Cells alter their tRNA abundance to selectively regulate protein synthesis during stress conditions.

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.

Discovering Putative Prion-Like Proteins in Plasmodium falciparum: A Computational and Experimental Analysis.

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.

Characterization of Amyloid Cores in Prion Domains.

Amyloids consist of repetitions of a specific polypeptide chain in a regular cross-ß-sheet conformation. Amyloid propensity is largely determined by the protein sequence, the aggregation process being nucleated by specific and short segments. Prions are special amyloids that become self-perpetuating after aggregation. Prions are responsible for neuropathology in mammals, but they can also be functional, as in yeast prions. The conversion of these last proteins to the prion state is driven by prion forming domains (PFDs), which are generally large, intrinsically disordered, enriched in glutamines/asparagines and depleted in hydrophobic residues. The self-assembly of PFDs has been thought to rely mostly on their particular amino acid composition, rather than on their sequence. Instead, we have recently proposed that specific amyloid-prone sequences within PFDs might be key to their prion behaviour. Here, we demonstrate experimentally the existence of these amyloid stretches inside the PFDs of the canonical Sup35, Swi1, Mot3 and Ure2 prions. These sequences self-assemble efficiently into highly ordered amyloid fibrils, that are functionally competent, being able to promote the PFD amyloid conversion in vitro and in vivo. Computational analyses indicate that these kind of amyloid stretches may act as typical nucleating signals in a number of different prion domains.

Computational analysis of candidate prion-like proteins in bacteria and their role.

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.

Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors.

BACKGROUND: The amyloid-ß peptide (Aß42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aß42 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 Aß42 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.

The effect of amyloidogenic peptides on bacterial aging correlates with their intrinsic aggregation propensity.

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 ß 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.

AGGRESCAN: method, application, and perspectives for drug design.

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.

Contribution of disulfide bonds to stability, folding, and amyloid fibril formation: the PI3-SH3 domain case.

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.

Biological role of bacterial inclusion bodies: a model for amyloid aggregation.

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.

Linking amyloid protein aggregation and yeast survival.

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 ß 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 role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils.

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 ß-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, ß-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.

Modulation of Abeta42 fibrillogenesis by glycosaminoglycan structure.

The role of amyloid ß (Aß) peptide in the onset and progression of Alzheimer's disease is linked to the presence of soluble Aß species. Sulfated glycosaminoglycans (GAGs) promote Aß 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 Aß(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable Aß(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-Å separating Aß(42) monomers within protofibrillar ß-sheets. Incubation of Aß(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, Aß(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 Aß(42) fibrils induced by PVS and Aß(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 Aß(42) fibrils trapping soluble toxic forms.

Protein folding and aggregation in bacteria.

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.

Protein aggregation profile of the bacterial cytosol.

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.

Amyloids in bacterial inclusion bodies.

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.

Studies on bacterial inclusion bodies.

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.

Recent structural and computational insights into conformational diseases.

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.

The in vivo and in vitro aggregation properties of globular proteins correlate with their conformational stability: the SH3 case.

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.

AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides.

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.