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Bacteria use protein-protein interactions to infect their hosts and hijack fundamental pathways, which ensures their survival and proliferation. Hence, the infectious capacity of the pathogen is closely related to its ability to interact with host proteins. Here, we show that hubs in the host-pathogen interactome are isolated in the pathogen network by adapting the geometry of the interacting interfaces. An imperfect mimicry of the eukaryotic interfaces allows pathogen proteins to actively bind to the host's target while preventing deleterious effects on the pathogen interactome. Understanding how bacteria recognize eukaryotic proteins may pave the way for the rational design of new antibiotic molecules., Competing Interests: The authors have declared that no competing interests exist.

Proteins and RNAs assemble in membrane-less organelles that organize intracellular spaces and regulate biochemical reactions. The ability of proteins and RNAs to form condensates is encoded in their sequences, yet it is unknown which domains drive the phase separation (PS) process and what are their specific roles. Here, we systematically investigated the human and yeast proteomes to find regions promoting condensation. Using advanced computational methods to predict the PS propensity of proteins, we designed a set of experiments to investigate the contributions of Prion-Like Domains (PrLDs) and RNA-binding domains (RBDs). We found that one PrLD is sufficient to drive PS, whereas multiple RBDs are needed to modulate the dynamics of the assemblies. In the case of stress granule protein Pub1 we show that the PrLD promotes sequestration of protein partners and the RBD confers liquid-like behaviour to the condensate. Our work sheds light on the fine interplay between RBDs and PrLD to regulate formation of membrane-less organelles, opening up the avenue for their manipulation., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Localized infections or disruption of the skin barrier can enable the entry of bacteria into the bloodstream, possibly leading to acute inflammation and sepsis. There is currently no holistic view on how bacteria can survive and spread in the bloodstream. In this context, we combined transposon mutagenesis, gene-expression profiling and a protein interaction network analysis to examine how uropathogenic Escherichia coli can proliferate in blood. Our results indicate that, upon migration from the urea to serum, E. coli reacts to the osmolarity difference, triggering a transcriptomic response in order to express survival genes. The proteins codified by these genes are precisely organized at the interactome level and specifically target short linear motifs located in disordered regions of host proteins. Such a coordinated response helps to explain how bacteria can adapt to and survive environmental changes within the host. Overall, our results provide a general framework for the study of bacteremia and reveal new targets for potential study as novel antimicrobials.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are incurable motor neuron diseases associated with muscle weakness, paralysis and respiratory failure. Accumulation of TAR DNA-binding protein 43 (TDP-43) as toxic cytoplasmic inclusions is one of the hallmarks of these pathologies. TDP-43 is an RNA-binding protein responsible for regulating RNA transcription, splicing, transport and translation. Aggregated TDP-43 does not retain its physiological function. Here, we exploit the ability of TDP-43 to bind specific RNA sequences to validate our hypothesis that the native partners of a protein can be used to interfere with its ability to self-assemble into aggregates. We propose that binding of TDP-43 to specific RNA can compete with protein aggregation. This study provides a solid proof of concept to the hypothesis that natural interactions can be exploited to increase protein solubility and could be adopted as a more general rational therapeutic strategy., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Decoding the information in mRNA during protein synthesis relies on tRNA adaptors, the abundance of which can affect the decoding rate and translation efficiency. To determine whether cells alter tRNA abundance to selectively regulate protein expression, we quantified changes in the abundance of individual tRNAs at different time points in response to diverse stress conditions in Saccharomyces cerevisiae We found that the tRNA pool was dynamic and rearranged in a manner that facilitated selective translation of stress-related transcripts. Through genomic analysis of multiple data sets, stochastic simulations, and experiments with designed sequences of proteins with identical amino acids but altered codon usage, we showed that changes in tRNA abundance affected protein expression independently of factors such as mRNA abundance. We suggest that cells alter their tRNA abundance to selectively affect the translation rates of specific transcripts to increase the amounts of required proteins under diverse stress conditions., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Prions are a singular subset of proteins able to switch between a soluble conformation and a self-perpetuating amyloid state. Traditionally associated with neurodegenerative diseases, increasing evidence indicates that organisms exploit prion-like mechanisms for beneficial purposes. The ability to transit between conformations is encoded in the so-called prion domains, long disordered regions usually enriched in glutamine/asparagine residues. Interestingly, Plasmodium falciparum , the parasite that causes the most virulent form of malaria, is exceptionally rich in proteins bearing long Q/N-rich sequence stretches, accounting for roughly 30% of the proteome. This biased composition suggests that these protein regions might correspond to prion-like domains (PrLDs) and potentially form amyloid assemblies. To investigate this possibility, we performed a stringent computational survey for Q/N-rich PrLDs on P. falciparum . Our data indicate that ∼10% of P. falciparum protein sequences have prionic signatures, and that this subproteome is enriched in regulatory proteins, such as transcription factors and RNA-binding proteins. Furthermore, we experimentally demonstrate for several of the identified PrLDs that, despite their disordered nature, they contain inner short sequences able to spontaneously self-assemble into amyloid-like structures. Although the ability of these sequences to nucleate the conformational conversion of the respective full-length proteins should still be demonstrated, our analysis suggests that, as previously described for other organisms, prion-like proteins might also play a functional role in P. falciparum .

Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

Protein misfolding and aggregation have been associated with the onset of neurodegenerative disorders. Recent studies demonstrate that the aggregation process can result in a high diversity of protein conformational states, however the identity of the specific species responsible for the cellular damage is still unclear. Here, we use yeast as a model to systematically analyse the intracellular effect of expressing 21 variants of the amyloid-ß-peptide, engineered to cover a continuous range of intrinsic aggregation propensities. We demonstrate the existence of a striking negative correlation between the aggregation propensity of a given variant and the oxidative stress it elicits. Interestingly, each variant generates a specific distribution of protein assemblies in the cell. This allowed us to identify the aggregated species that remain diffusely distributed in the cytosol and are unable to coalesce into large protein inclusions as those causing the highest levels of oxidative damage. Overall, our results indicate that the formation of large insoluble aggregates may act as a protective mechanism to avoid cellular oxidative stress., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

To perform their functions proteins must interact with each other, but how these interactions influence bacterial infection remains elusive. Here we demonstrate that connectivity in the host-pathogen interactome is directly related to pathogen fitness during infection. Using Y. pestis as a model organism, we show that the centrality-lethality rule holds for pathogen fitness during infection but only when the host-pathogen interactome is considered. Our results suggest that the importance of pathogen proteins during infection is directly related to their number of interactions with the host. We also show that pathogen proteins causing an extensive rewiring of the host interactome have a higher impact in pathogen fitness during infection. Hence, we conclude that hubs in the host-pathogen interactome should be explored as promising targets for antimicrobial drug design.

From transcription, to transport, storage, and translation, RNA depends on association with different RNA-binding proteins (RBPs). Methods based on next-generation sequencing and protein mass-spectrometry have started to unveil genome-wide interactions of RBPs but many aspects still remain out of sight. How many of the binding sites identified in high-throughput screenings are functional? A number of computational methods have been developed to analyze experimental data and to obtain insights into the specificity of protein-RNA interactions. How can theoretical models be exploited to identify RBPs? In addition to oligomeric complexes, protein and RNA molecules can associate into granular assemblies whose physical properties are still poorly understood. What protein features promote granule formation and what effects do these assemblies have on cell function? Here, we describe the newest in silico, in vitro, and in vivo advances in the field of protein-RNA interactions. We also present the challenges that experimental and computational approaches will have to face in future studies. WIREs RNA 2016, 7:793-810. doi: 10.1002/wrna.1378 For further resources related to this article, please visit the WIREs website., (© 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.)

