to produce a volume mesh approximation. The protein aggregations were subsequently segmented based on an iso-value and blended with the resulting volume mesh. The results obtained were consistent with qualitative observations in literature, allowing non-biased, reliable and high throughput protein aggregates quantification. This may lead to a significant improvement on neurodegenerative diseases treatment planning and interventions prevention.

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

    PubMed

    Govers, Sander K; Aertsen, Abram

    2015-11-20

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

  14. Difference in aggregation between functional and toxic amyloids studied by atomistic simulations

    NASA Astrophysics Data System (ADS)

    Carballo Pacheco, Martin; Ismail, Ahmed E.; Strodel, Birgit

    Amyloids are highly structured protein aggregates, normally associated with neurodegenerative diseases such as Alzheimer's disease. In recent years, a number of nontoxic amyloids with physiologically normal functions, called functional amyloids, have been found. It is known that soluble small oligomers are more toxic than large fibrils. Thus, we study with atomistic explicit-solvent molecular dynamics simulations the oligomer formation of the amyloid- β peptide Aβ25 - 35, associated with Alzheimer's disease, and two functional amyloid-forming tachykinin peptides: kassinin and neuromedin K. Our simulations show that monomeric peptides in extended conformations aggregate faster than those in collapsed hairpin-like conformations. In addition, we observe faster aggregation by functional amyloids than toxic amyloids, which could explain their lack of toxicity.

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

    PubMed

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

    2014-08-01

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

  16. Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics.

    PubMed

    Bajardi-Taccioli, Adriana; Blum, Andrew; Xu, Chongfeng; Sosic, Zoran; Bergelson, Svetlana; Feschenko, Marina

    2015-10-01

    Recycling of antibodies and Fc containing therapeutic proteins by the neonatal Fc receptor (FcRn) is known to prolong their persistence in the bloodstream. Fusion of Fc fragment of IgG1 to other proteins is one of the strategies to improve their pharmacokinetic properties. Accurate measurement of Fc-FcRn binding provides information about the strength of this interaction, which in most cases correlates with serum half-life of the protein. It can also offer insight into functional integrity of Fc region. We investigated FcRn binding activity of a large set of Fc-fusion samples after thermal stress by the method based on AlphaScreen technology. An unexpected significant increase in FcR binding was found to correlate with formation of aggregates in these samples. Monomer purified from a thermally-stressed sample had normal FcRn binding, confirming that its Fc portion was intact. Experiments with aggregates spiked into a sample with low initial aggregation level, demonstrated strong correlation between the level of aggregates and FcRn binding. This correlation varied significantly in different methods. By introducing modifications to the assay format we were able to minimize the effects of aggregated species on FcRn binding, which should prevent masking functional changes of Fc-fusion protein. Biolayer interferometry (BLI) was used as an alternative method to measure FcRn binding. Both optimized AlphaScreen- and BLI-based assays were sensitive to structural changes in Fc portion of the molecule, such as oxidation of methionines 252 and 428, and therefore suitable for characterization of FcRn binding.

  17. Light scattering / SEC study of iota-carageenan aggregation

    NASA Astrophysics Data System (ADS)

    Sharp, Erica; Thomson, Robert; Norwood, David

    2003-11-01

    Carrageenans are believed to change conformation from random coil to helix when varying temperature and/or salt concentration. It is a point of contention as to whether this helix is formed from one coil or two. We present a study of this conformation change using viscometry and multi-angle laser light scattering (MALLS) with size-exclusion chromatography (SEC). Light scattering results show a clear increase in molecular weight and radius of gyration within 30 minutes of adding NaCl to high concentrations of iota-carrageenan, which may be interpreted as the conformation change described. However, estimates of persistence length ( ˜ R_G^2 / M_W) show no strong variation when increasing added salt. This suggests that the molecular weight increase reflects an aggregation of polymer molecules instead of a change in conformation. The iota-carrageenan molecules continue to aggregate slowly (hours to days depending on NaCl concentration). The final molecular weight of the aggregates depends on salt concentration, but the aggregation time seems to depend only on polymer concentration. If samples mixed in pure water are injected while the SEC system flows a NaCl mobile phase, we see effects typical of salting a linear polyelectrolyte.

  18. Development of dual targeting inhibitors against aggregations of amyloid-β and tau protein.

    PubMed

    Fuse, Shinichiro; Matsumura, Keisuke; Fujita, Yuki; Sugimoto, Hachiro; Takahashi, Takashi

    2014-10-01

    Aggregations of both amyloid-β (Aβ) and hyper-phosphorylated tau proteins are recognized as key pathological manifestations of Alzheimer's disease (AD). Agents that inhibit both those forms of aggregation show promise as drug candidates. Seventeen oligo heteroaromatic compounds were rapidly synthesized via a one-pot, 3- or 4-component coupling procedure. Evaluations showed that compounds E16 and E18 were the most potent inhibitors of Aβ and tau aggregations (E16: IC50s = 0.38, 0.29 μM against Aβ, tau, respectively, E18: IC50s = 0.55, 0.30 μM against Aβ, tau, respectively).

  19. Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3{zeta} protein

    SciTech Connect

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

    2009-05-22

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

  20. Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins.

    PubMed

    Lea, Wendy A; O'Neil, Pierce T; Machen, Alexandra J; Naik, Subhashchandra; Chaudhri, Tapan; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T; Burns, Joshua R; Baldwin, Michael R; Khar, Karen R; Karanicolas, John; Fisher, Mark T

    2016-09-01

    Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies caused by protein folding defects or deleterious kinetic transitions. Current methods of examining binding of a ligand to these marginally stable native states are limited because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, and multidomain proteins) and metastable proteins (e.g., low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein, immobilized on a BLI biosensor, to increasing denaturant concentrations (urea or GuHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remains is detected by an increased level of GroEL binding. Because this kinetic denaturant pulse is brief, the amplitude of binding of GroEL to the immobilized protein depends on the duration of the exposure to the denaturant, the concentration of the denaturant, wash times, and the underlying protein unfolding-refolding kinetics; fixing all other parameters and plotting the GroEL binding amplitude versus denaturant pulse concentration result in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein manifests as a decreased level of GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant

  1. Salt- and pH-induced desorption: Comparison between non-aggregated and aggregated mussel adhesive protein, Mefp-1, and a synthetic cationic polyelectrolyte.

    PubMed

    Krivosheeva, Olga; Dedinaite, Andra; Claesson, Per M

    2013-10-15

    Mussel adhesive proteins are of great interest in many applications due to their ability to bind strongly to many types of surfaces under water. Effective use such proteins, for instance the Mytilus edulis foot protein - Mefp-1, for surface modification requires achievement of a large adsorbed amount and formation of a layer that is resistant towards desorption under changing conditions. In this work we compare the adsorbed amount and layer properties obtained by using a sample containing small Mefp-1 aggregates with that obtained by using a non-aggregated sample. We find that the use of the sample containing small aggregates leads to higher adsorbed amount, larger layer thickness and similar water content compared to what can be achieved with a non-aggregated sample. The layer formed by the aggregated Mefp-1 was, after removal of the protein from bulk solution, exposed to aqueous solutions with high ionic strength (up to 1M NaCl) and to solutions with low pH in order to reduce the electrostatic surface affinity. It was found that the preadsorbed Mefp-1 layer under all conditions explored was significantly more resistant towards desorption than a layer built by a synthetic cationic polyelectrolyte with similar charge density. These results suggest that the non-electrostatic surface affinity for Mefp-1 is larger than for the cationic polyelectrolyte.

  2. A Fiber Optic Probe for Monitoring Protein Aggregation, Nucleation, and Crystallization

    NASA Technical Reports Server (NTRS)

    Ansari, Rafat R.; Suh, Kwang I.; Arabshahi, Alireza; Wilson, William W.; Bray, Terry L.; DeLucas, Lawrence J.

    1996-01-01

    Protein crystals are experimentally grown in hanging drops in microgravity experiments on-board the Space Shuttle orbiter. The technique of dynamic light scattering (DLS) can be used to monitor crystal growth process in hanging droplets (approx. 30 (L)) in microgravity experiments, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. In this paper we demonstrate that such experiments are now feasible. We apply a newly developed fiber optic probe to various earth and space (micro- gravity) bound protein crystallization system configurations to test its capability. These include conventional batch (cuvette or capillary) systems, hanging drop method in a six-pack hanging drop vapor diffusion apparatus (HDVDA), a modified HDVDA for temperature- induced nucleation and aggregation studies, and a newly envisioned dynamically controlled vapor diffusion system (DCVDS) configuration. Our compact system exploits the principles of DLS and offers a fast (within a few seconds) means of quantitatively and non-invasively monitoring the various growth stages of protein crystallization. In addition to DLS capability, the probe can also be used for performing single-angle static light scattering measurements. It utilizes extremely low levels of laser power (approx. few (W)) without a need of having any optical alignment and vibration isolation. The compact probe is also equipped with a miniaturized microscope for visualization of macroscopic protein crystals. This new optical diagnostic system opens up enormous opportunity for exploring new ways to grow good quality crystals suitable for x-ray crystallographic analysis and may help develop a concrete scientific basis for understanding the process of crystallization.

  3. Identification of key amino acids responsible for the distinct aggregation properties of microtubule-associated protein 2 and tau.

    PubMed

    Xie, Ce; Soeda, Yoshiyuki; Shinzaki, Yuki; In, Yasuko; Tomoo, Koji; Ihara, Yasuo; Miyasaka, Tomohiro

    2015-10-01

    The carboxyl-terminal sequence of tau composes the framework for its intracellular inclusions that appear in diverse neurodegenerative disorders known as tauopathies. However, microtubule-associated protein 2 (MAP2), which contains a homologous carboxyl-terminal sequence of tau, is undetectable in the mature tau inclusions. The mechanisms underlying this phenomenon have remained largely unknown. Here, we show that tau and MAP2 have different aggregation properties: tau aggregates to form filaments but MAP2 remains to be granules. Exchanging (221) YKPV(224) of tau (0N3R) near the PHF6 motif for (340) TKKI(343) of MAP2c profoundly changed aggregation properties, suggesting that the YKPV motif is important for filament formation, whereas the TKKI motif is for granule formation. Thus, these minimal sequences may determine the different fates of tau and MAP2 in the formation of inclusions in tauopathies. Tau and microtubule-associated protein 2 (MAP2) are homologous microtubule-associated proteins in neurons. So far, it is largely unknown why tau but not MAP2 is selectively involved in the filamentous inclusions (neurofibrillary tangles, NFT) formation in tauopathies, including Alzheimer's disease. In this study, we found that the difference of only two amino acids in tau and MAP2 sequences may determine their different fates in tauopathies. These results may lead to the elucidation of tau deregulation in pathological conditions.

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

    PubMed

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

    2015-01-01

    Mammalian expression systems are used routinely for the production of recombinant proteins as therapeutic molecules as well as research tools. Transient expression has become increasingly popular in recent years due to its rapid timeline and improvements in expression level. While improvements to transient expression systems have focused mainly on the level of protein expression, the aspect of protein quality has received little attention. The removal of undesirable products, such as aggregation, depends primarily on purification, requiring additional cumbersome steps, which can lead to a lower product yield and longer timelines. In this study, we show that reducing the level of transcription by transfecting at a lower gene dose improves the quality of secreted molecules prone to aggregation. For gene dosing to have this effect, it is critical for the carrier DNA to be an empty vector containing the same elements as the gene containing plasmid. This approach can be used in combination with a temperature shift to hypothermic conditions during production to enhance the effect. The observed improvements not only minimized aggregation levels, but also generated products with overall superior quality, including more homogeneous signal peptide cleavage and N-linked glycosylation profiles. These techniques have produced a similar improvement in product quality with a variety of other molecules, suggesting that this may be a general approach to enhance product quality from transient expression systems. PMID:25395220

  5. Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

    SciTech Connect

    Yamagishi, Nobuyuki; Goto, Kazumasa; Nakagawa, Satomi; Saito, Youhei; Hatayama, Takumi

    2010-09-10

    Hsp105{alpha} and Hsp105{beta} are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105{alpha} is expressed constitutively in the cytoplasm of cells, while Hsp105{beta}, an alternatively spliced form of Hsp105{alpha}, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105{beta} but also Hsp105{alpha} accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105{alpha} and Hsp105{beta} with changes in the nuclear localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105{alpha} and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105{alpha} and Hsp105{beta} on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105{alpha} and Hsp105{beta} suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.

  6. Oxidation of sarcoplasmic proteins during processing of Cantonese sausage in relation to their aggregation behaviour and in vitro digestibility.

    PubMed

    Sun, Weizheng; Zhao, Mouming; Yang, Bao; Zhao, Haifeng; Cui, Chun

    2011-07-01

    The physicochemical changes of sarcoplasmic proteins, especially oxidation behaviour, were measured to determine their mechanism of action on in vitro protein digestibility during Cantonese sausage processing. The results indicated that carbonyl level increased (p<0.05) during the process. The fluorescence loss of tryptophan residues was a direct consequence of the oxidative degradation. All the parameters of protein aggregation were highly (p<0.05) correlated with carbonyl level and protein surface hydrophobicity (H(0)), indicating that protein oxidation and thermal denaturation could induce protein aggregation, leading to secondary structural changes. The analysis of in vitro digestibility showed no correlation between pepsin activity and protein oxidation, due to the biphasic response of sarcoplasmic proteins toward proteolysis. However, a highly significant (p<0.05) correlation was observed with trypsin and α-chymotrypsin activity, indicating that protein oxidation induced the changes in H(0), protein aggregation and secondary structure, which further influenced in vitro digestibility. PMID:21353394

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

    PubMed Central

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

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  9. Oil as reaction medium for glycation, oxidation, denaturation, and aggregation of whey protein systems of low water activity.

