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1.
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.  相似文献   

2.
Polyglutamine (polyQ) repeat-containing proteins are widespread in the human proteome but only nine of them are associated with highly incapacitating neurodegenerative disorders. The genetic expansion of the polyQ tract in disease-related proteins triggers a series of events resulting in neurodegeneration. The polyQ tract plays the leading role in the aggregation mechanism, but other elements modulate the aggregation propensity in the context of the full-length proteins, as implied by variations in the length of the polyQ tract required to trigger the onset of a given polyQ disease. Intrinsic features such as the presence of aggregation-prone regions (APRs) outside the polyQ segments and polyQ-flanking sequences, which synergistically participate in the aggregation process, are emerging for several disease-related proteins. The inherent polymorphic structure of polyQ stretches places the polyQ proteins in a central position in protein–protein interaction networks, where interacting partners may additionally shield APRs or reshape the aggregation course. Expansion of the polyQ tract perturbs the cellular homeostasis and contributes to neuronal failure by modulating protein–protein interactions and enhancing toxic oligomerization. Post-translational modifications further regulate self-assembly either by directly altering the intrinsic aggregation propensity of polyQ proteins, by modulating their interaction with different macromolecules or by modifying their withdrawal by the cell quality control machinery. Here we review the recent data on the multifaceted aggregation pathways of disease-related polyQ proteins, focusing on ataxin-3, the protein mutated in Machado-Joseph disease. Further mechanistic understanding of this network of events is crucial for the development of effective therapies for polyQ diseases.  相似文献   

3.
Intrinsically disordered (ID) proteins function in the absence of a unique stable structure and appear to challenge the classic structure-function paradigm. The extent to which ID proteins take advantage of subtle conformational biases to perform functions, and whether signals for such mechanism can be identified in proteome-wide studies is not well understood. Of particular interest is the polyproline II (PII) conformation, suggested to be highly populated in unfolded proteins. We experimentally determine a complete calorimetric propensity scale for the PII conformation. Projection of the scale into representative eukaryotic proteomes reveals significant PII bias in regions coding for ID proteins. Importantly, enrichment of PII in ID proteins, or protein segments, is also captured by other PII scales, indicating that this enrichment is robustly encoded and universally detectable regardless of the method of PII propensity determination. Gene ontology (GO) terms obtained using our PII scale and other scales demonstrate a consensus for molecular functions performed by high PII proteins across the proteome. Perhaps the most striking result of the GO analysis is conserved enrichment (P < 10−8) of phosphorylation sites in high PII regions found by all PII scales. Subsequent conformational analysis reveals a phosphorylation-dependent modulation of PII, suggestive of a conserved “tunability” within these regions. In summary, the application of an experimentally determined polyproline II (PII) propensity scale to proteome-wide sequence analysis and gene ontology reveals an enrichment of PII bias near disordered phosphorylation sites that is conserved throughout eukaryotes.  相似文献   

4.
5.
Numerous studies have demonstrated that the propensity of a protein to form amyloids or amorphous aggregates is encoded by its amino acid sequence. This led to the emergence of several computational programs to predict amyloidogenicity from amino acid sequences. However, a growing number of studies indicate that an accurate prediction of the protein aggregation can only be achieved when also accounting for the overall structural context of the protein, and the likelihood of transition between the initial state and the aggregate. Here, we describe a computational pipeline called TAPASS, which was designed to do just that. The pipeline assigns each residue of a protein as belonging to a structured region or an intrinsically disordered region (IDR). For this purpose, TAPASS uses either several state-of-the-art programs for prediction of IDRs, of transmembrane regions and of structured domains or the artificial intelligence program AlphaFold. In the next step, this assignment is crossed with amyloidogenicity prediction. As a result, TAPASS allows the detection of Exposed Amyloidogenic Regions (EARs) located within intrinsically disordered regions (IDRs) and carrying high amyloidogenic potential. TAPASS can substantially improve the prediction of amyloids and be used in proteome-wide analysis to discover new amyloid-forming proteins. Its results, combined with clinical data, can create individual risk profiles for different amyloidoses, opening up new opportunities for personalised medicine. The architecture of the pipeline is designed so that it makes it easy to add new individual predictors as they become available. TAPASS can be used through the web interface (https://bioinfo.crbm.cnrs.fr/index.php?route=tools&tool=32).  相似文献   