Cell-to-cell variation in gene expression levels (noise) generates phenotypic diversity and is an important phenomenon in evolution, development and disease. TATA-box binding protein (TBP) is an essential factor that is required at virtually every eukaryotic promoter to initiate transcription. While the presence of a TATA-box motif in the promoter has been strongly linked with noise, the molecular mechanism driving this relationship is less well understood. Through an integrated analysis of multiple large-scale data sets, computer simulation and experimental validation in yeast, we provide molecular insights into how noise arises as an emergent property of variable binding affinity of TBP for different promoter sequences, competition between interaction partners to bind the same surface on TBP (to either promote or disrupt transcription initiation) and variable residence times of TBP complexes at a promoter. These determinants may be fine-tuned under different conditions and during evolution to modulate eukaryotic gene expression noise.

Systemic inflammation and infections are associated with neurodegenerative diseases. Unfortunately, the molecular bases of this link are still largely undiscovered. We, therefore, review how inflammatory processes can imbalance membrane homeostasis and theorize how this may have an effect on the aggregation behavior of the proteins implicated in such diseases. Specifically, we describe the processes that generate such imbalances at the molecular level, and try to understand how they affect protein folding and localization. Overall, current knowledge suggests that microglia pro-inflammatory mediators can generate membrane damage, which may have an impact in terms of triggering or accelerating disease manifestation.

Prion proteins were initially associated with diseases such as Creutzfeldt Jakob and transmissible spongiform encephalopathies. However, deeper research revealed them as versatile tools, exploited by the cells to execute fascinating functions, acting as epigenetic elements or building membrane free compartments in eukaryotes. One of the most intriguing properties of prion proteins is their ability to propagate a conformational assembly, even across species. In this context, it has been observed that bacterial amyloids can trigger the formation of protein aggregates by interacting with host proteins. As our life is closely linked to bacteria, either through a parasitic or symbiotic relationship, prion-like proteins produced by bacterial cells might play a role in this association. Bioinformatics is helping us to understand the factors that determine conformational conversion and infectivity in prion-like proteins. We have used PrionScan to detect prion domains in 839 different bacteria proteomes, detecting 2200 putative prions in these organisms. We studied this set of proteins in order to try to understand their functional role and structural properties. Our results suggest that these bacterial polypeptides are associated to peripheral rearrangement, macromolecular assembly, cell adaptability, and invasion. Overall, these data could reveal new threats and therapeutic targets associated to infectious diseases.

The formation of aggregates by misfolded proteins is thought to be inherently toxic, affecting cell fitness. This observation has led to the suggestion that selection against protein aggregation might be a major constraint on protein evolution. The precise fitness cost associated with protein aggregation has been traditionally difficult to evaluate. Moreover, it is not known if the detrimental effect of aggregates on cell physiology is generic or depends on the specific structural features of the protein deposit. In bacteria, the accumulation of intracellular protein aggregates reduces cell reproductive ability, promoting cellular aging. Here, we exploit the cell division defects promoted by the intracellular aggregation of Alzheimer's-disease-related amyloid β peptide in bacteria to demonstrate that the fitness cost associated with protein misfolding and aggregation is connected to the protein sequence, which controls both the in vivo aggregation rates and the conformational properties of the aggregates. We also show that the deleterious impact of protein aggregation on bacterial division can be buffered by molecular chaperones, likely broadening the sequential space on which natural selection can act. Overall, the results in the present work have potential implications for the evolution of proteins and provide a robust system to experimentally model and quantify the impact of protein aggregation on cell fitness., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders., Results: Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest., Conclusions: Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides.

Protein aggregation underlies the development of an increasing number of conformational human diseases of growing incidence, such as Alzheimer's and Parkinson's diseases. Furthermore, the accumulation of recombinant proteins as intracellular aggregates represents a critical obstacle for the biotechnological production of polypeptides. Also, ordered protein aggregates constitute novel and versatile nanobiomaterials. Consequently, there is an increasing interest in the development of methods able to forecast the aggregation properties of polypeptides in order to modulate their intrinsic solubility. In this context, we have developed AGGRESCAN, a simple and fast algorithm that predicts aggregation-prone segments in protein sequences, compares the aggregation properties of different proteins or protein sets and analyses the effect of mutations on protein aggregation propensities.

