Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins.     

Emerging ideas on the molecular basis of protein and peptide aggregation

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

A review of acquired thermotolerance, heat-shock proteins, and molecular chaperones in archaea

Abstract: Acquired thermotolerance, the associated synthesis of heat-shock proteins (HSPs) under stress conditions, and the role of HSPs as molecular chaperones under normal growth conditions have been studied extensively in eukaryotes and bacteria, whereas research in these areas in archaea is only beginning. All organisms have evolved a variety of strategies for coping with high-temperature stress, and among these strategies is the increased synthesis of HSPs. The facts that both high temperatures and chemical stresses induce the HSPs and that some of the HSPs recognize and bind to unfolded proteins in vitro have led to the theory that the function of HSPs is to prevent protein aggregation in vivo. The facts that some HSPs are abundant under normal growth conditions and that they assist in protein folding in vitro have led to the theory that they assist protein folding in vivo; in this role, they are referred to as molecular chaperones. The limited research on acquired thermotolerance, HSPs, and molecular chaperones in archaea, particularly the hyperthermophilic archaea, suggests that these extremophiles provide a new perspective in these areas of research, both because they are members of a separate phylogenetic domain and because they have evolved to live under extreme conditions.     

Effects of glycerol on the compaction and stability of the wild type and mutated rabbit muscle creatine kinase

All organisms have developed detect, repair, regulation, and stabilization mechanisms to survive from cellular and molecular damage induced by diverse stresses. Among them, the accumulation of osmolytes is a common mechanism evolved by cells to maintain cell volume and stabilize macromolecules against various environmental stresses. The molecular mechanisms by which osmolytes stabilize proteins and prevent aggregation have been well-established. However, little is known about the effects of osmolytes on mutated or damaged proteins. In this research, we investigated the effects of glycerol on the activity, structure, and stability of the wild type (WT) and D54G CK under normal and extreme (high temperature) conditions. It was found that glycerol had similar effects on the suppression of the aggregation during the refolding of both proteins. Under native conditions, the effect of glycerol on the mutated protein was more obvious than on the WT protein. Glycerol could efficiently force the mutated protein to fold to a state close to the WT protein, and thus stabilize the native state of the mutated protein. Glycerol could also protect both the WT and mutated proteins against heat-induced denaturation. However, the change in the transition free energy of heat-induced inactivation of the WT protein was larger than that of the mutated protein. These results suggested that glycerol might have differential effects on the changes of the chemical potential and the transition free energy of the WT and mutated proteins.     

On the Role of Aggregation Prone Regions in Protein Evolution,Stability, and Enzymatic Catalysis: Insights from Diverse Analyses

The various roles that aggregation prone regions (APRs) are capable of playing in proteins are investigated here via comprehensive analyses of multiple non-redundant datasets containing randomly generated amino acid sequences, monomeric proteins, intrinsically disordered proteins (IDPs) and catalytic residues. Results from this study indicate that the aggregation propensities of monomeric protein sequences have been minimized compared to random sequences with uniform and natural amino acid compositions, as observed by a lower average aggregation propensity and fewer APRs that are shorter in length and more often punctuated by gate-keeper residues. However, evidence for evolutionary selective pressure to disrupt these sequence regions among homologous proteins is inconsistent. APRs are less conserved than average sequence identity among closely related homologues (≥80% sequence identity with a parent) but APRs are more conserved than average sequence identity among homologues that have at least 50% sequence identity with a parent. Structural analyses of APRs indicate that APRs are three times more likely to contain ordered versus disordered residues and that APRs frequently contribute more towards stabilizing proteins than equal length segments from the same protein. Catalytic residues and APRs were also found to be in structural contact significantly more often than expected by random chance. Our findings suggest that proteins have evolved by optimizing their risk of aggregation for cellular environments by both minimizing aggregation prone regions and by conserving those that are important for folding and function. In many cases, these sequence optimizations are insufficient to develop recombinant proteins into commercial products. Rational design strategies aimed at improving protein solubility for biotechnological purposes should carefully evaluate the contributions made by candidate APRs, targeted for disruption, towards protein structure and activity.     

Correlation between the structural stability and aggregation propensity of proteins

Protein aggregation, being an outcome of improper protein folding, is largely dependent on the folding kinetics of a protein. Previous studies have reported a positive correlation between the stability of the secondary structural elements of a protein and their rate of folding/unfolding. In this in silico study, the secondary and tertiary structures of proteins a) that form inclusion bodies on overexpression in Escherichia coli, b) that form amyloid fibrils and c) that are soluble on overexpression in E. coli are analyzed for certain features that are known to be associated with structural stability. The study revealed that the soluble proteins seem to have a higher rate of folding (based on contact order) and a lower percentage of exposed hydrophobic residues as compared to the inclusion body forming or amyloidogenic proteins. The soluble proteins also seem to have a more favored helix and strand composition (based on the known secondary structural propensities of amino acids). The secondary structure analyses also reveal that the evolutionary pressure is directed against protein aggregation. This understanding of the positive correlation between structural stability and solubility, along with the other parameters known to influence aggregation, could be exploited in the design of mutations aimed at reducing the aggregation propensity of the proteins.     