    PubMed

    Potes, Naritchaya; Kerry, Joseph P; Roos, Yrjö H

    2013-04-17

    Whey protein isolate (WPI)-oil (75:25) and WPI-oil-(glucose-fructose) (45:15:40) as models of high-protein systems containing either olive (OO) or sunflower oil (SO) were stored at 20 or 40 °C to investigate component interactions. The indicators of protein oxidation (carbonyl content) and aggregation (total sulfhydryl content) and heats of protein denaturation and aggregation were investigated. Highest levels of disulfide bonding and carbonyls in WPI-OO formed during the first 2 weeks of storage concomitantly with enhanced protein aggregation. WPI-OO and WPI-SO systems (prestorage) showed increased protein denaturation temperature. The WPI proteins showed higher heat sensitivity with OO or SO at 40 °C, and the system with OO showed preaggregated protein as found from decreased heats of protein aggregation. OO or SO in WPI-oil-(glucose-fructose) systems reduced heats of protein aggregation. Lipid oxidation products and nonenzymatic browning reactions in glucose-fructose-containing systems decreased the solubility of solids and increased protein aggregation, hydrophobicity, and hardening of structure. PMID:23517062

  10. Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability.

    PubMed

    Camilloni, Carlo; Sala, Benedetta Maria; Sormanni, Pietro; Porcari, Riccardo; Corazza, Alessandra; De Rosa, Matteo; Zanini, Stefano; Barbiroli, Alberto; Esposito, Gennaro; Bolognesi, Martino; Bellotti, Vittorio; Vendruscolo, Michele; Ricagno, Stefano

    2016-01-01

    A wide range of human diseases is associated with mutations that, destabilizing proteins native state, promote their aggregation. However, the mechanisms leading from folded to aggregated states are still incompletely understood. To investigate these mechanisms, we used a combination of NMR spectroscopy and molecular dynamics simulations to compare the native state dynamics of Beta-2 microglobulin (β2m), whose aggregation is associated with dialysis-related amyloidosis, and its aggregation-resistant mutant W60G. Our results indicate that W60G low aggregation propensity can be explained, beyond its higher stability, by an increased average protection of the aggregation-prone residues at its surface. To validate these findings, we designed β2m variants that alter the aggregation-prone exposed surface of wild-type and W60G β2m modifying their aggregation propensity. These results allowed us to pinpoint the role of dynamics in β2m aggregation and to provide a new strategy to tune protein aggregation by modulating the exposure of aggregation-prone residues. PMID:27150430

  11. Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability

    PubMed Central

    Camilloni, Carlo; Sala, Benedetta Maria; Sormanni, Pietro; Porcari, Riccardo; Corazza, Alessandra; De Rosa, Matteo; Zanini, Stefano; Barbiroli, Alberto; Esposito, Gennaro; Bolognesi, Martino; Bellotti, Vittorio; Vendruscolo, Michele; Ricagno, Stefano

    2016-01-01

    A wide range of human diseases is associated with mutations that, destabilizing proteins native state, promote their aggregation. However, the mechanisms leading from folded to aggregated states are still incompletely understood. To investigate these mechanisms, we used a combination of NMR spectroscopy and molecular dynamics simulations to compare the native state dynamics of Beta-2 microglobulin (β2m), whose aggregation is associated with dialysis-related amyloidosis, and its aggregation-resistant mutant W60G. Our results indicate that W60G low aggregation propensity can be explained, beyond its higher stability, by an increased average protection of the aggregation-prone residues at its surface. To validate these findings, we designed β2m variants that alter the aggregation-prone exposed surface of wild-type and W60G β2m modifying their aggregation propensity. These results allowed us to pinpoint the role of dynamics in β2m aggregation and to provide a new strategy to tune protein aggregation by modulating the exposure of aggregation-prone residues. PMID:27150430

  12. Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability

    NASA Astrophysics Data System (ADS)

    Camilloni, Carlo; Sala, Benedetta Maria; Sormanni, Pietro; Porcari, Riccardo; Corazza, Alessandra; De Rosa, Matteo; Zanini, Stefano; Barbiroli, Alberto; Esposito, Gennaro; Bolognesi, Martino; Bellotti, Vittorio; Vendruscolo, Michele; Ricagno, Stefano

    2016-05-01

    A wide range of human diseases is associated with mutations that, destabilizing proteins native state, promote their aggregation. However, the mechanisms leading from folded to aggregated states are still incompletely understood. To investigate these mechanisms, we used a combination of NMR spectroscopy and molecular dynamics simulations to compare the native state dynamics of Beta-2 microglobulin (β2m), whose aggregation is associated with dialysis-related amyloidosis, and its aggregation-resistant mutant W60G. Our results indicate that W60G low aggregation propensity can be explained, beyond its higher stability, by an increased average protection of the aggregation-prone residues at its surface. To validate these findings, we designed β2m variants that alter the aggregation-prone exposed surface of wild-type and W60G β2m modifying their aggregation propensity. These results allowed us to pinpoint the role of dynamics in β2m aggregation and to provide a new strategy to tune protein aggregation by modulating the exposure of aggregation-prone residues.

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

    PubMed

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

    2016-01-06

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

  14. The structural basis for optimal performance of oligothiophene-based fluorescent amyloid ligands: conformational flexibility is essential for spectral assignment of a diversity of protein aggregates.

    PubMed

    Klingstedt, Therése; Shirani, Hamid; Åslund, K O Andreas; Cairns, Nigel J; Sigurdson, Christina J; Goedert, Michel; Nilsson, K Peter R

    2013-07-29

    Protein misfolding diseases are characterized by deposition of protein aggregates, and optical ligands for molecular characterization of these disease-associated structures are important for understanding their potential role in the pathogenesis of the disease. Luminescent conjugated oligothiophenes (LCOs) have proven useful for optical identification of a broader subset of disease-associated protein aggregates than conventional ligands, such as thioflavin T and Congo red. Herein, the molecular requirements for achieving LCOs able to detect nonthioflavinophilic Aβ aggregates or non-congophilic prion aggregates, as well as spectrally discriminate Aβ and tau aggregates, were investigated. An anionic pentameric LCO was subjected to chemical engineering by: 1) replacing thiophene units with selenophene or phenylene moieties, or 2) alternating the anionic substituents along the thiophene backbone. In addition, two asymmetric tetrameric ligands were generated. Overall, the results from this study identified conformational freedom and extended conjugation of the conjugated backbone as crucial determinants for obtaining superior thiophene-based optical ligands for sensitive detection and spectral assignment of disease-associated protein aggregates.

  15. 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

  16. The Protein Oxidation Repair Enzyme Methionine Sulfoxide Reductase A Modulates Aβ Aggregation and Toxicity In Vivo

    PubMed Central

    Minniti, Alicia N.; Arrazola, Macarena S.; Bravo-Zehnder, Marcela; Ramos, Francisca; Inestrosa, Nibaldo C.

    2015-01-01

    Abstract Aims: To examine the role of the enzyme methionine sulfoxide reductase A-1 (MSRA-1) in amyloid-β peptide (Aβ)-peptide aggregation and toxicity in vivo, using a Caenorhabditis elegans model of the human amyloidogenic disease inclusion body myositis. Results: MSRA-1 specifically reduces oxidized methionines in proteins. Therefore, a deletion of the msra-1 gene was introduced into transgenic C. elegans worms that express the Aβ-peptide in muscle cells to prevent the reduction of oxidized methionines in proteins. In a constitutive transgenic Aβ strain that lacks MSRA-1, the number of amyloid aggregates decreases while the number of oligomeric Aβ species increases. These results correlate with enhanced synaptic dysfunction and mislocalization of the nicotinic acetylcholine receptor ACR-16 at the neuromuscular junction (NMJ). Innovation: This approach aims at modulating the oxidation of Aβ in vivo indirectly by dismantling the methionine sulfoxide repair system. The evidence presented here shows that the absence of MSRA-1 influences Aβ aggregation and aggravates locomotor behavior and NMJ dysfunction. The results suggest that therapies which boost the activity of the Msr system could have a beneficial effect in managing amyloidogenic pathologies. Conclusion: The absence of MSRA-1 modulates Aβ-peptide aggregation and increments its deleterious effects in vivo. Antioxid. Redox Signal. 22, 48–62. PMID:24988428

  17. The Chaperone-Like Activity of α-Synuclein Attenuates Aggregation of Its Alternatively Spliced Isoform, 112-Synuclein In Vitro: Plausible Cross-Talk between Isoforms in Protein Aggregation

    PubMed Central

    Manda, Krishna Madhuri; Yedlapudi, Deepthi; Korukonda, Srikanth; Bojja, Sreedhar; Kalivendi, Shasi V.

    2014-01-01

    Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn) has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD). Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synclein (112-syn) in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45°C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT) staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37°C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates. PMID:24892822

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  1. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC.

  2. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC. PMID:16550298

  3. Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

    PubMed

    Valiente-Gabioud, Ariel A; Miotto, Marco C; Chesta, María E; Lombardo, Verónica; Binolfi, Andres; Fernández, Claudio O

    2016-05-17

    The aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aβ) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aβ, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the

  4. Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

    PubMed

    Valiente-Gabioud, Ariel A; Miotto, Marco C; Chesta, María E; Lombardo, Verónica; Binolfi, Andres; Fernández, Claudio O

    2016-05-17

    The aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aβ) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aβ, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the

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

    PubMed

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

    2015-01-01

    Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum. PMID:25178969

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

    PubMed

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

    2010-11-01

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

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

    PubMed

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

    2015-01-01

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

  8. Molecular Dynamics Simulations to Clarify the Concentration Dependency of Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Nishikawa, Naohiro; Sakae, Yoshitake; Okamoto, Yuko

    We examined the concentration dependency of amyloid protein aggregation by using several molecular dynamics simulations, which were performed with different concentrations for each system. For these simulations, we used a fragment of amyloid-β, which is believed to be the cause of Alzheimer's disease, as our simulation system. We found that high concentration of amyloid peptides promotes the formation of β-structures which is the origin of amyloid fibrils.

  9. Ice-induced partial unfolding and aggregation of an integral membrane protein.

    PubMed

    Garber Cohen, Iona P; Castello, Pablo R; González Flecha, F Luis

    2010-11-01

    Although the deleterious effects of ice on water-soluble proteins are well established, little is known about the freeze stability of membrane proteins. Here we explore this issue through a combined kinetic and spectroscopic approach using micellar-purified plasma membrane calcium pump as a model. The ATPase activity of this protein significantly diminished after freezing using a slow-cooling procedure, with the decrease in the activity being an exponential function of the storage time at 253K, with t(½)=3.9±0.6h. On the contrary, no significant changes on enzyme activity were detected when a fast cooling procedure was performed. Regardless of the cooling rate, successive freeze-thaw cycles produced an exponential decrease in the Ca(2+)-ATPase activity, with the number of cycles at which the activity was reduced to half being 9.2±0.3 (fast cooling) and 3.7±0.2 (slow cooling). PAGE analysis showed that neither degradation nor formation of SDS-stable aggregates of the protein takes place during protein inactivation. Instead, the inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, far UV circular dichroism, and 1-anilino-naphtalene-8-sulfonate binding. This inactive protein undergoes, in a later stage, a further irreversible transformation leading to large aggregates.

  10. The challenging SO2-mediated chemical build-up of protein aggregates in wines.

    PubMed

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

    2016-02-01

    Despite the extensive research performed during the last decades, the multifactorial mechanism responsible for white wine protein haze formation is not fully characterized. A model is proposed, which is essentially based on two postulates: the experimental identification of sulfur dioxide as the non-proteinaceous factor, and the inference from reliable data available in the literature of the dynamic chemistry played by wine protein sulfhydryl groups. Unlike other reducing agents, addition of SO2 to must/wine upon heating cleaves intraprotein disulfide bonds, hinders thiol-disulfide exchange during protein interactions, and leads to formation of novel interprotein disulfide bonds. These bonds are ultimately responsible for wine protein aggregation following a nucleation-growth kinetic model, as shown by Dynamic Light Scattering experiments. The model was tested in wine model solution (using total and fractionated wine proteins) and validated under real wine conditions. The results achieved may open the way to develop techniques that will find wide application in the wine industry.