Aims: The failure of proteins to fold or to remain folded very often leads to their deposition into amyloid fibrils and is the origin of a variety of human diseases. Accordingly, mutations that destabilize the native conformation are associated with pathological phenotypes in several protein models. Protein backbone cyclization by disulfide bond crosslinking strongly reduces the entropy of the unfolded state and, usually, increases protein stability. The effect of protein cyclization on the thermodynamic and kinetics of folding has been extensively studied, but little is know on its effect on aggregation reactions., Results: The SRC homology 3 domain (SH3) of p85α subunit of bovine phosphatidyl-inositol-3'-kinase (PI3-SH3) domain is a small globular protein, whose folding and amyloid properties are well characterized. Here we describe the effect of polypeptide backbone cyclization on both processes., Innovation: We show that a cyclized PI3-SH3 variant is more stable, folds faster, aggregates slower, and forms conformationally and functionally different amyloid fibrils than the wild-type domain., Conclusion: Disulfide bridges may act as key molecular determinants of both productive protein folding and deleterious aggregation reactions.

Inclusion bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form inclusion bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of inclusion bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make inclusion bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Intrinsically disordered proteins (IDPs) are enriched in signaling and regulatory functions because disordered segments permit interaction with several proteins and hence the re-use of the same protein in multiple pathways. Understanding IDP regulation is important because altered expression of IDPs is associated with many diseases. Recent studies show that IDPs are tightly regulated and that dosage-sensitive genes encode proteins with disordered segments. The tight regulation of IDPs may contribute to signaling fidelity by ensuring that IDPs are available in appropriate amounts and not present longer than needed. The altered availability of IDPs may result in sequestration of proteins through non-functional interactions involving disordered segments (i.e., molecular titration), thereby causing an imbalance in signaling pathways. We discuss the regulation of IDPs, address implications for signaling, disease and drug development, and outline directions for future research., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Protein aggregation and amyloid formation lie behind an increasing number of human diseases. Here we describe the application of an "aggregation reporter", in which the test protein is fused to dihydrofolate reductase, as a general method to assess the intracellular solubility of amyloid proteins in eukaryotic background. Because the aggregation state of the target protein is linked directly to yeast cells survival in the presence of methotrexate, protein solubility can be monitored in vivo without the requirement of a functional assay for the protein of interest. In addition, the approach allows the in vivo visualization of the cellular location and aggregated state of the target protein. To demonstrate the applicability of the assay in the screening of genes or compounds that modulate amyloid protein aggregation in living cells, we have used as models the Alzheimer's amyloid β peptide, polyglutamine expansions of huntingtin, α-synuclein and non-aggregating variants thereof. Moreover, the anti-aggregational properties of small molecules and the effects of the yeast protein quality control machinery have also been evaluated using this method.

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The role of amyloid β (Aβ) peptide in the onset and progression of Alzheimer's disease is linked to the presence of soluble Aβ species. Sulfated glycosaminoglycans (GAGs) promote Aβ fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42-residue fragment Aβ(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable Aβ(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-Å separating Aβ(42) monomers within protofibrillar β-sheets. Incubation of Aβ(42) with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2- to 5-fold, whereas in the presence of the cationic polysaccharide chitosan, Aβ(42) fibrillar species were reduced by 25% and sensitivity to protease degradation increased ∼3-fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between Aβ(42) fibrils induced by PVS and Aβ(42) fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from -11.2 to -13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of -7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble Aβ(42) fibrils trapping soluble toxic forms.