6.
Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.  相似文献   

7.
8.
Experimental determination of the key features of the free energy landscapes of proteins, which dictate their adeptness to fold correctly, or propensity to misfold and aggregate and which are modulated upon a change from physiological to aggregation-prone conditions, is a difficult challenge. In this study, sub-millisecond kinetic measurements of the folding and unfolding of the mouse prion protein reveal how the free energy landscape becomes more complex upon a shift from physiological (pH 7) to aggregation-prone (pH 4) conditions. Folding and unfolding utilize the same single pathway at pH 7, but at pH 4, folding occurs on a pathway distinct from the unfolding pathway. Moreover, the kinetics of both folding and unfolding at pH 4 depend not only on the final conditions but also on the conditions under which the processes are initiated. Unfolding can be made to switch to occur on the folding pathway by varying the initial conditions. Folding and unfolding pathways appear to occupy different regions of the free energy landscape, which are separated by large free energy barriers that change with a change in the initial conditions. These barriers direct unfolding of the native protein to proceed via an aggregation-prone intermediate previously identified to initiate the misfolding of the mouse prion protein at low pH, thus identifying a plausible mechanism by which the ruggedness of the free energy landscape of a protein may modulate its aggregation propensity.  相似文献   

9.
Protein aggregation is the phenomenon of protein self-association potentially leading to detrimental effects on physiology, which is closely related to numerous human diseases such as Alzheimer's and Parkinson's disease. Despite progress in understanding the mechanism of protein aggregation, how natural selection against protein aggregation acts on subunits of protein complexes and on proteins with different contributions to organism fitness remains largely unknown. Here, we perform a proteome-wide analysis by using an experimentally validated algorithm TANGO and utilizing sequence, interactomic and phenotype-based functional genomic data from yeast, fly, and nematode. We find that proteins that are capable of forming homooligomeric complex have lower aggregation propensity compared with proteins that do not function as homooligomer. Further, proteins that are essential to the fitness of an organism have lower aggregation propensity compared with nonessential ones. Our finding suggests that the selection force against protein aggregation acts across different hierarchies of biological system.  相似文献   

10.
Khare SD  Wilcox KC  Gong P  Dokholyan NV 《Proteins》2005,61(3):617-632
Diverse point mutations in the enzyme Cu, Zn superoxide dismutase (SOD1) are linked to its aggregation in the familial form of the disease amyotrophic lateral sclerosis. The disease-associated mutations are known to destabilize the protein, but the structural basis of the aggregation of the destabilized protein and the structure of aggregates are not well understood. Here, we investigate in silico the sequence and structural determinants of SOD1 aggregation: (1) We identify sequence fragments in SOD1 that have a high aggregation propensity, using only the sequence of SOD1, and (2) we perform molecular dynamics simulations of the SOD1 dimer folding and misfolding. In both cases, we identify identical regions of the protein as having high propensity to form intermolecular interactions. These regions correspond to the N- and C-termini, and two crossover loops and two beta-strands in the Greek-key native fold of SOD1. Our results suggest that the high aggregation propensity of mutant SOD1 may result from a synergy of two factors: the presence of highly amyloidogenic sequence fragments ("hot spots"), and the presence of these fragments in regions of the protein that are structurally most likely to form intermolecular contacts under destabilizing conditions. Therefore, we postulate that the balance between the self-association of aggregation-prone sequences and the specific structural context of these sequences in the native state determines the aggregation propensity of proteins.  相似文献   

11.
《MABS-AUSTIN》2013,5(6):580-582
Monoclonal antibodies represent the fastest growing class of pharmaceuticals. A major problem, however, is that the proteins are susceptible to aggregation at the high concentration commonly used during manufacturing and storage. Our recent publication describes a technology based on molecular simulations to identify aggregation-prone regions of proteins in silico. The technology, called spatial aggregation propensity (SAP), identifies hot-spots for aggregation based on the dynamic exposure of spatially-adjacent hydrophobic amino acids. Monoclonal antibodies (mAbs) in which patches with high-SAP scores are changed to patches with significantly reduced SAP scores via a single mutation are more stable than wild type, thus validating the SAP method for mapping aggregation-prone regions on proteins. We propose that the SAP technology will be useful for protein stabilization, and as a screening tool to bridge discovery and development of protein-based therapeutics by a rational assessment of the developability of candidate protein drugs.  相似文献   