  11. Soy protein nanoparticle aggregates as pickering stabilizers for oil-in-water emulsions.

    PubMed

    Liu, Fu; Tang, Chuan-He

    2013-09-18

    In recent years, there have been increasing interests in developing food-grade Pickering stabilizers, due to their potential applications in formulations of novel functional foods. The present work was to investigate the potential of soy proteins to be developed into a kind of Pickering-like stabilizer for oil-in-water emulsions. The nanoparticle aggregates of soy protein isolate (SPI) were formed by sequential treatments of heating at 95 °C for 15 min and then electrostatic screening with NaCl addition. The particle size and microstructure of these aggregates were characterized using dynamic light scattering and atomic force microscopy, indicating that the fabricated nanoparticle aggregates were ∼100 nm in size with more surface hydrophobic nature (relative to unheated SPI). The influence of particle concentration (c; 0.5-6.0%, w/w) and increasing oil fraction (ϕ; in the range 0.2-0.6) on the droplet size and coalescence and/or creaming stability of the emulsions stabilized by these nanoparticle aggregates was investigated. The results showed that, at ϕ = 0.2, increasing the c resulted in a progressive but slight decrease in droplet size, and improved the stability against coalescence and creaming; at a specific c, the creaming stability was progressively increased by increasing the ϕ, with better improvement observed at a higher c (e.g., 6.0% vs 2.0%). The improvement of creaming stability was largely associated with the formation of a gel-like network that could entrap the oil droplets within the network. The observations are generally consistent with those observed for the conventional Pickering emulsions, confirming that soy proteins could be applied as a kind of effective Pickering-like stabilizer. The finding may have important implications for the design and fabrication of protein-based emulsion formulations, and even for the development of soy protein products with some unique functions. To the authors' knowledge, this is the first work to report

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

    PubMed

    Pandey, R B; Farmer, B L

    2014-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

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

    PubMed

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

    2016-10-15

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

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

    PubMed

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

    2016-10-15

    The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8 (E8), 9 (E9), 10 (E10) and 11 (E11)), followed by acid precipitation. The obtained protein isolates were freeze dried. The protein isolates E8 and E9 resulted in a lower protein yield as well as less protein denaturation. These isolates also had a higher protein purity, more protein bands at higher molecular weights, and a higher protein solubility in the pH range of 3-4.5, compared to the isolates E10 and E11. Heating the 10%w/w protein isolate suspensions E8 and E9 led to increased aggregation, and semi-solid gels with a dense microstructure were formed. The isolate suspensions E10 and E11, on the other hand, aggregated less, did not form self-supporting gels and had loose particle arrangements. We conclude that extraction pH plays an important role in determining the functionality of quinoa protein isolates. PMID:27173553

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

    PubMed Central

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

    2010-01-01

    The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations. PMID:20094032

  17. Early aggregation studies of diabetic amyloid in solution

    NASA Astrophysics Data System (ADS)

    Singh, Sadanand; de Pablo, Juan

    2011-03-01

    Islet amyloid polypeptide (IAPP, also known as amylin) is responsible for pancreatic amyloid deposits in type II diabetes. The deposits, as well as intermediates in their assembly, are cytotoxic to pancreatic β -cells and contribute to the loss of β -cell mass associated with type II diabetes. To better understand the mechanism and cause of such aggregation, molecular simulations with explicit solvent models were used to compare monomer structure and early aggregation mechanism. Using free-energy maps generated~through~a variety of novel, enhanced sampling free-energy calculation techniques, we have found that, in water, the peptide adopts three major structures. One has a small α -helix at the N-terminus and a small β -hairpin at the other end. The second and the most stable one, is a complete β -hairpin, and the third is a random coil structure. Transition Path Sampling simulations along with reaction coordinate analysis reveal that the peptide follows a ``zipping mechanism'' in folding from α -helical to β -hairpin state. From studies of the dimerization of monomers in water, we have found that the early aggregation proceeds by conversion of all α -helical configurations to β -hairpins, and by two β -hairpins coming together to form a parallel β -sheet. Several aspects of the proposed mechanism have been verified by concerted 2D IR experimental measurements, thereby adding credence to the validity of our predictions.

  18. Aggregation of model asphaltenes: a molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Costa, J. L. L. F. S.; Simionesie, D.; Zhang, Z. J.; Mulheran, P. A.

    2016-10-01

    Natural asphaltenes are defined as polyaromatic compounds whose chemical composition and structure are dependent on their geological origin and production history, hence are regarded as complex molecules with aromatic cores and aliphatic tails that occur in the heaviest fraction of crude oil. The aggregation of asphaltenes presents a range of technical challenges to the production and processing of oil. In this work we study the behaviour of the model asphaltene-like molecule hexa-tert-butylhexa-peri-hexabenzocoronene (HTBHBC) using molecular dynamics simulation. It was found that the regular arrangement of the tert-butyl side chains prevents the formation of strongly-bound dimers by severely restricting the configurational space of the aggregation pathway. In contrast, a modified molecule with only 3 side chains is readily able to form dimers. This work therefore confirms the influence of the molecular structure of polyaromatic compounds on their aggregation mechanism, and reveals the unexpected design rules required for model systems that can mimic the behavior of asphaltenes.

  19. Aggregation of model asphaltenes: a molecular dynamics study.

    PubMed

    Costa, J L L F S; Simionesie, D; Zhang, Z J; Mulheran, P A

    2016-10-01

    Natural asphaltenes are defined as polyaromatic compounds whose chemical composition and structure are dependent on their geological origin and production history, hence are regarded as complex molecules with aromatic cores and aliphatic tails that occur in the heaviest fraction of crude oil. The aggregation of asphaltenes presents a range of technical challenges to the production and processing of oil. In this work we study the behaviour of the model asphaltene-like molecule hexa-tert-butylhexa-peri-hexabenzocoronene (HTBHBC) using molecular dynamics simulation. It was found that the regular arrangement of the tert-butyl side chains prevents the formation of strongly-bound dimers by severely restricting the configurational space of the aggregation pathway. In contrast, a modified molecule with only 3 side chains is readily able to form dimers. This work therefore confirms the influence of the molecular structure of polyaromatic compounds on their aggregation mechanism, and reveals the unexpected design rules required for model systems that can mimic the behavior of asphaltenes. PMID:27465036

  20. Rational design of solution additives for the prevention of protein aggregation.

    PubMed

    Baynes, Brian M; Trout, Bernhardt L

    2004-09-01

    We have developed a statistical-mechanical model of the effect of solution additives on protein association reactions. This model incorporates solvent radial distribution functions obtained from all-atom molecular dynamics simulations of particular proteins into simple models of protein interactions. In this way, the effects of additives can be computed along the entire association/dissociation reaction coordinate. We used the model to test our hypothesis that a class of large solution additives, which we term "neutral crowders," can slow protein association and dissociation by being preferentially excluded from protein-protein encounter complexes, in a manner analogous to osmotic stress. The magnitude of this proposed "gap effect" was probed for two simple model systems: the association of two spheres and the association of two planes. Our results suggest that for a protein of 20 A radius, an 8 A additive can increase the free energy barrier for association and dissociation by as much as 3-6 kcal/mol. Because the proposed gap effect is present only for reactions involving multiple molecules, it can be exploited to develop novel additives that affect protein association reactions although having little or no effect on unimolecular reactions such as protein folding. This idea has many potential applications in areas such as the stabilization of proteins against aggregation during folding and in pharmaceutical formulations.

  1. Study of the aggregation of human insulin Langmuir monolayer.

    PubMed

    Liu, Wei; Johnson, Sheba; Micic, Miodrag; Orbulescu, Jhony; Whyte, Jeffrey; Garcia, Andrew R; Leblanc, Roger M

    2012-02-21

    The human insulin (HI) Langmuir monolayer at the air-water interface was systematically investigated in the presence and absence of Zn(II) ions in the subphase. HI samples were dissolved in acidic (pH 2) and basic (pH 9) aqueous solutions and then spread at the air-water interface. Spectroscopic data of aqueous solutions of HI show a difference in HI conformation at different pH values. Moreover, the dynamics of the insulin protein showed a dependence on the concentration of Zn(II) ions. In the absence of Zn(II) ions in the subphase, the acidic and basic solutions showed similar behavior at the air-water interface. In the presence of Zn(II) ions in the subphase, the surface pressure-area and surface potential-area isotherms suggest that HI may aggregate at the air-water interface. It was observed that increasing the concentration of Zn(II) ions in the acidic (pH 2) aqueous solution of HI led to an increase of the area at a specific surface pressure. It was also seen that the conformation of HI in the basic (pH 9) medium had a reverse effect (decrease in the surface area) with the increase of the concentration of Zn(II) ions in solution. From the compression-decompression cycles we can conclude that the aggregated HI film at air-water interface is not stable and tends to restore a monolayer of monomers. These results were confirmed from UV-vis and fluorescence spectroscopy analysis. Infrared reflection-absorption and circular dichroism spectroscopy techniques were used to determine the secondary structure and orientation changes of HI by zinc ions. Generally, the aggregation process leads to a conformation change from α-helix to β-strand and β-turn, and at the air-water interface, the aggregation process was likewise seen to induce specific orientations for HI in the acidic and basic media. A proposed surface orientation model is presented here as an explanation to the experimental data, shedding light for further research on the behavior of insulin as a Langmuir

  2. Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate.

    PubMed

    Tao-Cheng, J-H

    2007-12-12

    Although it has been suggested that presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis. Here, I propose that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses. The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, the SV membrane proteins labeled the clear vesicles in the aggregate as well as many other SV-like and pleiomorphic vesicular structures in the axons, and synapsin I labeling was associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were approximately 0.22 by 0.13 microm in average dimensions and contain one to two DCV and five to six small clear vesicles. Serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.

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

    PubMed Central

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

    2014-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates

    PubMed Central

    Hong, Paula; Koza, Stephan; Bouvier, Edouard S. P.

    2012-01-01

    In recent years, the use and number of biotherapeutics has increased significantly. For these largely protein-based therapies, the quantitation of aggregates is of particular concern given their potential effect on efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). In the following review we will outline the history and background of SEC for the analysis of proteins. We will also discuss the instrumentation for these analyses, including the use of different types of detectors. Method development for protein analysis by SEC will also be outlined, including the effect of mobile phase and column parameters (column length, pore size). We will also review some of the applications of this mode of separation that are of particular importance to protein biopharmaceutical development and highlight some considerations in their implementation. PMID:23378719

  6. Structural and Aggregation Study of Protic Ionic Liquids

    NASA Astrophysics Data System (ADS)

    Mattedi, S.; Martin-Pastor, M.; Iglesias, M.

    2011-12-01

    In this work there were studied structural and agreggation aspects of ionic liquids formed by the reaction between ethanolamines with low chain organic acids using NMR techniques. Three ionic liquids composed of pentanoic acid and (mono-, di- and tri-) ethanol amine were studied by 1H, and 13C solution NMR methods. NMR assisted the chemical and quantitative characterization of these three ionic liquids and provided insight in their structural arrangement of their components in the ionic liquid medium. The obtained results could be used to understand the structure and aggregation pattern of these ionic liquids and helps in the development of possible industrial applications.

  7. Rosin Surfactant QRMAE Can Be Utilized as an Amorphous Aggregate Inducer: A Case Study of Mammalian Serum Albumin.

    PubMed

    Ishtikhar, Mohd; Chandel, Tajjali Ilm; Ahmad, Aamir; Ali, Mohd Sajid; Al-Lohadan, Hamad A; Atta, Ayman M; Khan, Rizwan Hasan

    2015-01-01

    Quaternary amine of diethylaminoethyl rosin ester (QRMAE), chemically synthesized biocompatible rosin based cationic surfactant, has various biological applications including its use as a food product additive. In this study, we examined the amorphous aggregation behavior of mammalian serum albumins at pH 7.5, i.e., two units above their isoelectric points (pI ~5.5), and the roles played by positive charge and hydrophobicity of exogenously added rosin surfactant QRMAE. The study was carried out on five mammalian serum albumins, using various spectroscopic methods, dye binding assay, circular dichroism and electron microscopy. The thermodynamics of the binding of mammalian serum albumins to cationic rosin modified surfactant were established using isothermal titration calorimetry (ITC). It was observed that a suitable molar ratio of protein to QRMAE surfactant enthusiastically induces amorphous aggregate formation at a pH above two units of pI. Rosin surfactant QRMAE-albumins interactions revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions that play an important role towards the formation of hydrophobic interactions-driven amorphous aggregate. Amorphous aggregation of proteins is associated with varying diseases, from the formation of protein wine haze to the expansion of the eye lenses in cataract, during the expression and purification of recombinant proteins. This study can be used for the design of novel biomolecules or drugs with the ability to neutralize factor(s) responsible for the aggregate formation, in addition to various other industrial applications.