Proteins might experience many conformational changes and interactions during their lifetimes, from their synthesis at ribosomes to their controlled degradation. Because, in most cases, only folded proteins are functional, protein folding in bacteria is tightly controlled genetically, transcriptionally, and at the protein sequence level. In addition, important cellular machinery assists the folding of polypeptides to avoid misfolding and ensure the attainment of functional structures. When these redundant protective strategies are overcome, misfolded polypeptides are recruited into insoluble inclusion bodies. The protein embedded in these intracellular deposits might display different conformations including functional and beta-sheet-rich structures. The latter assemblies are similar to the amyloid fibrils characteristic of several human neurodegenerative diseases. Interestingly, bacteria exploit the same structural principles for functional properties such as adhesion or cytotoxicity. Overall, this review illustrates how prokaryotic organisms might provide the bedrock on which to understand the complexity of protein folding and aggregation in the cell.

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

Protein misfolding and aggregation into amyloid structures are associated with dozens of human diseases. Recent studies have provided compelling evidence for the existence of highly ordered, amyloid-like conformations in the insoluble inclusion bodies produced during heterologous protein expression in bacteria. Thus, amyloid aggregation seems to be an omnipresent process in both eukaryotic and prokaryotic organisms. Amyloid formation inside cell factories raises important safety concerns with regard to the toxicity and infectivity of recombinant proteins. Yet such findings also suggest that prokaryotic cells could be useful systems for studying how and why proteins aggregate in vivo, and they could also provide a biologically relevant background for screening therapeutic approaches to pathologic protein deposition.

The field of protein misfolding and aggregation has become an extremely active area of research in recent years. Of particular interest is the deposition of polypeptides into inclusion bodies inside bacterial cells. One reason for this interest is that protein aggregation constitutes a major bottleneck in protein production and restricts the spectrum of protein-based drugs available for commercialization. Additionally, prokaryotic cells could provide a simple yet powerful system for studying the formation and prevention of toxic aggregates, such as those responsible for a number of degenerative diseases. Here, we review recent work that has challenged our understanding of the structure and physiology of inclusion bodies and provided us with a new view of intracellular protein deposition, which has important implications in microbiology, biomedicine and biotechnology.

Protein misfolding and deposition underlie an increasing number of debilitating human disorders and constitute a problem of major concern in biotechnology. In the last years, in vitro studies have provided valuable insights into the physicochemical principles underlying protein aggregation. Nevertheless, information about the determinants of protein deposition within the cell is scarce and only a few systematic studies comparing in vitro and in vivo data have been reported. Here, we have used the SH3 domain of alpha-spectrin as a model globular protein in an attempt to understand the relationship between protein aggregation in the test-tube and in the more complex cellular environment. The investigation of the aggregation in Escherichia coli of this domain and a large set of mutants, together with the analysis of their sequential and conformational properties allowed us to evaluate the contribution of different polypeptidic factors to the cellular deposition of globular proteins. The data presented here suggest that the rules that govern in vitro protein aggregation are also valid in in vivo contexts. They also provide relevant insights into intracellular protein deposition in both conformational diseases and recombinant protein production.

Protein aggregation correlates with the development of several deleterious human disorders such as Alzheimer's disease, Parkinson's disease, prion-associated transmissible spongiform encephalopathies and type II diabetes. The polypeptides involved in these disorders may be globular proteins with a defined 3D-structure or natively unfolded proteins in their soluble conformations. In either case, proteins associated with these pathogeneses all aggregate into amyloid fibrils sharing a common structure, in which beta-strands of polypeptide chains are perpendicular to the fibril axis. Because of the prominence of amyloid deposits in many of these diseases, much effort has gone into elucidating the structural basis of protein aggregation. A number of recent experimental and theoretical studies have significantly increased our understanding of the process. On the one hand, solid-state NMR, X-ray crystallography and single molecule methods have provided us with the first high-resolution 3D structures of amyloids, showing that they exhibit conformational plasticity and are able to adopt different stable tertiary folds. On the other hand, several computational approaches have identified regions prone to aggregation in disease-linked polypeptides, predicted the differential aggregation propensities of their genetic variants and simulated the early, crucial steps in protein self-assembly. This review summarizes these findings and their therapeutic relevance, as by uncovering specific structural or sequential targets they may provide us with a means to tackle the debilitating diseases linked to protein aggregation.