12.
Human α1-acid glycoprotein (AGP) is a positive acute phase plasma protein containing two disulfide bridges. Structural studies have shown that under specific conditions AGP undergoes aggregation. In this study, we analysed the nature of AGP's aggregates formed under reducing and non-reducing conditions at pH 5.5 and at relatively low temperatures. Thioflavin T and Congo red spectroscopic analyses indicated the presence of cross-β structures in both unreduced and reduced AGP aggregates. In these samples amyloid-like fibrils were detected by transmission electron microscopy. The fibrils are branched and bent and present in very large amount in reduced AGP. Kinetics of AGP fibrillation proceeds without a lag phase and the rate constants of cross-β formation are linearly dependent on AGP concentration and result higher under reducing conditions. The data suggest a possible downhill mechanism of polymerization with a first-order monomer concentration dependence. Bioinformatics tools highlighted an extended region that sheathes one side of the molecule containing aggregation-prone regions. Reducing conditions make the extended region less constricted, allowing greater exposure of aggregation-prone regions, thus explaining the higher propensity of AGP to aggregate and fibrillate.  相似文献   

13.
The toxic accumulation of misfolded proteins as inclusions, fibrils, or aggregates is a hallmark of many neurodegenerative diseases. However, how molecular chaperones, such as heat shock protein 70 kDa (Hsp70) and heat shock protein 90 kDa (Hsp90), defend cells against the accumulation of misfolded proteins remains unclear. The ATP-dependent foldase function of both Hsp70 and Hsp90 actively transitions misfolded proteins back to their native conformation. By contrast, the ATP-independent holdase function of Hsp70 and Hsp90 prevents the accumulation of misfolded proteins. Foldase and holdase functions can protect against the toxicity associated with protein misfolding, yet we are only beginning to understand the mechanisms through which they modulate neurodegeneration. This review compares recent structural findings regarding the binding of Hsp90 to misfolded and intrinsically disordered proteins, such as tau, α-synuclein, and Tar DNA-binding protein 43. We propose that Hsp90 and Hsp70 interact with these proteins through an extended and dynamic interface that spans the surface of multiple domains of the chaperone proteins. This contrasts with many other Hsp90–client protein interactions for which only a single bound conformation of Hsp90 is proposed. The dynamic nature of these multidomain interactions allows for polymorphic binding of multiple conformations to vast regions of Hsp90. The holdase functions of Hsp70 and Hsp90 may thus allow neuronal cells to modulate misfolded proteins more efficiently by reducing the long-term ATP running costs of the chaperone budget. However, it remains unclear whether holdase functions protect cells by preventing aggregate formation or can increase neurotoxicity by inadvertently stabilizing deleterious oligomers.  相似文献   

14.
Ángyán AF  Perczel A  Gáspári Z 《FEBS letters》2012,586(16):2468-2472
Present-day proteins are believed to have evolved features to reduce the risk of aggregation. However, proteins can emerge de novo by translation of non-coding DNA segments. In this study we assess the aggregation, disorder and transmembrane propensity of protein sequences generated by translating random nucleotide sequences of varying GC-content. Potential de novo random-sequence proteins translated from regions with GC content between 40% and 60% do not show stronger aggregation propensity than existing ones and exhibit similar tendency to be disordered. We suggest that de novo emerging proteins do not mean an unavoidable aggregation threat to evolving organisms.  相似文献   

15.
S100 proteins are small dimeric calcium-binding proteins which control cell cycle, growth and differentiation via interactions with different target proteins. Intrinsic disorder is a hallmark among many signaling proteins and S100 proteins have been proposed to contain disorder-prone regions. Interestingly, some S100 proteins also form amyloids: S100A8/A9 forms fibrils in prostatic inclusions and S100A6 fibrillates in vitro and seeds SOD1 aggregation. Here we report a study designed to investigate whether β-aggregation is a feature extensive to more members of S100 family. In silico analysis of seven human S100 proteins revealed a direct correlation between aggregation and intrinsic disorder propensity scores, suggesting a relationship between these two independent properties. Averaged position-specific analysis and structural mapping showed that disorder-prone segments are contiguous to aggregation-prone regions and that whereas disorder is prominent on the hinge and target protein-interaction regions, segments with high aggregation propensity are found in ordered regions within the dimer interface. Acidic conditions likely destabilize the seven S100 studied by decreasing the shielding of aggregation-prone regions afforded by the quaternary structure. In agreement with the in silico analysis, hydrophobic moieties become accessible as indicated by strong ANS fluorescence. ATR-FTIR spectra support a structural inter-conversion from α-helices to intermolecular β-sheets, and prompt ThT-binding takes place with no noticeable lag phase. Dot blot analysis using amyloid conformational antibodies denotes a high diversity of conformers; subsequent analysis by TEM shows fibrils as dominant species. Altogether, our data suggests that β-aggregation and disorder-propensity are related properties in S100 proteins, and that the onset of aggregation is likely triggered by loss of protective tertiary and quaternary interactions.  相似文献   