  8. Rosin Surfactant QRMAE Can Be Utilized as an Amorphous Aggregate Inducer: A Case Study of Mammalian Serum Albumin

    PubMed Central

    Ishtikhar, Mohd; Chandel, Tajjali Ilm; Ahmad, Aamir; Ali, Mohd Sajid; Al-lohadan, Hamad A.; Atta, Ayman M.; Khan, Rizwan Hasan

    2015-01-01

    Quaternary amine of diethylaminoethyl rosin ester (QRMAE), chemically synthesized biocompatible rosin based cationic surfactant, has various biological applications including its use as a food product additive. In this study, we examined the amorphous aggregation behavior of mammalian serum albumins at pH 7.5, i.e., two units above their isoelectric points (pI ~5.5), and the roles played by positive charge and hydrophobicity of exogenously added rosin surfactant QRMAE. The study was carried out on five mammalian serum albumins, using various spectroscopic methods, dye binding assay, circular dichroism and electron microscopy. The thermodynamics of the binding of mammalian serum albumins to cationic rosin modified surfactant were established using isothermal titration calorimetry (ITC). It was observed that a suitable molar ratio of protein to QRMAE surfactant enthusiastically induces amorphous aggregate formation at a pH above two units of pI. Rosin surfactant QRMAE-albumins interactions revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions that play an important role towards the formation of hydrophobic interactions-driven amorphous aggregate. Amorphous aggregation of proteins is associated with varying diseases, from the formation of protein wine haze to the expansion of the eye lenses in cataract, during the expression and purification of recombinant proteins. This study can be used for the design of novel biomolecules or drugs with the ability to neutralize factor(s) responsible for the aggregate formation, in addition to various other industrial applications. PMID:26418451

  9. Rosin Surfactant QRMAE Can Be Utilized as an Amorphous Aggregate Inducer: A Case Study of Mammalian Serum Albumin.

    PubMed

    Ishtikhar, Mohd; Chandel, Tajjali Ilm; Ahmad, Aamir; Ali, Mohd Sajid; Al-Lohadan, Hamad A; Atta, Ayman M; Khan, Rizwan Hasan

    2015-01-01

    Quaternary amine of diethylaminoethyl rosin ester (QRMAE), chemically synthesized biocompatible rosin based cationic surfactant, has various biological applications including its use as a food product additive. In this study, we examined the amorphous aggregation behavior of mammalian serum albumins at pH 7.5, i.e., two units above their isoelectric points (pI ~5.5), and the roles played by positive charge and hydrophobicity of exogenously added rosin surfactant QRMAE. The study was carried out on five mammalian serum albumins, using various spectroscopic methods, dye binding assay, circular dichroism and electron microscopy. The thermodynamics of the binding of mammalian serum albumins to cationic rosin modified surfactant were established using isothermal titration calorimetry (ITC). It was observed that a suitable molar ratio of protein to QRMAE surfactant enthusiastically induces amorphous aggregate formation at a pH above two units of pI. Rosin surfactant QRMAE-albumins interactions revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions that play an important role towards the formation of hydrophobic interactions-driven amorphous aggregate. Amorphous aggregation of proteins is associated with varying diseases, from the formation of protein wine haze to the expansion of the eye lenses in cataract, during the expression and purification of recombinant proteins. This study can be used for the design of novel biomolecules or drugs with the ability to neutralize factor(s) responsible for the aggregate formation, in addition to various other industrial applications. PMID:26418451

  10. A coarse grained protein model with internal degrees of freedom. Application to α-synuclein aggregation

    NASA Astrophysics Data System (ADS)

    Ilie, Ioana M.; den Otter, Wouter K.; Briels, Wim J.

    2016-02-01

    Particles in simulations are traditionally endowed with fixed interactions. While this is appropriate for particles representing atoms or molecules, objects with significant internal dynamics—like sequences of amino acids or even an entire protein—are poorly modelled by invariable particles. We develop a highly coarse grained polymorph patchy particle with the ultimate aim of simulating proteins as chains of particles at the secondary structure level. Conformational changes, e.g., a transition between disordered and β-sheet states, are accommodated by internal coordinates that determine the shape and interaction characteristics of the particles. The internal coordinates, as well as the particle positions and orientations, are propagated by Brownian Dynamics in response to their local environment. As an example of the potential offered by polymorph particles, we model the amyloidogenic intrinsically disordered protein α-synuclein, involved in Parkinson's disease, as a single particle with two internal states. The simulations yield oligomers of particles in the disordered state and fibrils of particles in the "misfolded" cross-β-sheet state. The aggregation dynamics is complex, as aggregates can form by a direct nucleation-and-growth mechanism and by two-step-nucleation through conversions between the two cluster types. The aggregation dynamics is complex, with fibrils formed by direct nucleation-and-growth, by two-step-nucleation through the conversion of an oligomer and by auto-catalysis of this conversion.

  11. The Hexapeptide Repeated Segment LIAGY is a Hot Spot of Aggregation of the Pseudomonas syringae Ice Nucleation Protein.

    PubMed

    Di Martino, Patrick

    2016-01-01

    Ice nucleation proteins (INPs) form oligomeric structures by self-assembly and aggregation. We looked for the presence of potential aggregating sequences inside the INP from Pseudomonas syringae by a computational approach with the AGGRESCAN, FOMDAMYLOID and TANGO softwares. A total of 38 hot spots of aggregation were predicted in the INP sequence: 7 localized in the Nterminal domain, 2 in the C-terminal region, 28 in the highly repetitive central (HRC) region and 1 shared between the HRC and the Carboxyl-terminus regions of the protein. All the hot spots of aggregation identified in the HRC domain overlapped a 8-residue low fidelity repeat including a LIAGYrelated sequence. We confirmed the predictions by an experimental approach using synthetic peptides corresponding to different parts of the INP central sequence, absorbance spectroscopy and fluorescence spectroscopy in the presence of Congo red (CR) or Thioflavin T (ThT), respectively. Peptide 620-SFIIAGYG-627 predicted to aggregate by the three softwares induced an increase in fluorescence of ThT. Peptide 729-GFKSILTAGY-738 predicted to aggregate by AGGRESCAN and FOLDAMYLOID induced a shift in the maximum of absorbance of CR. Peptide 1124-SVLTAGA-1130 predicted to aggregate only by TANGO did not interfere with CR absorbance or ThT fluorescence. In conclusion, the use of three aggregation prediction algorithms and two biochemical assays showed that the hexapeptide repeated segment LIAGY, previously shown to form a hairpin loop may be involved in the aggregation of the P. syringae INP. PMID:26548995

  12. [A method for studying intravascular platelet aggregation in vitro].

    PubMed

    Ikonnikova, E I; Chernousova, L A; Moshkina, I R

    1999-06-01

    A simple available method for evaluating intravascular platelet aggregation is proposed. It consists in graphic recording of disaggregation of platelet-rich citrate plasma, which indicates the degree of intravascular aggregation. Intravascular aggregation is notably increased in coronary patients and negligible in normal subjects. The method may be used for the diagnosis of diseases with a high thrombogenic risk.

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

    PubMed

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

    2014-11-01

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

  14. Macrophage uptake of low-density lipoprotein bound to aggregated C-reactive protein: possible mechanism of foam-cell formation in atherosclerotic lesions.

    PubMed Central

    Fu, Tao; Borensztajn, Jayme

    2002-01-01

    Foam cells found in atherosclerotic lesions are believed to derive from macrophages that take up aggregated low-density lipoprotein (LDL) particles bound to the extracellular matrix of arterial walls. C-reactive protein (CRP) is an acute-phase protein found in atherosclerotic lesions, which when immobilized on a solid phase, can bind and cluster LDL particles in a calcium-dependent manner. In the present study, we examined whether CRP-bound aggregated LDL could be taken up by macrophages in culture. CRP molecules were aggregated in the presence of calcium and immobilized on the surface of polystyrene microtitre wells. Human LDL added to the wells bound to and aggregated on the immobilized CRP, also in a calcium-dependent manner. On incubation with macrophages, the immobilized CRP-bound LDL aggregates were readily taken up by the cells, as demonstrated by immunofluorescence microscopy, by the cellular accumulation of cholesterol and by the overexpression of adipophilin. Immunofluorescence microscopy and flow-cytometry analysis established that the uptake of the LDL-CRP complex was not mediated by the CRP receptor CD32. These observations with immobilized CRP and LDL, approximating the conditions that exist in the extracellular matrix of the arterial wall, thus suggest that CRP may contribute to the formation of foam cells in atherosclerotic lesions by causing the aggregation of LDL molecules that are then taken up by macrophages through a CD32-independent pathway. PMID:12033985

  15. Small heat shock proteins protect against {alpha}-synuclein-induced toxicity and aggregation

    SciTech Connect

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

    2006-12-22

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

  16. Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain.

    PubMed

    Nezis, Ioannis P; Simonsen, Anne; Sagona, Antonia P; Finley, Kim; Gaumer, Sébastien; Contamine, Didier; Rusten, Tor Erik; Stenmark, Harald; Brech, Andreas

    2008-03-24

    P62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.

  17. Identification of the chromophores involved in aggregation-dependent energy quenching of the monomeric photosystem II antenna protein Lhcb5.

    PubMed

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

    2010-09-01

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

  18. Application of a kosmotrope-based solubility assay to multiple protein therapeutic classes indicates broad use as a high-throughput screen for protein therapeutic aggregation propensity.

    PubMed

    Yamniuk, Aaron P; Ditto, Noah; Patel, Mehul; Dai, Jun; Sejwal, Preeti; Stetsko, Paul; Doyle, Michael L

    2013-08-01

    Aggregation propensity is a critical attribute of protein therapeutics that can influence production, manufacturing, delivery, and potential activity and safety (immunogenicity). It is therefore imperative to select molecules with low aggregation propensity in the early stages of drug discovery to mitigate the risk of delays or failure in clinical development. Although many biophysical methods have been developed to characterize protein aggregation, most established methods are low-throughput, requiring large quantities of protein, lengthy assay times, and/or significant upstream sample preparation, which can limit application in early candidate screening. To avoid these limitations, we developed a reliable method to characterize aggregation propensity, by measuring the relative solubility of protein therapeutic candidates in the presence of the kosmotropic salt ammonium sulfate. Manual bench-scale and automated plate-based methods were applied to different protein therapeutic formats including Adnectins, domain antibodies, PEGylated Adnectins, Fc fusion proteins, and monoclonal antibodies. The kosmotrope solubility data agreed well with the aggregation propensity observed by established methods, while being amenable to high-throughput screening because of speed, simplicity, versatility and low protein material requirements. The results suggest that kosmotrope-based solubility assessment has broad applicability to selecting protein therapeutic candidates with low aggregation propensity and high "developability" to progress into development.

  19. The neuronal extracellular matrix restricts distribution and internalization of aggregated Tau-protein.

    PubMed

    Suttkus, A; Holzer, M; Morawski, M; Arendt, T

    2016-01-28

    Alzheimer's disease (AD) is a chronic degenerative disorder characterized by fibrillary aggregates of Aß and Tau-protein. Formation and progression of these pathological hallmarks throughout the brain follow a specific spatio-temporal pattern which provides the basis for neuropathological staging. Previously, we could demonstrate that cortical and subcortical neurons are less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called 'perineuronal net' (PN). PNs are composed of large aggregating chondroitin sulfate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R. Recently, PN-associated neurons were shown to be better protected against iron-induced neurodegeneration compared to neurons without PN, indicating a neuroprotective function. Here, we investigated the role of PNs in distribution and internalization of exogenous Tau-protein by using organotypic slice cultures of wildtype mice as well as mice lacking the ECM-components aggrecan, HAPLN1 or tenascin-R. We could demonstrate that PNs restrict both distribution and internalization of Tau. Accordingly, PN-ensheathed neurons were less frequently affected by Tau-internalization, than neurons without PN. Finally, the PNs as well as their three investigated components were shown to modulate the processes of distribution as well as internalization of Tau. PMID:26621125

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-11-01

    Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers. PMID:26282322

  2. Ethanol extract of Brazilian propolis ameliorates cognitive dysfunction and suppressed protein aggregations caused by hyperhomocysteinemia.

    PubMed

    Miyazaki, Yuta; Sugimoto, Yasushi; Fujita, Akikazu; Kanouchi, Hiroaki

    2015-01-01

    Homocysteine (Hcy) has been proposed to be a risk factor for cognitive dysfunction. We investigated the effects and the underlying mechanisms of action of propolis, which has antioxidant activity on Hcy-induced oxidative stress in vitro and in vivo. For the in vitro assays, neuroblastoma SH-SY5Y and glioblastoma U-251MG cells were cultured with Hcy and various concentrations of propolis. Cell death and reactive oxygen species production were significantly suppressed by propolis in dose-dependent manner, compared with Hcy alone. For the in vivo assays, mice were fed a propolis-containing diet and Hcy thiolactone in water. Cognitive function was evaluated using the Morris water maze test. Propolis suppressed cognitive dysfunction caused by hyperhomocysteinemia. Accumulation of aggregated protein in brain was accelerated in hyperhomocysteinemia, and the accumulation was suppressed by propolis. Hyperhomocysteinemia, however, did not enhance the oxidative stress in brain. In vitro amyloid formation assay showed that Hcy accelerated lysozyme aggregation and propolis inhibited the aggregation.

  3. Adsorption of a Protein Monolayer via Hydrophobic Interactions Prevents Nanoparticle Aggregation under Harsh Environmental Conditions

    PubMed Central

    Dominguez-Medina, Sergio; Blankenburg, Jan; Olson, Jana; Landes, Christy F.; Link, Stephan

    2013-01-01

    We find that citrate-stabilized gold nanoparticles aggregate and precipitate in saline solutions below the NaCl concentration of many bodily fluids and blood plasma. Our experiments indicate that this is due to complexation of the citrate anions with Na+ cations in solution. A dramatically enhanced colloidal stability is achieved when bovine serum albumin is adsorbed to the gold nanoparticle surface, completely preventing nanoparticle aggregation under harsh environmental conditions where the NaCl concentration is well beyond the isotonic point. Furthermore, we explore the mechanism of the formation of this albumin ‘corona’ and find that monolayer protein adsorption is most likely ruled by hydrophobic interactions. As for many nanotechnology-based biomedical and environmental applications, particle aggregation and sedimentation are undesirable and could substantially increase the risk of toxicological side-effects, the formation of the BSA corona presented here provides a low-cost bio-compatible strategy for nanoparticle stabilization and transport in highly ionic environments. PMID:23914342

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2012-03-23

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

  8. Study of aggregation in polysaccharides by triple detection HPLC

    NASA Astrophysics Data System (ADS)

    Norwood, David

    2005-11-01

    It has been observed that various species of the polysaccharide carrageenan undergo a change in conformation under varying conditions of temperature and added salt. The change is from a random coil (under conditions of high temperature or low salt) to a helix (under conditions of low temperature or high salt). It is a point of contention as to whether the helix is formed of one coil wrapping upon itself or two coils wrapping around one another. We present the results of a study of this conformation change using viscometry and multi-angle laser light scattering (MALLS) incorporating size-exclusion chromatography (SEC). In previous research, our batch light scattering results showed a clear increase in molecular weight and radius of gyration when NaCl was added to varying concentrations of iota-carrageenan, which could be interpreted as the conformation change described. However, estimates of persistence length (˜ RG^2 / MW) calculated using MALLS data show no strong variation when increasing added salt. This suggests that the molecular weight increase reflects an aggregation of polymer molecules instead of a change in conformation. However, SEC results show very little variation in molecular weight and radius of gyration, and instead, show behavior typical of linear polyelectrolytes. Resolution of these different results suggests a loose aggregation of polymer molecules that preserves the overall structure rather than a significant change in polymer architecture. We present viscometry and dynamic light scattering results that confirm these conclusions.