Peptidic self-assembled nanostructures are said to have a wide range of applications in nanotechnology, yet the mechanistic details of hierarchical self-assembly are still poorly understood. The Phe-Phe recognition motif of the Alzheimer's Abeta peptide is the smallest peptide able to assemble into higher-order structures. Here, we show that the Ile-Phe dipeptide analog is also able to self-associate in aqueous solution as a transparent, thermoreversible gel formed by a network of fibrillar nanostructures that exhibit strong birefringence upon Congo red binding. Besides, a second dipeptide Val-Phe, differing only in a methyl group from the former, is unable to self-assemble. The detailed analysis of the differential polymeric behavior of these closely related molecules provides insight into the forces triggering the first steps in self-assembly processes such as amyloid formation.

Background: Protein aggregation correlates with the development of several debilitating human disorders of growing incidence, such as Alzheimer's and Parkinson's diseases. On the biotechnological side, protein production is often hampered by the accumulation of recombinant proteins into aggregates. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. AGGRESCAN is a web-based software for the prediction of aggregation-prone segments in protein sequences, the analysis of the effect of mutations on protein aggregation propensities and the comparison of the aggregation properties of different proteins or protein sets., Results: AGGRESCAN is based on an aggregation-propensity scale for natural amino acids derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities. It also provides new insights into the differential aggregation properties displayed by globular proteins, natively unfolded polypeptides, amyloidogenic proteins and proteins found in bacterial inclusion bodies., Conclusion: By identifying aggregation-prone segments in proteins, AGGRESCAN http://bioinf.uab.es/aggrescan/ shall facilitate (i) the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases and (ii) the anticipation of aggregation phenomena during storage or recombinant production of bioactive polypeptides or polypeptide sets.

Increasing evidence indicates that protein aggregation in bacteria does not necessarily imply loss of biological activity. Here, we have investigated the effect of growth-temperature on both the activity and stability of the inclusion bodies formed by a point-mutant of Abeta42 Alzheimer peptide, using green fluorescent protein as a reporter. The activity in the aggregates inversely correlates with the temperature. In contrast, inclusion bodies become more stable in front of chemical denaturation and proteolysis when temperature increases. Overall, the data herein open new perspectives in protein production, while suggesting a kinetic competition between protein folding and aggregation during recombinant protein expression.

Recent data show that protein aggregation as bacterial inclusion bodies does not necessarily imply loss of biological activity. Here, we investigate the effect of a large set of single-point mutants of an aggregation-prone protein on its specific activity once deposited in inclusion bodies. The activity of such aggregates significantly correlates with the predicted aggregation rates for each mutant, suggesting that rationally tuning the kinetic competition between folding and aggregation might result in highly active, inclusion bodies. The exploration of this technology during recombinant protein production would have a significant biotechnological value.

Protein misfolding and deposition underlie an increasing number of debilitating human disorders. Alzheimer's disease is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain, composed primarily of the 42 amino acid human beta-amyloid peptide (Abeta42). Disease-linked mutations in Abeta42 occur in or near a central hydrophobic cluster comprising residues 17-21. We exploited the ability of green fluorescent protein to act as a reporter of the aggregation of upstream fused Abeta42 variants to characterize the effects of a large set of single-point mutations at the central position of this hydrophobic sequence as well as substitutions linked to early onset of the disease located in or close to this region. The aggregational properties of the different protein variants clearly correlated with changes in the intrinsic physicochemical properties of the side chains at the point of mutation. Reduction in hydrophobicity and beta-sheet propensity resulted in an increase of in vivo fluorescence indicating disruption of aggregation, as confirmed by the in vitro analysis of synthetic Abeta42 variants. The results confirm the key role played by the central hydrophobic stretch on Abeta42 deposition and support the hypothesis that sequence tunes the aggregation propensities of polypeptides.