A comparison of genomic coding sequences for feather and scale keratins: structural and evolutionary implications.

DNA sequences have been obtained for embryonic chick feather and scale keratin genes. Strong homologies exist between the protein coding regions of the two gene types and between the deduced amino acid sequences of the keratin proteins. Scale keratins are larger than feather keratins and the size difference is mainly attributable to four 13-amino acid repeats between residues 77 and 128 which compose a peptide sequence rich in glycine and tyrosine. The strong similarities between the two peptide structures for feather and scale in the homologous regions suggests a similar conformation within the protein filaments. A likely consequence is that the additional repeat region of the scale protein is located externally to the core filament. Tissue-specific features of filament aggregation may be attributable to this one striking sequence difference between the constituent proteins. It is believed that the genes share a common ancestry and that feather-like keratin genes may have evolved from a scale keratin gene by a single deletion event.     

The Unfolded State of the Murine Prion Protein and Properties of Single-point Mutants Related to Human Prion Diseases

The prion protein can exist both in a normal cellular isoform and in a pathogenic conformational isoform. The latter is responsible for the development of different neurodegenerative diseases, for example Creutzfeldt-Jakob disease or fatal familial insomnia. To convert the native benign state of the protein into a highly ordered fibrillar aggregate, large-scale rearrangements of the tertiary structure are necessary during the conversion process and intermediates that are at least partially unfolded are present during fibril formation. In addition to the sporadic conversion into the pathogenic isoform, more than 20 familial diseases are known that are caused by single point mutations increasing the probability of aggregation and neurodegeneration. Here, we demonstrate that the chemically denatured states of the mouse and human prion proteins have very similar structural and dynamic characteristics. Initial studies on the single point mutants E196K, F198S, V203I and R208H of the oxidized mouse construct, which are related to human prion diseases, reveal significant differences in the rate of aggregation. Aggregation for mutants V203I and R208H is slower than it is for the wild type, and the constructs E196K and F198S show accelerated aggregation. These differences in aggregation behaviour are not correlated with the thermal stability of the mutants, indicating different mechanisms promoting the conformational conversion process.     

Primary amine coding as a path to comparative proteomics

Various isotope coding strategies are being used today in the field of comparative proteomics. This article specifically reviews the strengths and limitations of various N-termini-directing strategies. N-termini-directed coding strategy allows for use of different chromatographic enrichment techniques. Since N-termini-directed coding strategies are global in nature, they can be utilized in studying PTMs as well as protein expression. Using different N-termini-directed coding strategies, both relative and absolute quantification of proteins can be achieved either in the MS mode or in the MS/MS mode. The review ends with the conclusion that significant improvements have been made in the last decade. Among various issues, a need still exists for a better understanding of the kinetic issues in proteomics, relative protein pool sizes for different proteins and the issue of stimulus-induced changes in protein aggregation. Another critical issue that needs to be addressed in great detail is the role of PTMs in regulation.     

昆虫免疫识别与病原物免疫逃避机理研究进展

昆虫在长期进化过程中形成复杂的天然免疫系统,病原识别是启动下游免疫反应的第一步,这一过程主要是由不同的模式识别蛋白来完成的。目前发现并鉴定的昆虫模式识别蛋白主要包括肽聚糖识别蛋白、类免疫球蛋白、β-1,3-葡聚糖结合蛋白、C型凝集素及具多功能的载脂蛋白等,不同的蛋白种类具有不同的结构、功能及识别对象。与昆虫免疫识别相对应的是,不同昆虫病原物在进化过程中发展出不同策略的免疫逃避能力,以战胜宿主免疫而致病或最终杀死昆虫。本文就昆虫免疫过程中不同模式识别蛋白的结合对象、结构与功能,以及逐渐兴起的病原物通过分子伪装等进行免疫逃避的研究进展进行了综述。并在此基础上,作者就昆虫免疫与昆虫病理研究的发展方向进行了展望,认为只有当两方面研究相结合时,才能更好地揭示昆虫宿主与病原物之间免疫与抗免疫的动态相互作用过程。     

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

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

Folding and aggregation are selectively influenced by the conformational preferences of the alpha-helices of muscle acylphosphatase

The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.     