  9. Study of adsorption of Neon on open Carbon nanohorns aggregates

    NASA Astrophysics Data System (ADS)

    Ziegler, Carl Andrew

    Adsorption isotherms can be used to determine surface area of a substrate and the heat released when adsorption occurs. Our measurements are done determining the equilibrium pressures corresponding to a given amount of gas adsorbed on a substrate at constant temperature. The adsorption studies were done on aggregates of open dahlia-like carbon nanohorns. The nanohorns were oxidized for 9 hours at 550 °C to open them up and render their interior space accessible for adsorption. Volumetric adsorption measurements of Ne were performed at twelve different temperatures between 19 K and 48 K. The isotherms showed two substeps. The first substep corresponds to adsorption on the high energy binding sites in the interior of the nanohorns, near the tip. The second substep corresponds to low energy binding sites both on the outside of the nanotubes and inside the nanotube away from the tip. The isosteric heat measurements obtained from the isotherm data also shows these two distinct substeps. The effective surface area of the open nanotubes was determined from the isotherms using the point-B method. The isosteric heat and surface area data for neon on open nanohorns were compared to two similar experiments of neon adsorbed on aggregates of closed nanohorns.

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

    PubMed

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development. PMID:26338058

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

    PubMed

    Luo, Shen; Zhang, Baolin

    2015-01-01

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

  12. Neuronal aggregates: formation, clearance and spreading

    PubMed Central

    Lim, Junghyun; Yue, Zhenyu

    2015-01-01

    Summary Proteostasis is maintained by multiple cellular pathways, including protein synthesis, quality control and degradation. An imbalance of neuronal proteostasis, associated with protein misfolding and aggregation, leads to proteinopathies or neurodegeneration. While genetic variations and protein modifications contribute to aggregate formation, components of the proteostasis network dictate the fate of protein aggregates. Here we provide an overview of proteostasis pathways and their interplay (particularly autophagy) with the metabolism of disease-related proteins. We review recent studies on neuronal activity-mediated regulation of proteostasis and transcellular propagation of protein aggregates in the nervous system. Targeting proteostasis pathways therapeutically remains an attractive but challenging task. PMID:25710535

  13. Structural Studies of Protein-Surfactant Complexes

    SciTech Connect

    Chodankar, S. N.; Aswal, V. K.; Wagh, A. G.

    2008-03-17

    The structure of protein-surfactant complexes of two proteins bovine serum albumin (BSA) and lysozyme in presence of anionic surfactant sodium dodecyl sulfate (SDS) has been studied using small-angle neutron scattering (SANS). It is observed that these two proteins form different complex structures with the surfactant. While BSA protein undergoes unfolding on addition of surfactant, lysozyme does not show any unfolding even up to very high surfactant concentrations. The unfolding of BSA protein is caused by micelle-like aggregation of surfactant molecules in the complex. On the other hand, for lysozyme protein there is only binding of individual surfactant molecules to protein. Lysozyme in presence of higher surfactant concentrations has protein-surfactant complex structure coexisting with pure surfactant micelles.

  14. Monte Carlo simulations of protein amyloid formation reveal origin of sigmoidal aggregation kinetics.

    PubMed

    Linse, Björn; Linse, Sara

    2011-07-01

    Severe conditions and lack of cure for many amyloid diseases make it highly desired to understand the underlying principles of formation of fibrillar aggregates (amyloid). Here, amyloid formation from peptides was studied using Monte Carlo simulations. Systems of 20, 50, 100, 200 or 500 hexapeptides were simulated. Association kinetics were modeled equal for fibrillar and other (inter- and intra-peptide) contacts and assumed to be faster the lower the effective contact order, which represents the distance in space. Attempts to form contacts were thus accepted with higher probability the lower the effective contact order, whereby formation of new contacts next to preexisting ones is favored by shorter physical separation. Kinetic discrimination was invoked by using two different life-times for formed contacts. Contacts within amyloid fibrils were assumed to have on average longer life-time than other contacts. We find that the model produces fibrillation kinetics with a distinct lag phase, and that the fibrillar contacts need to dissociate on average 5-20 times slower than all other contacts for the fibrillar structure to dominate at equilibrium. Analysis of the species distribution along the aggregation process shows that no other intermediate is ever more populated than the dimer. Instead of a single nucleation event there is a concomitant increase in average aggregate size over the whole system, and the occurrence of multiple parallel processes makes the process more reproducible the larger the simulated system. The sigmoidal shape of the aggregation curves arises from cooperativity among multiple interactions within each pair of peptides in a fibril. A governing factor is the increasing probability as the aggregation process proceeds of neighboring reinforcing contacts. The results explain the very strong bias towards cross β-sheet fibrils in which the possibilities for cooperativity among interactions involving neighboring residues and the repetitive use of

  15. LMP1 mediates multinuclearity through downregulation of shelterin proteins and formation of telomeric aggregates

    PubMed Central

    Lajoie, Valérie; Lemieux, Bruno; Sawan, Bassem; Lichtensztejn, Daniel; Lichtensztejn, Zelda; Wellinger, Raymund; Mai, Sabine

    2015-01-01

    Hodgkin lymphoma (HL) and Burkitt lymphoma are both germinal center–derived B-cell lymphomas. To assess the consequences of permanent latent membrane protein 1 (LMP1) expression as observed in tumor cells of Epstein-Barr virus (EBV) –associated HL, we analyzed 3-dimensional (3D) telomere dynamics and measured the expression of shelterin proteins at the transcriptional and translational level and their topographic distribution in the EBV-negative Burkitt cell line BJAB stably transfected with an inducible LMP1 system. Stable LMP1 expression led to a highly significant increase of multinucleated cells, nuclear volume, and 3D telomeric aggregates when compared with the LMP1-suppressed BJAB controls. Most importantly, LMP1 induced a significant downregulation of the shelterin components TRF1, TRF2, and POT1 at the transcriptional and translational level, and this downregulation was reversed after resuppression of LMP1. In addition, as revealed by spectral karyotyping, LMP1 induced “outré” giant cells and hypoploid “ghost” cells. This LMP1-induced multinucleation was blocked upon LMP1-independent TRF2 expression. These results show that LMP1-dependent deregulation of telomere stability and nuclear organization via shelterin downregulation, in particular TRF2, favors chromosomal rearrangements. We speculate that telomeric aggregates and ongoing breakage-bridge-fusion cycles lead to disturbed cytokinesis and finally to multinuclearity, as observed in EBV-associated HL. PMID:25568351

  16. The inhibitory effects of Escherichia coli maltose binding protein on β-amyloid aggregation and cytotoxicity.

    PubMed

    Sharoar, Md Golam; Shahnawaz, Md; Islam, Md Imamul; Ramasamy, Vijay Sankar; Shin, Song Yub; Park, Il-Seon

    2013-10-01

    The aggregation of β-amyloid (Aβ) peptide from its monomeric to its fibrillar form importantly contributes to the development of Alzheimer's disease. Here, we investigated the effects of Escherichia coli maltose binding protein (MBP), which has been previously used as a fusion protein, on Aβ42 fibrillization, in order to improve understanding of the self-assembly process and the cytotoxic mechanism of Aβ42. MBP, at a sub-stoichiometric ratio with respect to Aβ42, was found to have chaperone-like inhibitory effects on β-sheet fibril formation, due to the accumulation of Aβ42 aggregates by sequestration of active Aβ42 species as Aβ42-MBP complexes. Furthermore, MBP increased the lag time of Aβ42 polymerization, decreased the growth rate of fibril extension, and suppressed Aβ42 mediated toxicity in human neuroblastoma SH-SY5Y cells. It appears that MBP decreases the active concentration of Aβ42 by sequestering it as Aβ42-MBP complex, and that this sequestration suppresses ongoing nucleation and retards the growth rate of Aβ42 species required for fibril formation. We speculate that inhibition of the growth rate of potent Aβ42 species by MBP suppresses Aβ42-mediated toxicity in SH-SY5Y cells. PMID:23948569

  17. The role of thiol oxidative stress response in heat-induced protein aggregate formation during thermotolerance in Bacillus subtilis.

    PubMed

    Runde, Stephanie; Molière, Noël; Heinz, Anja; Maisonneuve, Etienne; Janczikowski, Armgard; Elsholz, Alexander K W; Gerth, Ulf; Hecker, Michael; Turgay, Kürşad

    2014-03-01

    Using Bacillus subtilis as a model organism, we investigated thermotolerance development by analysing cell survival and in vivo protein aggregate formation in severely heat-shocked cells primed by a mild heat shock. We observed an increased survival during severe heat stress, accompanied by a strong reduction of heat-induced cellular protein aggregates in cells lacking the ClpXP protease. We could demonstrate that the transcription factor Spx, a regulatory substrate of ClpXP, is critical for the prevention of protein aggregate formation because its regulon encodes redox chaperones, such as thioredoxin, required for protection against thiol-specific oxidative stress. Consequently B. subtilis cells grown in the absence of oxygen were more protected against severe heat shock and much less protein aggregates were detected compared to aerobically grown cells. The presented results indicate that in B. subtilis Spx and its regulon plays not only an important role for oxidative but also for heat stress response and thermotolerance development. In addition, our experiments suggest that the protection of misfolded proteins from thiol oxidation during heat shock can be critical for the prevention of cellular protein aggregation in vivo.

  18. The Non-native Helical Intermediate State May Accumulate at Low pH in the Folding and Aggregation Landscape of the Intestinal Fatty Acid Binding Protein.

    PubMed

    Sarkar-Banerjee, Suparna; Chowdhury, Sourav; Paul, Simanta Sarani; Dutta, Debashis; Ghosh, Anisa; Chattopadhyay, Krishnananda

    2016-08-16

    There has been widespread interest in studying early intermediate states and their roles in protein folding. The interest in intermediate states has been further emphasized in the recent literature because of their implications for protein aggregation. Unfortunately, direct kinetic characterization of intermediates has been difficult because of the limited time resolutions offered by the kinetic techniques and the heterogeneity of the folding and aggregation landscape. Even in equilibrium experiments, the characterization of intermediate states could be difficult because (a) their populations in equilibrium could be low and/or (b) they lack any specific biochemical or biophysical signatures for their identification. In this paper, we have used fluorescence correlation spectroscopy to study the nature of a low-pH intermediate state of the intestinal fatty acid binding protein, a small protein with predominantly β-sheet structure. Our results have shown that the pH 3 intermediate diffuses faster than the folded protein and has strong helix forming propensity. These behaviors support Lim's hypothesis according to which even an entirely β-sheet protein would form helical bundles at the early stage. Using dynamic light scattering and thioflavin T binding measurements, we have observed that the pH 3 intermediate is prone to aggregation. We believe that early helix formation is the result of a local effect, which originates from the interaction of the neighboring amino acids around the hydrophobic core residues. This early intermediate reorganizes subsequently, and this structural reorganization is initiated by the destabilizing interactions induced by the distant residues, unfavorable entropic costs, and steric constraints of the hydrophobic side chains. Mutational analyses show further that the increase in the hydrophobicity in the hydrophobic core region increases the population of the α-helical intermediate, enhancing the aggregation propensity of the protein

  19. SENSITIVITY ANALYSIS OF AGGREGATED ENVIRONMENTAL INDICES WITH A CASE-STUDY OF THE MID-ATLANTIC REGION

    EPA Science Inventory

    Environmental indicators are often aggregated into a single index for various purposes in environmental studies. Aggregated indices derived from the same data set can differ, usually because the aggregated indices' sensitivities are not thoroughly analyzed. Furthermore, if a sens...

  20. Study of colloidal particle Brownian aggregation by low-coherence fiber optic dynamic light scattering.

    PubMed

    Xia, Hui; Pang, Ru Yi; Zhang, Rui; Miao, Cai Xia; Wu, Xiao Yun; Hou, Xue Shun; Zhong, Cheng

    2012-06-15

    The aggregation kinetics of particles in dense polystyrene latex suspensions is studied by low-coherence fiber optic dynamic light scattering. Low-coherence fiber optic dynamic light scattering is used to measure the hydrodynamic radius of the aggregates. The aggregation kinetics data obtained can be fitted into a single exponential function, which is the characteristic of slow aggregation. It is found that the aggregation rate of particles increased with higher electrolyte levels and with larger particle concentrations. The experimental results can be explained by use of the Derjaruin-Landau-Verwey-Overbeer (DLVO) theory. PMID:22446146

  1. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.