16.
Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed protein misfolding disorders that are characterized by the neuronal accumulation of protein aggregates. Manipulation of the cellular stress-response involving induction of heat shock proteins (Hsps) in differentiated neurons offers a therapeutic strategy to counter conformational changes in neuronal proteins that trigger pathogenic cascades resulting in neurodegenerative diseases. Hsps are protein repair agents that provide a line of defense against misfolded, aggregation-prone proteins. These proteins are not induced in differentiated neurons by conventional heat shock. We have found that celastrol, a quinine methide triterpene, induced expression of a wider set of Hsps, including Hsp70B', in differentiated human neurons grown in tissue culture compared to cultured rodent neuronal cells. Hence the beneficial effect of celastrol against human neurodegenerative diseases may exceed its potential in rodent models of these diseases.  相似文献   

17.
Protein aggregation is linked to a growing list of diseases, but it is also an intrinsic property of polypeptides, because the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including the M14B subfamily carboxypeptidases. We show here that the monomeric transthyretin-like domain of human carboxypeptidase D aggregates under close to physiological conditions into amyloid structures, with the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic β-strands in the native state. These structural elements should serve for functional/structural purposes, because they have not been purged out by evolution, but at the same time they put proteins like carboxypeptidase D at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.  相似文献   

18.
19.
High-affinity antibodies are critical for numerous diagnostic and therapeutic applications, yet their utility is limited by their variable propensity to aggregate either at low concentrations for antibody fragments or high concentrations for full-length antibodies. Therefore, determining the sequence and structural features that differentiate aggregation-resistant antibodies from aggregation-prone ones is critical to improving their activity. We have investigated the molecular origins of antibody aggregation for human V(H) domain antibodies that differ only in the sequence of the loops containing their complementarity determining regions (CDRs), yet such antibodies possess dramatically different aggregation propensities in a manner not correlated with their conformational stabilities. We find the propensity of these antibodies to aggregate after being transiently unfolded is not a distributed property of the CDR loops, but can be localized to aggregation hotspots within and near the first CDR (CDR1). Moreover, we have identified a triad of charged mutations within CDR1 and a single charged mutation adjacent to CDR1 that endow the poorly soluble variant with the desirable biophysical properties of the aggregation-resistant antibody. Importantly, we find that several other charged mutations in CDR1, non-CDR loops and the antibody scaffold are incapable of preventing aggregation. We expect that our identification of aggregation hotspots that govern antibody aggregation within and proximal to CDR loops will guide the design and selection of antibodies that not only possess high affinity and conformational stability, but also extreme resistance to aggregation.  相似文献   

20.
Lee Y  Zhou T  Tartaglia GG  Vendruscolo M  Wilke CO 《Proteomics》2010,10(23):4163-4171
We analyze the relationship between codon usage bias and residue aggregation propensity in the genomes of four model organisms, Escherichia coli, yeast, fly, and mouse, as well as the archaeon Halobacterium species NRC-1. Using the Mantel-Haenszel procedure, we find that translationally optimal codons associate with aggregation-prone residues. Our results are qualitatively and quantitatively similar to those of an earlier study where we found an association between translationally optimal codons and buried residues. We also combine the aggregation-propensity data with solvent-accessibility data. Although the resulting data set is small, and hence statistical power low, results indicate that the association between optimal codons and aggregation-prone residues exists both at buried and at exposed sites. By comparing codon usage at different combinations of sites (exposed, aggregation-prone sites versus buried, non-aggregation-prone sites; buried, aggregation-prone sites versus exposed, non-aggregation-prone sites), we find that aggregation propensity and solvent accessibility seem to have independent effects of (on average) comparable magnitude on codon usage. Finally, in fly, we assess whether optimal codons associate with sites at which amino acid substitutions lead to an increase in aggregation propensity, and find only a very weak effect. These results suggest that optimal codons may be required to reduce the frequency of translation errors at aggregation-prone sites that coincide with certain functional sites, such as protein-protein interfaces. Alternatively, optimal codons may be required for rapid translation of aggregation-prone regions.  相似文献   

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