Hsp70 from Cyprinion macrostomus macrostomus and Garra rufa obtuse: Stability and stability-dependent activity

Two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, tolerate adverse conditions in the Kangal hot springs and cope with multiple stressors such as food deprivation, extreme temperature, toxins, protein degradation, hypoxia, and microbial damage. These fish have evolved strategies to counteract the stressors including the induction of heat shock proteins (Hsps). Hsps play an essential role in maintaining cellular homeostasis, and one of the key proteins in the mechanism is Hsp70. Hsp70 itself is exposed to the same stressors as all other proteins, and, hence, the stability of Hsp70 was investigated. For this purpose, Hsp70 ATPase activity was determined at different urea concentrations. It was found that the protein maintains considerable ATP hydrolysis activity at higher denaturant conditions. Temperature effects on the substrate peptide binding showed that Hsp70s bind prominently at elevated temperatures. Furthermore, temperature effects on Hsp70 aggregation indicated that the presence of nucleotides decreases the aggregation process. The present work has determined the stability and activity of cmHsp70 and grHsp70 themselves under extreme conditions. The stability of the Hsp70 proteins maintains substrate proteins in the native state, which may aid in the adaptation of the fish species to the hot spring environment.     

Beta edge strands in protein structure prediction and aggregation

It is well established that recognition between exposed edges of beta-sheets is an important mode of protein-protein interaction and can have pathological consequences; for instance, it has been linked to the aggregation of proteins into a fibrillar structure, which is associated with a number of predominantly neurodegenerative disorders. A number of protective mechanisms have evolved in the edge strands of beta-sheets, preventing the aggregation and insolubility of most natural beta-sheet proteins. Such mechanisms are unfavorable in the interior of a beta-sheet. The problem of distinguishing edge strands from central strands based on sequence information alone is important in predicting residues and mutations likely to be involved in aggregation, and is also a first step in predicting folding topology. Here we report support vector machine (SVM) and decision tree methods developed to classify edge strands from central strands in a representative set of protein domains. Interestingly, rules generated by the decision tree method are in close agreement with our knowledge of protein structure and are potentially useful in a number of different biological applications. When trained on strands from proteins of known structure, using structure-based (Dictionary of Secondary Structure in Proteins) strand assignments, both methods achieved mean cross-validated, prediction accuracies of approximately 78%. These accuracies were reduced when strand assignments from secondary structure prediction were used. Further investigation of this effect revealed that it could be explained by a significant reduction in the accuracy of standard secondary structure prediction methods for edge strands, in comparison with central strands.     

Role of arginine in protein refolding, solubilization, and purification

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.     

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

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. Proteins 2016; 84:254–266. © 2015 Wiley Periodicals, Inc.     

Guiding protein aggregation with macromolecular crowding

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.     

Molecular genetics of disease resistance in cereals

AIMS: This Botanical Briefing attempts to summarize what is currently known about the molecular bases of disease resistance in cereal species and suggests future research directions. SCOPE: An increasing number of resistance (R) genes have been isolated from rice, maize, wheat and barley that encode both structurally related and unique proteins. This R protein diversity may be attributable to the different modus operandi employed by pathogen species in some cases, but it is also a consequence of multiple defence strategies being employed against phytopathogens. Mutational analysis of barley has identified additional genes required for activation of an R gene-mediated defence response upon pathogen infection. In some instances very closely related barley R proteins require different proteins for defence activation, demonstrating that, within a single plant species, multiple resistance signalling pathways and different resistance strategies have evolved to confer protection against a single pathogen species. Despite the apparent diversity of cereal resistance mechanisms, some of the additional molecules required for R protein function are conserved amongst cereal and dicotyledonous species and even other eukaryotic species. Thus the derivation of functional homologues and interacting partner proteins from other species is contributing to the understanding of resistance signalling in cereals. The potential and limit of utilizing the rice genome sequence for further R gene isolation from cereal species is also considered, as are the new biotechnological possibilities for disease control arising from R gene isolation. CONCLUSIONS: Molecular analyses in cereals have further highlighted the complexity of plant-pathogen co-evolution and have shown that numerous active and passive defence strategies are employed by plants against phytopathogens. Many advances in understanding the molecular basis of disease resistance in cereals have focused on monogenic resistance traits. Future research targets are likely to include less experimentally tractable, durable polygenic resistances and nonhost resistance mechanisms.     

Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies

Monoclonal antibodies (mAbs) are powerful therapeutics, and their characterization has drawn considerable attention and urgency. Unlike small-molecule drugs (150–600 Da) that have rigid structures, mAbs (∼150 kDa) are engineered proteins that undergo complicated folding and can exist in a number of low-energy structures, posing a challenge for traditional methods in structural biology. Mass spectrometry (MS)-based biophysical characterization approaches can provide structural information, bringing high sensitivity, fast turnaround, and small sample consumption. This review outlines various MS-based strategies for protein biophysical characterization and then reviews how these strategies provide structural information of mAbs at the protein level (intact or top-down approaches), peptide, and residue level (bottom-up approaches), affording information on higher order structure, aggregation, and the nature of antibody complexes.