    PubMed

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  2. αA-Crystallin Peptide 66SDRDKFVIFLDVKHF80 Accumulating in Aging Lens Impairs the Function of α-Crystallin and Induces Lens Protein Aggregation

    PubMed Central

    Santhoshkumar, Puttur; Raju, Murugesan; Sharma, K. Krishna

    2011-01-01

    Background The eye lens is composed of fiber cells that are filled with α-, β- and γ-crystallins. The primary function of crystallins is to maintain the clarity of the lens through ordered interactions as well as through the chaperone-like function of α-crystallin. With aging, the chaperone function of α-crystallin decreases, with the concomitant accumulation of water-insoluble, light-scattering oligomers and crystallin-derived peptides. The role of crystallin-derived peptides in age-related lens protein aggregation and insolubilization is not understood. Methodology/Principal Findings We found that αA-crystallin-derived peptide, 66SDRDKFVIFLDVKHF80, which accumulates in the aging lens, can inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. Age-related change in the concentration of αA-(66-80) peptide was estimated by mass spectrometry. The interaction of the peptide with native crystallin was studied by multi-angle light scattering and fluorescence methods. High molar ratios of peptide-to-crystallin were favourable for aggregation and precipitation. Time-lapse recordings showed that, in the presence of αA-(66-80) peptide, α-crystallin aggregates and functions as a nucleus for protein aggregation, attracting aggregation of additional α-, β- and γ-crystallins. Additionally, the αA-(66-80) peptide shares the principal properties of amyloid peptides, such as β-sheet structure and fibril formation. Conclusions/Significance These results suggest that crystallin-derived peptides such as αA-(66-80), generated in vivo, can induce age-related lens changes by disrupting the structure and organization of crystallins, leading to their insolubilization. The accumulation of such peptides in aging lenses may explain a novel mechanism for age-related crystallin aggregation and cataractogenesis. PMID:21552534

  3. Large-scale analysis of macromolecular crowding effects on protein aggregation using a reconstituted cell-free translation system

    PubMed Central

    Niwa, Tatsuya; Sugimoto, Ryota; Watanabe, Lisa; Nakamura, Shugo; Ueda, Takuya; Taguchi, Hideki

    2015-01-01

    Proteins must fold into their native structures in the crowded cellular environment, to perform their functions. Although such macromolecular crowding has been considered to affect the folding properties of proteins, large-scale experimental data have so far been lacking. Here, we individually translated 142 Escherichia coli cytoplasmic proteins using a reconstituted cell-free translation system in the presence of macromolecular crowding reagents (MCRs), Ficoll 70 or dextran 70, and evaluated the aggregation propensities of 142 proteins. The results showed that the MCR effects varied depending on the proteins, although the degree of these effects was modest. Statistical analyses suggested that structural parameters were involved in the effects of the MCRs. Our dataset provides a valuable resource to understand protein folding and aggregation inside cells. PMID:26500644

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

    SciTech Connect

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

    2009-04-01

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

  5. Location Trumps Length: Polyglutamine-Mediated Changes in Folding and Aggregation of a Host Protein

    PubMed Central

    Tobelmann, Matthew D.; Murphy, Regina M.

    2011-01-01

    Expanded CAG diseases are progressive neurodegenerative disorders in which specific proteins have an unusually long polyglutamine stretch. Although these proteins share no other sequence or structural homologies, they all aggregate into intracellular inclusions that are believed to be pathological. We sought to determine what impact the position and number of glutamines have on the structure and aggregation of the host protein, apomyoglobin. Variable-length polyQ tracts were inserted either into the loop between the C- and D-helices (QnCD) or at the N-terminus (QnNT). The QnCD mutants lost some α-helix and gained unordered and/or β-sheet in a length-dependent manner. These mutants were partially unfolded and rapidly assembled into soluble chain-like oligomers. In sharp contrast, the QnNT mutants largely retained wild-type tertiary structure but associated into long, fibrillar aggregates. Control proteins with glycine-serine repeats (GS8CD and GS8NT) were produced. GS8CD exhibited similar structural perturbations and aggregation characteristics to an analogously sized Q16CD, indicating that the observed effects are independent of amino acid composition. In contrast to Q16NT, GS8NT did not form fibrillar aggregates. Thus, soluble oligomers are produced through structural perturbation and do not require polyQ, whereas classic fibrils arise from specific polyQ intermolecular interactions in the absence of misfolding. PMID:21641323

  6. Study of the durability of demolition aggregates on pavements

    NASA Astrophysics Data System (ADS)

    Bachir, Melbouci; Fazia, Boudjemia

    2009-06-01

    This work is an experimental contribution to the study of durability of various materials coming from the demolition of buildings for their use in the flexible infrastructures of pavements. However, these pavements undergo traffic loading which causes, in the long time permanent deformations and settlements thus generating significant degradations of materials. Various recent works aim at characterizing these materials. This work falls under this scope and gives an approach of the mechanical behavior and the response of these last to repetitive loadings. After identification of materials and the determination of the characteristics of hardness and durability, we studied their mechanical behavior with cyclic shearing on these three materials (aggregates of concrete, brick and concrete block). These tests enabled us to analyze the mechanisms which occur through the various parameters related to loading (the amplitude and the repetition of loading, the parameters related to materials (the water content, the density and the grain size distribution). The study of the durability of these materials consists on quantifying the degradation rate of the grains after the various tests and the grain size distribution which governs the amplitude of crushing.

  7. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  8. Kirkwood–Buff theory of molecular and protein association, aggregation, and cellular crowding

    PubMed Central

    Gee, Moon Bae; Smith, Paul E.

    2009-01-01

    An analysis of the effect of a cosolvent on the association of a solute in solution using the Kirkwood–Buff theory of solutions is presented. The approach builds on the previous results of Ben-Naim by extending the range of applicability to include any number of components at finite concentrations in both closed and semiopen systems. The derived expressions, which are exact, provide a foundation for the analysis and rationalization of cosolvent effects on molecular and biomolecular equilibria including protein association, aggregation, and cellular crowding. A slightly different view of cellular crowding is subsequently obtained. In particular, it is observed that the addition of large cosolvents still favors the associated form even when traditional excluded volume effects are absent. PMID:19894976

  9. Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation

    PubMed Central

    Schultz, Sebastian Wolfgang; Nilsson, K. Peter R.; Westermark, Gunilla Torstensdotter

    2011-01-01

    Background Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death. Methodology/Principal Findings We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elavC155,Gal4 showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures. Conclusions/Significance These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology. PMID:21695120

  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. Mammalian ribosomal and chaperone protein RPS3A counteracts α-synuclein aggregation and toxicity in a yeast model system.

    PubMed

    De Graeve, Stijn; Marinelli, Sarah; Stolz, Frank; Hendrix, Jelle; Vandamme, Jurgen; Engelborghs, Yves; Van Dijck, Patrick; Thevelein, Johan M

    2013-11-01

    Accumulation of aggregated forms of αSyn (α-synuclein) into Lewy bodies is a known hallmark associated with neuronal cell death in Parkinson's disease. When expressed in the yeast Saccharomyces cerevisiae, αSyn interacts with the plasma membrane, forms inclusions and causes a concentration-dependent growth defect. We have used a yeast mutant, cog6Δ, which is particularly sensitive to moderate αSyn expression, for screening a mouse brain-specific cDNA library in order to identify mammalian proteins that counteract αSyn toxicity. The mouse ribosomal and chaperone protein RPS3A was identified as a suppressor of αSyn [WT (wild-type) and A53T] toxicity in yeast. We demonstrated that the 50 N-terminal amino acids are essential for this function. The yeast homologues of RPS3A were not effective in suppressing the αSyn-induced growth defect, illustrating the potential of our screening system to identify modifiers that would be missed using yeast gene overexpression as the first screening step. Co-expression of mouse RPS3A delayed the formation of αSyn-GFP inclusions in the yeast cells. The results of the present study suggest that the recently identified extraribosomal chaperonin function of RPS3A also acts on the neurodegeneration-related protein αSyn and reveal a new avenue for identifying promising candidate mammalian proteins involved in αSyn functioning.

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

    PubMed

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

    2015-09-01

    therapeutics. A key basis for the commonality between viral and neurodegenerative disease aggregation is a broader definition of assembly as more than just simple aggregation, particularly suited for the crowded cytoplasm. The assembly machines are collections of proteins that catalytically accelerate an assembly reaction that would occur spontaneously but too slowly to be relevant in vivo. Being an enzyme complex with a functional allosteric site, appropriated for a non-physiological purpose (e.g. viral infection or conformational disease), these assembly machines present a superior pharmacological target because inhibition of their active site will amplify an effect on their substrate reaction. Here, we present this hypothesis based on recent proof-of-principle studies against Aβ assembly relevant in Alzheimer's disease.

  13. Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase

    PubMed Central

    Burchett, Gina G.; Folsom, Charles G.; Lane, Kimberly T.

    2015-01-01

    β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins. PMID:26121040

  14. Study on the structure of topsoil aggregates for Mu Us sandy land in China

    NASA Astrophysics Data System (ADS)

    Li, Z. H.; Zhang, Q. H.; Tian, H. W.; Xie, Y. H.

    2016-08-01

    It is significantly important to study the topsoil aggregates occurrence for land use, recovery at regional scale and protecting from topsoil sandification. Based on land sampling through experimental analysis and correlation statistics, this paper studies on the characteristics and the correlation of different habitat topsoil aggregates in Mu Us region. The results show: The soil aggregates are mainly micro aggregates in the range of 0.5∼0.25mm in this area. The contents of soil aggregates are different in different depths which are 0cm>10cm>20cm respectively. The contents of soil aggregates are also different in different habitats, and the contents of >1mm soil aggregates are highest 4.27% and 3.55% in poplar woodland and Calamagrostisepigeios grassland sampled at the 0cm depth. After analyzing the stability of soil aggregates, the development of soil aggregates is not sufficient, and the stability of soil aggregates is poor. The soil structure is gradually optimized in the better area of vegetation protection or restoration.

  15. O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

    NASA Astrophysics Data System (ADS)

    Marotta, Nicholas P.; Lin, Yu Hsuan; Lewis, Yuka E.; Ambroso, Mark R.; Zaro, Balyn W.; Roth, Maxwell T.; Arnold, Don B.; Langen, Ralf; Pratt, Matthew R.

    2015-11-01

    Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

  16. Effect of cysteine on lowering protein aggregation and subsequent hardening of whey protein isolate (WPI) protein bars in WPI/buffer model systems.

    PubMed

    Zhu, Dan; Labuza, Theodore P

    2010-07-14

    Whey protein isolate (WPI) bar hardening without and with cysteine (Cys) or N-ethylmaleimide (NEM) was investigated in model systems (WPI/buffer = 6:4, by weight, pH 6.8, a(w) approximately 0.97) in an accelerated shelf-life test (ASLT) at 45 degrees C over a period of up to 35 days. The formation of insoluble aggregates as determined by solubility and the structural rearrangement of WPI protein aggregates as observed by SEM were responsible for the WPI bars' hardening. As corroborated by electrophoresis analysis, both beta-lactoglobulin (beta-lg) and alpha-lactalbumin (alpha-la) were involved in the formation of aggregates via the thiol-disulfide interchange reaction and/or noncovalent interactions. The former force dominated the bar hardening at an earlier stage, whereas the latter force played a role for the long-term hardening. In comparison with the control bar without Cys, the thiol-disulfide interchange reaction was significantly reduced by Cys (WPI/Cys = 0.05), increased by Cys (WPI/Cys = 0.25), and inhibited by NEM (WPI/NEM = 2). Therefore, bar hardening was significantly delayed by Cys (WPI/Cys = 0.05) and NEM but accelerated by Cys (WPI/Cys = 0.25).

  17. Elevated Hapln2 Expression Contributes to Protein Aggregation and Neurodegeneration in an Animal Model of Parkinson's Disease

    PubMed Central

    Wang, Qinqin; Zhou, Qinbo; Zhang, Shuzhen; Shao, Wei; Yin, Yanqing; Li, Yandong; Hou, Jincan; Zhang, Xinhua; Guo, Yongshun; Wang, Xiaomin; Gu, Xiaosong; Zhou, Jiawei

    2016-01-01

    Parkinson's disease (PD), the second most common age-associated progressive neurodegenerative disorder, is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SN). The pathogenesis of PD and the mechanisms underlying the degeneration of DA neurons are still not fully understood. Our previous quantitative proteomics study revealed that hyaluronan and proteoglycan binding link protein 2 (Hapln2) is one of differentially expressed proteins in the substantia nigra tissues from PD patients and healthy control subjects. However, the potential role of Hapln2 in PD pathogenesis remains elusive. In the present study, we characterized the expression pattern of Hapln2. In situ hybridization revealed that Hapln2 mRNA was widely expressed in adult rat brain with high abundance in the substantia nigra. Immunoblotting showed that expression levels of Hapln2 were markedly upregulated in the substantia nigra of either human subjects with Parkinson's disease compared with healthy control. Likewise, there were profound increases in Hapln2 expression in neurotoxin 6-hydroxydopamine-treated rat. Overexpression of Hapln2 in vitro increased vulnerability of MES23.5 cells, a dopaminergic cell line, to 6-hydroxydopamine. Moreover, Hapln2 overexpression led to the formation of cytoplasmic aggregates which were co-localized with ubiquitin and E3 ligases including Parkin, Gp78, and Hrd1 in vitro. Endogenous α-synuclein was also localized in Hapln2-containing aggregates and ablation of Hapln2 led to a marked decrease of α-synuclein in insoluble fraction compared with control. Thus, Hapln2 is identified as a novel factor contributing to neurodegeneration in PD. Our data provides new insights into the cellular mechanism underlying the pathogenesis in PD.

  18. Elevated Hapln2 Expression Contributes to Protein Aggregation and Neurodegeneration in an Animal Model of Parkinson's Disease.

    PubMed

    Wang, Qinqin; Zhou, Qinbo; Zhang, Shuzhen; Shao, Wei; Yin, Yanqing; Li, Yandong; Hou, Jincan; Zhang, Xinhua; Guo, Yongshun; Wang, Xiaomin; Gu, Xiaosong; Zhou, Jiawei

    2016-01-01

    Parkinson's disease (PD), the second most common age-associated progressive neurodegenerative disorder, is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SN). The pathogenesis of PD and the mechanisms underlying the degeneration of DA neurons are still not fully understood. Our previous quantitative proteomics study revealed that hyaluronan and proteoglycan binding link protein 2 (Hapln2) is one of differentially expressed proteins in the substantia nigra tissues from PD patients and healthy control subjects. However, the potential role of Hapln2 in PD pathogenesis remains elusive. In the present study, we characterized the expression pattern of Hapln2. In situ hybridization revealed that Hapln2 mRNA was widely expressed in adult rat brain with high abundance in the substantia nigra. Immunoblotting showed that expression levels of Hapln2 were markedly upregulated in the substantia nigra of either human subjects with Parkinson's disease compared with healthy control. Likewise, there were profound increases in Hapln2 expression in neurotoxin 6-hydroxydopamine-treated rat. Overexpression of Hapln2 in vitro increased vulnerability of MES23.5 cells, a dopaminergic cell line, to 6-hydroxydopamine. Moreover, Hapln2 overexpression led to the formation of cytoplasmic aggregates which were co-localized with ubiquitin and E3 ligases including Parkin, Gp78, and Hrd1 in vitro. Endogenous α-synuclein was also localized in Hapln2-containing aggregates and ablation of Hapln2 led to a marked decrease of α-synuclein in insoluble fraction compared with control. Thus, Hapln2 is identified as a novel factor contributing to neurodegeneration in PD. Our data provides new insights into the cellular mechanism underlying the pathogenesis in PD. PMID:27601993

  19. Elevated Hapln2 Expression Contributes to Protein Aggregation and Neurodegeneration in an Animal Model of Parkinson's Disease

    PubMed Central

    Wang, Qinqin; Zhou, Qinbo; Zhang, Shuzhen; Shao, Wei; Yin, Yanqing; Li, Yandong; Hou, Jincan; Zhang, Xinhua; Guo, Yongshun; Wang, Xiaomin; Gu, Xiaosong; Zhou, Jiawei

    2016-01-01

    Parkinson's disease (PD), the second most common age-associated progressive neurodegenerative disorder, is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SN). The pathogenesis of PD and the mechanisms underlying the degeneration of DA neurons are still not fully understood. Our previous quantitative proteomics study revealed that hyaluronan and proteoglycan binding link protein 2 (Hapln2) is one of differentially expressed proteins in the substantia nigra tissues from PD patients and healthy control subjects. However, the potential role of Hapln2 in PD pathogenesis remains elusive. In the present study, we characterized the expression pattern of Hapln2. In situ hybridization revealed that Hapln2 mRNA was widely expressed in adult rat brain with high abundance in the substantia nigra. Immunoblotting showed that expression levels of Hapln2 were markedly upregulated in the substantia nigra of either human subjects with Parkinson's disease compared with healthy control. Likewise, there were profound increases in Hapln2 expression in neurotoxin 6-hydroxydopamine-treated rat. Overexpression of Hapln2 in vitro increased vulnerability of MES23.5 cells, a dopaminergic cell line, to 6-hydroxydopamine. Moreover, Hapln2 overexpression led to the formation of cytoplasmic aggregates which were co-localized with ubiquitin and E3 ligases including Parkin, Gp78, and Hrd1 in vitro. Endogenous α-synuclein was also localized in Hapln2-containing aggregates and ablation of Hapln2 led to a marked decrease of α-synuclein in insoluble fraction compared with control. Thus, Hapln2 is identified as a novel factor contributing to neurodegeneration in PD. Our data provides new insights into the cellular mechanism underlying the pathogenesis in PD. PMID:27601993

  20. Study of platelet aggregation in acute coronary syndrome with special reference to metabolic syndrome

    PubMed Central

    Paul, Rudrajit; Banerjee, Amit K; Guha, Shantanu; Chaudhuri, Utpal; Ghosh, Srabani; Mondal, Jayati; Bandyopadhyay, Ramtanu

    2013-01-01

    Background/Context: Antiplatelet drug resistance increases the risk of adverse events like stent thrombosis in acute coronary syndrome (ACS). Metabolic syndrome (MS) is a prothrombotic state and presence of MS further increases the risk of antiplatelet drug resistance. Aims and Objectives: We studied platelet aggregation characteristics in patients of ACS for aspirin or clopidogrel resistance. We studied the relation of drug resistance with blood markers like high sensitivity C-reactive protein (hsCRP). We also studied for any relation of drug resistance with presence of MS. Materials and Methods: We studied platelet aggregation characteristics by optical aggregometry using platelet-rich plasma (PRP) of patients. Collagen (2 μg/mL) and adenosine diphosphate (ADP; 10 μmol) were used. Greater than 50% aggregation in PRP of patients was taken as an evidence of drug resistance. Suitable blood tests were done including newer risk markers like hsCRP, apolipoprotein B, and fibrinogen. Statistical test: Statistical tests included Student's t-test and Kendall's rank correlation coefficient. Results: We had a total of 94 patients of ACS with 47 (50%) having MS. MS patients showed higher blood levels of hsCRP and fibrinogen. Twenty-eight (59.5%) patients with MS showed antiplatelet drug resistance compared to 12 patients without MS. Serum fibrinogen showed strongest correlation with drug resistance. HsCRP levels showed correlation with aspirin resistance (r = 0.53) only in the MS group. Discussion and Conclusion: We found significantly high prevalence of antiplatelet drug resistance. Aspirin and clopidogrel resistance was comparable. MS was a significant risk factor for drug resistance. The prothrombotic and proinflammatory markers showed strong correlation with drug resistance. A larger randomized trial is needed to better characterize this clinical problem. PMID:24083147

  1. 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.

  2. Petrography study on altered flint aggregate by alkali-silica reaction

    SciTech Connect

    Bulteel, D. . E-mail: bulteel@ensm-douai.fr; Rafai, N.; Degrugilliers, P.; Garcia-Diaz, E.

    2004-11-15

    The aim of our study is to improve our understanding of an alkali-silica reaction (ASR) via petrography. We used a chemical concrete subsystem: flint aggregate, portlandite and KOH. The altered flint aggregate is followed by optical microscopy and scanning electron microscopy (SEM) before and after acid treatment at different intervals. After acid treatment, the observations showed an increase in aggregate porosity and revealed internal degradation of the aggregate. This degradation created amorphous zones. Before acid treatment, the analyses on polished sections by scanning electron microscopy coupled with energy dispersive spectroscopy (EDS) enabled visualization of K{sup +} and Ca{sup 2+} penetration into the aggregate. The appearance of amorphous zones and penetration of positive ions into the aggregate are correlated with the increase in the molar fraction of silanol sites. This degradation is specific to the alkali-silica reaction.

  3. Organic carbon, water repellency and soil stability to slaking under different crops and managements: a case study at aggregate and intra-aggregate scales

    NASA Astrophysics Data System (ADS)

    Jordán, A.; García-Moreno, J.; Gordillo-Rivero, Á. J.; Zavala, L. M.; Cerdà, A.

    2014-08-01

    This research studies the distribution of organic C and intensity of water repellency in soil aggregates with different size and in the interior of aggregates from Mediterranean soils under different crops (apricot, citrus and wheat) and management (conventional tilling and no tilling/mulching). For this, undisturbed aggregates were sampled and carefully divided in size fractions (0.25-0.5, 0.5-1, 1-2, 2-5, 5-10 and 10-15 mm) or peeled to obtain separated aggregate layers (exterior, transitional and interior). Organic C content in the fine earth fraction of soils under different crops did not show important variations, although it increased significantly from conventionally tilled to mulched soils. The distribution of organic C content in aggregates with different size varied among soils under different crops, generally increasing with decreasing size. At the intra-aggregate level, organic C concentrated preferably in the exterior layer of aggregates from conventionally tilled soils, probably because of recent organic inputs or leachates. In the case of mulched soils, higher concentrations were observed, but no significant differences among aggregate regions were found. The intensity of water repellency, determined by the ethanol method, did not show great variations among crops, but increased significantly from conventionally tilled to mulched soils. Coarser aggregates were generally wettable, while finer aggregates showed slight water repellency. Regardless of variations in the distribution of organic C in aggregate layers from conventionally tilled soils, great or significant differences in the distribution of water repellency at the intra-aggregate level were not found in any case. Finally, the intensity of water repellency was much more important than the concentration of organic C in the stability to slaking of aggregates.

  4. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses

    PubMed Central

    Åslund, Andreas; Sigurdson, Christina J.; Klingstedt, Therése; Grathwohl, Stefan; Bolmont, Tristan; Dickstein, Dara L.; Glimsdal, Eirik; Prokop, Stefan; Lindgren, Mikael; Konradsson, Peter; Holtzman, David M.; Hof, Patrick R.; Heppner, Frank L.; Gandy, Samuel; Jucker, Mathias; Aguzzi, Adriano; Hammarström, Per; Nilsson, K. Peter R.

    2010-01-01

    Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying cerebral amyloidoses. Here we report the chemical design of pentameric thiophene derivatives, denoted luminescent conjugated oligothiophenes (LCOs), which could be used for real-time visualization of cerebral protein aggregates in transgenic mouse models of neurodegenerative diseases by multiphoton microscopy. One of the LCOs, p-FTAA, showed conformation-dependent optical properties and could be utilized for ex vivo spectral assignment of distinct prion deposits from two mouse-adapted prion strains. p-FTAA also revealed staining of transient soluble pre-fibrillar non-thioflavinophilic Aβ- assemblies during in vitro fibrillation of Aβ peptides. In brain tissue samples, Aβ deposits and neurofibrillary tangles (NFTs) were readily identified by a strong fluorescence from p-FTAA and the LCO staining showed complete co-localization with conventional antibodies (6E10 and AT8), indicating that p-FTAA detects all the immuno-positive aggregated proteinaceous species in Alzheimer disease, but with significantly shorter imaging time (100 fold) compared to immunofluorescence. In addition, a patchy islet-like staining of individual Aβ plaque was unveiled by the anti-oligomer A11 antibody during co-staining with p-FTAA, suggesting that pre-fibrillar species are likely an intrinsic component of Aβ plaques in human brain. The major hallmarks of Alzheimer’s disease, namely Aβ aggregates versus NFTs could also be distinguished due to distinct emission spectra from p-FTAA. Overall, we demonstrate that LCOs can be utilized as powerful practical research tools for studying protein aggregation diseases and facilitate the study of amyloid origin, evolution and maturation, Aβ−tau interactions and pathogenesis both ex vivo and in vivo. PMID:19624097

  5. The DUB-ious lack of ALIS in Salmonella infection: a Salmonella deubiquitinase regulates the autophagy of protein aggregates.

    PubMed

    Thomas, Mair; Mesquita, Francisco S; Holden, David W

    2012-12-01

    Ubiquitinated aggregates are formed in eukaryotic cells in response to several external stimuli, including exposure to bacterial lipopolysaccharide (LPS). Although Salmonella enterica serovar Typhimurium (S. Typhimurium) LPS has been shown to induce aggresome-like induced structures (ALIS) in macrophages, these have not been described in S. Typhimurium-infected macrophages. Given that LPS is present in infection, this suggests that S. Typhimurium might suppress the formation of ALIS. We found that S. Typhimurium induces the formation of ubiquitinated aggregates in epithelial cells and macrophages, but that their presence is masked by the deubiquitinase (DUB) activity of the S. Typhimurium virulence protein, SseL. SseL deubiquitinates SQSTM1/p62-bound proteins found in S. Typhimurium-induced aggregates and ALIS, and reduces the recruitment of autophagic components. While the functions of ALIS and other ubiquitinated aggregates remain unclear, they serve to sequester cytosolic proteins under a variety of stress conditions and are suggested to be involved in host immune defense. During infection, the deubiquitinase activity of SseL reduces autophagic flux in infected cells and favors bacterial replication. This is a new example of how a bacterial pathogen counteracts the autophagy pathway through the action of a translocated virulence protein.

  6. Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO{sub 2} nanoparticles

    SciTech Connect

    Chen Min; Mikecz, Anna von . E-mail: mikecz@uni-duesseldorf.de

    2005-04-15

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

  7. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  8. Disruption of neurofilament network with aggregation of light neurofilament protein: a common pathway leading to motor neuron degeneration due to Charcot-Marie-Tooth disease-linked mutations in NFL and HSPB1.

    PubMed

    Zhai, Jinbin; Lin, Hong; Julien, Jean-Pierre; Schlaepfer, William W

    2007-12-15

    Mutations in neurofilament light (NFL) subunit and small heat-shock protein B1 (HSPB1) cause autosomal-dominant axonal Charcot-Marie-Tooth disease type 2E (CMT2E) and type 2F (CMT2F). Previous studies have shown that CMT mutations in NFL and HSPB1 disrupt NF assembly and cause aggregation of NFL protein. In this study, we investigate the role of aggregation of NFL protein in the neurotoxicity of CMT mutant NFL and CMT mutant HSPB1 in motor neurons. We find that expression of CMT mutant NFL leads to progressive degeneration and loss of neuronal viability of cultured motor neurons. Degenerating motor neurons show fragmentation and loss of neuritic processes associated with disruption of NF network and aggregation of NFL protein. Co-expression of wild-type HSPB1 diminishes aggregation of CMT mutant NFL, induces reversal of CMT mutant NFL aggregates and reduces CMT mutant NFL-induced loss of motor neuron viability. Like CMT mutant NFL, expression of S135F CMT mutant HSPB1 also leads to progressive degeneration of motor neurons with disruption of NF network and aggregation of NFL protein. Further studies show that wild-type and S135F mutant HSPB1 associate with wild-type and CMT mutant NFL and that S135F mutant HSPB1 has dominant effect on disruption of NF assembly and aggregation of NFL protein. Finally, we show that deletion of NFL markedly reduces degeneration and loss of motor neuron viability induced by S135F mutant HSPB1. Together, our data support the view that disruption of NF network with aggregation of NFL is a common triggering event of motor neuron degeneration in CMT2E and CMT2F disease.

  9. A numerical study of soot aggregate formation in a laminar coflow diffusion flame

    SciTech Connect

    Zhang, Q.; Thomson, M.J.; Guo, H.; Liu, F.; Smallwood, G.J.

    2009-03-15

    Soot aggregate formation in a two-dimensional laminar coflow ethylene/air diffusion flame is studied with a pyrene-based soot model, a detailed sectional aerosol dynamics model, and a detailed radiation model. The chemical kinetic mechanism describes polycyclic aromatic hydrocarbon formation up to pyrene, the dimerization of which is assumed to lead to soot nucleation. The growth and oxidation of soot particles are characterized by the HACA surface mechanism and pyrene-soot surface condensation. The mass range of the solid soot phase is divided into thirty-five discrete sections and two equations are solved in each section to model the formation of the fractal-like soot aggregates. The coagulation model is improved by implementing the aggregate coagulation efficiency. Several physical processes that may cause sub-unitary aggregate coagulation efficiency are discussed. Their effects on aggregate structure are numerically investigated. The average number of primary soot particles per soot aggregate n{sub p} is found to be a strong function of the aggregate coagulation efficiency. Compared to the available experimental data, n{sub p} is well reproduced with a constant 20% aggregate coagulation efficiency. The predicted axial velocity, OH mole fraction, and C{sub 2}H{sub 2} mole fraction are validated against experimental data in the literature. Reasonable agreements are obtained. Finally, a sensitivity study of the effects of particle coalescence on soot volume fraction and soot aggregate nanostructure is conducted using a coalescence cutoff diameter method. (author)

  10. Sequence-dependent Internalization of Aggregating Peptides*

    PubMed Central

    Couceiro, José R.; Gallardo, Rodrigo; De Smet, Frederik; De Baets, Greet; Baatsen, Pieter; Annaert, Wim; Roose, Kenny; Saelens, Xavier; Schymkowitz, Joost; Rousseau, Frederic

    2015-01-01

    Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 μm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease. PMID:25391649

  11. Effects of different isoforms of apoE on aggregation of the α-synuclein protein implicated in Parkinson's disease.

    PubMed

    Emamzadeh, Fatemeh Nouri; Aojula, Harmesh; McHugh, Patrick C; Allsop, David

    2016-04-01

    Parkinson's disease is a progressive brain disorder due to the degeneration of dopaminergic neurons in the substantia nigra. The accumulation of aggregated forms of α-synuclein protein into Lewy bodies is one of the characteristic features of this disease although the pathological role of any such protein deposits in causing neurodegeneration remains elusive. Here, the effects of different apolipoprotein E isoforms (apoE2, apoE3, apoE4) on the aggregation of α-synuclein in vitro were examined using thioflavin T assays and also an immunoassay to detect the formation of multimeric forms. Our results revealed that the aggregation of α-synuclein is influenced by apoE concentration. At low concentrations of apoE (<15nM), all of the isoforms were able to increase the aggregation of α-synuclein (50μM), with apoE4 showing the greatest stimulatory effect. This is in contrast to a higher concentration (>15nM) of these isoforms, where a decrease in the aggregation of α-synuclein was noted. The data show that exceptionally low levels of apoE may seed α-syn aggregation, which could potentially lead to the pathogenesis of α-synuclein-induced neurodegeneration. On the other hand, higher levels of apoE could potentially lower the degree of α-synuclein aggregation and confer protection. The differential effects noted with apoE4 could explain why this particular isoform results in an earlier age of onset for Parkinson's disease.

  12. A native-like intermediate serves as a branching point between the folding and aggregation pathways of the mouse prion protein

    PubMed Central

    Honda, Ryo P.; Xu, Ming; Yamaguchi, Kei-ichi; Roder, Heinrich; Kuwata, Kazuo

    2015-01-01

    SUMMARY Transient folding intermediates and/or partially unfolded equilibrium states are thought to play a key role in the formation of protein aggregates. However, there is only indirect evidence linking accumulation of folding intermediates to aggregation, and the underlying mechanism remains to be elucidated. Here we show that a partially unfolded state of the prion protein accumulates both as a stable equilibrium state at acidic pH (A-state) and as a late folding intermediate. With a time resolution of approximately 60 μs, we systematically studied the kinetics of folding and unfolding, starting from various initial conditions including the U-, N-, and A-states. Quantitative modeling showed that the observed kinetic data are completely consistent with a sequential four-state mechanism where the A-state is a late folding intermediate. Combined with previous evidence linking A-state accumulation to aggregation, the results indicate that this native-like state serves as a branching point between the folding and aggregation pathways. PMID:26256540

  13. A role for a Hevea latex lectin-like protein in mediating rubber particle aggregation and latex coagulation.

    PubMed

    Wititsuwannakul, Rapepun; Pasitkul, Piyaporn; Kanokwiroon, Kamonwan; Wititsuwannakul, Dhirayos

    2008-01-01

    An in vitro aggregation of washed lutoid membrane and rubber particles, respectively, prepared from the bottom (lutoid) fraction and rubber layer of centrifuged fresh latex, leading to the formation of rubber coagulum necessary for a latex coagulation was demonstrated. A Triton X-100 extract of washed lutoid membrane proteins, isolated and prepared from the bottom fraction of centrifuged fresh latex was examined for its role in the latex coagulation process. It induced agglutination of rabbit erythrocytes, indicating the presence of a lectin-like protein. Hevea latex lectin-like protein (HLL) was purified to homogeneity by active chitin binding separation, followed by DEAE-Sepharose chromatography. Its M(r) analyzed by SDS-PAGE was 17 kDa, whereas that determined by gel filtration was 267 kDa. The HLL had a pI value of 7.2. Several glycoproteins were shown to inhibit the HLL-induced hemagglutination. The hemagglutinin activity of HLL was enhanced by Ca(2+). Of most interest was the finding that HLL strongly induced aggregation of the Hevea latex rubber particles (RP). This strong RP aggregation leads to latex coagulation, indicating the possibility that it is involved in the formation of the coagulum that plugs the latex vessel ends and stops the flow of latex upon tapping. In addition, the purified HLL also induced aggregation of RP taken from several other non-Hevea latex producing plants. This might indicate either a common or universal role of this lectin-like protein in RP aggregation and hence latex coagulation. This paper, for the first time, provides clear and unequivocal evidence for either a key biological role or physiological function of an endogenous latex lectin-like protein in the sequential process of latex coagulation. PMID:17897690

  14. Inactivation, aggregation, secondary and tertiary structural changes of germin-like protein in Satsuma mandarine with high polyphenol oxidase activity induced by ultrasonic processing.

    PubMed

    Huang, Nana; Cheng, Xi; Hu, Wanfeng; Pan, Siyi

    2015-02-01

    The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in α-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated PPO activity.

  15. RBC aggregation dynamics in autologous plasma and serum studied with double-channel optical tweezers

    NASA Astrophysics Data System (ADS)

    Lee, Kisung; Danilina, Anna; Potkin, Anton; Kinnunen, Matti; Priezzhev, Alexander; Meglinski, Igor

    2016-04-01

    Red blood cells aggregating and disaggregating forces were measured in the autologous plasma and serum using the double-channeled optical tweezers. A significant, three-fold decrease of the both forces was observed in the serum compared to the plasma. The results of this study help to better assess the RBC aggregation mechanism.

  16. Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins

    PubMed Central

    1994-01-01

    Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co- aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co- aggregated diI and IgE receptors. The results indicate that cross- linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre- existing membrane domains or causing their formation. PMID:8188747

  17. Get3 is a holdase chaperone and moves to deposition sites for aggregated proteins when membrane targeting is blocked

    PubMed Central

    Powis, Katie; Schrul, Bianca; Tienson, Heather; Gostimskaya, Irina; Breker, Michal; High, Stephen; Schuldiner, Maya; Jakob, Ursula; Schwappach, Blanche

    2013-01-01

    Summary The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis. PMID:23203805

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

    NASA Astrophysics Data System (ADS)

    Gadomski, A.; Kruszewska, N.

    2014-08-01

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

  19. An N-terminal nuclear export signal regulates trafficking and aggregation of Huntingtin (Htt) protein exon 1.

    PubMed

    Zheng, Zhiqiang; Li, Aimin; Holmes, Brandon B; Marasa, Jayne C; Diamond, Marc I

    2013-03-01

    Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity. PMID:23319588

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

    PubMed

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

    2016-02-15

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

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

    PubMed Central

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

    2016-01-01

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

  2. SUMO2/3 is associated with ubiquitinated protein aggregates in the mouse neocortex after middle cerebral artery occlusion

    PubMed Central

    Hochrainer, Karin; Jackman, Katherine; Benakis, Corinne; Anrather, Josef; Iadecola, Costantino

    2015-01-01

    Protein modifications cooperatively act to protect the proteome from cellular stress. Focal cerebral ischemia increases protein ubiquitination, resulting in formation of ubiquitin-rich aggregates. A concurrent elevation in small ubiquitin-related modifier (SUMO)-conjugated proteins has also been reported, but a potential connection to ubiquitin remains unexplored. Here we show that SUMO2/3 conjugates are present in postischemic ubiquitin-rich aggregates, physically associated with ubiquitin. The coaggregation of SUMO2/3 and ubiquitin is induced rapidly after ischemia, depends on reperfusion, and is also observed in the absence of ischemic damage. The association between SUMO and ubiquitin suggests overlapping functional roles after ischemia/reperfusion. PMID:25352045

  3. Protein crystallization studies

    NASA Technical Reports Server (NTRS)

    Lyne, James Evans

    1996-01-01

    The Structural Biology laboratory at NASA Marshall Spaceflight Center uses x-ray crystallographic techniques to conduct research into the three-dimensional structure of a wide variety of proteins. A major effort in the laboratory involves an ongoing study of human serum albumin (the principal protein in human plasma) and its interaction with various endogenous substances and pharmaceutical agents. Another focus is on antigenic and functional proteins from several pathogenic organisms including the human immunodeficiency virus (HIV) and the widespread parasitic genus, Schistosoma. My efforts this summer have been twofold: first, to identify clinically significant drug interactions involving albumin binding displacement and to initiate studies of the three-dimensional structure of albumin complexed with these agents, and secondly, to establish collaborative efforts to extend the lab's work on human pathogens.

  4. Application of an in vitro-amplification assay as a novel pre-screening test for compounds inhibiting the aggregation of prion protein scrapie

    PubMed Central

    Schmitz, Matthias; Cramm, Maria; Llorens, Franc; Candelise, Niccolò; Müller-Cramm, Dominik; Varges, Daniela; Schulz-Schaeffer, Walter J.; Zafar, Saima; Zerr, Inga

    2016-01-01

    In vitro amplification assays, such as real-time quaking-induced conversion (RT-QuIC) are used to detect aggregation activity of misfolded prion protein (PrP) in brain, cerebrospinal fluid (CSF) and urine samples from patients with a prion disease. We believe that the method also has a much broader application spectrum. In the present study, we applied RT-QuIC as a pre-screening test for substances that potentially inhibit the aggregation process of the cellular PrP (PrPC) to proteinase (PK)-resistant PrPres. We chose doxycycline as the test substance as it has been tested successfully in animal models and proposed in clinical studies as a therapeutic for prion diseases. The RT-QuIC-reaction was seeded with brain tissue or CSF from sCJD patients and doxycycline was then added in different concentrations as well as at different time points. In both experiments, we observed a dose- and time-dependent inhibition of the RT-QuIC seeding response and a decrease of PK resistant PrPres when doxycycline was added. In contrast, ampicillin or sucrose had no effect on the RT-QuIC seeding response. Our study is the first to apply RT-QuIC as a pre-screening assay for compounds inhibiting the PrP aggregation in vitro and confirms that doxycycline is an efficient inhibitor of the PrP aggregation process in RT-QuIC analysis. PMID:27385410

  5. Study on performance of concrete with over-burnt bricks aggregates and micro-silica admixture

    NASA Astrophysics Data System (ADS)

    Praveen, K.; Sathyan, Dhanya; Mini, K. M.

    2016-09-01

    Concrete is made by mixing cement, sand, aggregates and water in required proportion, where aggregates occupy the major volume. Addition of aggregates in concrete improves properties of concrete. With the natural resources depleting rapidly, limiting the use of natural resources and enhancing the use of waste materials is very important for sustainable development. Over-burnt bricks are a waste material which cannot be used in construction directly because of their irregular shape and dark colour. Use of over-burnt bricks helps to preserve natural aggregate source. The present study focuses on the effects of microsilica at various percentages as a partial cement replacement in concrete with over-burnt bricks as coarse aggregates. The mechanical properties of hardened concrete such as splitting tensile strength, flexural strength and compressive strength are studied and analyzed.

  6. Study of Optical Properties on Fractal Aggregation Using the GMM Method by Different Cluster Parameters