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Protein aggregation propensity is a crucial determinant of intracellular inclusion formation and quality control degradation

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Abstract

Protein aggregation is linked to many pathological conditions, including several neurodegenerative diseases. The aggregation propensities of proteins are thought to be controlled to a large extent by the physicochemical properties encoded in the primary sequence. We have previously exploited a set of amyloid β peptide (Aβ42) variants exhibiting a continuous gradient of intrinsic aggregation propensities to demonstrate that this rule applies in vivo in bacteria. In the present work we have characterized the behavior of these Aβ42 mutants when expressed in yeast. In contrast to bacteria, the intrinsic aggregation propensity is gated by yeast, in such a way that this property correlates with the formation of intracellular inclusions only above a specific aggregation threshold. Proteins displaying solubility levels above this threshold escape the inclusions formation pathway. In addition, the most aggregation-prone variants are selectively cleared by the yeast quality control degradation machinery. Thus, both inclusions formation and proteolysis target the same aggregation-prone variants and cooperate to minimize the presence of these potentially dangerous species in the cytosol. The demonstration that sorting to these pathways in eukaryotes is strongly influenced by protein primary sequence should facilitate the development of rational approaches to predict and hopefully prevent in vivo protein deposition.

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... The second component is a reporter (green fluorescent protein; GFP) that allows monitoring the integrity and the distribution/location of the protein in the cell (Fig 1B and D). The third component is a phase separation-promoting segment that leads to the formation of intracellular, insoluble protein deposits, which in our system is the 42-amino acid amyloid-b-peptide (Ab) Villar-Pique & Ventura, 2013;Sanchez de Groot et al, 2015;Fig 1D). ...
... Instead of using Ura3p sol as a control, we initially considered fusing Ura3p-GFP to a soluble variant of the Ab42 (Villar-Pique & Ventura, 2013; Sanchez de Groot et al, 2015; e.g., a non-fociforming variant). However, the different soluble variants are not always completely soluble in yeast, since after fractionation they are still found in the insoluble part and they form deposits in some of the stress environments investigated in the current work (Villar-Pique & Ventura, 2013). Hence, we decided that the addition of a soluble variant of Ab42 will not be a suitable control for generating the soluble version of Ura3p. ...
... The cellular behavior of Ab42 has been extensively studied in bacteria and yeast, both by others and by us Morell et al, 2011;Villar-Pique et al, 2012;Sanchez de Groot et al, 2015). In yeast, the formation of deposits/granules appears to protect cells against the toxic species (e.g., soluble oligomers; de Villar-Pique & Ventura, 2013;Sanchez de Groot et al, 2015). ...
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Phase separation of soluble proteins into insoluble deposits is associated with numerous diseases. However, protein deposits can also function as membrane-less compartments for many cellular processes. What are the fitness costs and benefits of forming such deposits in different conditions? Using a model protein that phase-separates into deposits, we distinguish and quantify the fitness contribution due to the loss or gain of protein function and deposit formation in yeast. The environmental condition and the cellular demand for the protein function emerge as key determinants of fitness. Protein deposit formation can influence cell-to-cell variation in free protein abundance between individuals of a cell population (i.e., gene expression noise). This results in variable manifestation of protein function and a continuous range of phenotypes in a cell population, favoring survival of some individuals in certain environments. Thus, protein deposit formation by phase separation might be a mechanism to sense protein concentration in cells and to generate phenotypic variability. The select-able phenotypic variability, previously described for prions, could be a general property of proteins that can form phase-separated assemblies and may influence cell fitness.
... We have previously exploited yeast to dissect the relationship between proteins aggregation in vivo and their physicochemical properties. For this purpose, we designed and expressed intracellularly 20 different variants of the amyloid-β-peptide (Aβ42) fused to GFP [39]. Later, we investigated the proteomic response caused by the expression of two individual proteins from this collection displaying different in vivo aggregation properties, one of them remaining diffusely distributed in the cytosol and the other one forming large PI in yeast cells. ...
... Yeast cells BY4741 (MAT a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) transformed as previously described [39,41] with pESC(-Ura) plasmid (Addgene) encoding for the Aβ42-GFP protein and 19 variants differing only in the residue in position 19th of the peptide, together with the variant F19D/L34P were grown overnight in glucose SC-URA medium at 30°C, and 100 µL was used to inoculate 5 mL of fresh medium. At an OD 590 of 0.5, cells were changed to a fresh raffinose SC-URA medium. ...
... This matches with the Q I GFP Dihydroethidium Overlay smaller and less fluorescent PI they exhibit, in comparison with the other aggregation-prone mutants (Fig. S4). In addition, we have previously shown that these observations are linked to the fact that Trp and Phe mutants are recognised by the protein quality machinery and degraded through autophagy [39], resulting in very low protein levels (Fig. 6A). All these observations suggest that the degradation of an aggregation-prone variant or sequestering it in PI may result in a similar prevention of oxidative stress. ...
Article
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Protein misfolding and aggregation has 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.
... In this context, yeast has arisen as a powerful model organism to understand not only the PQC machinery but also to address the pathological role of protein aggregation in human disease [9]. As a first attempt to unravel the yeast PQC response against protein misfolding, we recently expressed 20 GFP-fused peptides in yeast, derived from amyloid-b-peptide (Ab42), that cover a continuous range of aggregation propensities [10,11]. Interestingly, despite most of these peptides being highly insoluble, just some of them are recruited into foci. ...
... Interestingly, despite most of these peptides being highly insoluble, just some of them are recruited into foci. With this approach, we identified an aggregation propensity threshold above which the cell actively accumulates a protein into foci [11]. Here, we use two proteins from this collection, which are located on either side of the aggregation threshold, to decipher why protein foci are or are not formed in cells. ...
... The GFP-tagged peptides employed in this work are the Ab42 wild-type (Abwt) and the mutant Ab42 F19D (Abm), which includes a single substitution by a gatekeeper residue (aspartate) that disrupts a central hydrophobic stretch and reduces the aggregation propensity [10,11,15]. Actually, the presence of gatekeepers (charged residues and proline) flanking aggregation-prone regions is an evolutionary strategy to prevent anomalous protein self-assembly [16,17]. ...
Article
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Proteins adopt defined structures and are crucial to most cellular functions. Their misfolding and aggregation is associated with numerous degenerative human disorders such as type II diabetes, Huntington's or Alzheimer's diseases. Here, we aim to understand why cells promote the formation of protein foci. Comparison of two amyloid-β-peptide variants, mostly insoluble but differently recruited by the cell (inclusion body versus diffused), reveals small differences in cell fitness and proteome response. We suggest that the levels of oxidative stress act as a sensor to trigger protein recruitment into foci. Our data support a common cytoplasmic response being able to discern and react to the specific properties of polypeptides.
... Because all the Aβ-GFP fusions used were expressed from otherwise identical plasmids/promoters, reduced levels of Aβ42-GFP in cells likely stemmed from increased degradation of the insoluble aggregates of Aβ42-GFP in cells relative to Aβ40-GFP and AβEP-GFP. This hypothesis is consistent with other findings in yeast using Aβ fused to fluorescent reporters (Hamada et al., 2008;Morell et al., 2011;Villar-Pique and Ventura, 2013). The length of the linker sequence between an aggregation-prone domain and GFP influences the degree to which aggregation or misfolding inhibits the appearance of fluorescence. ...
... These findings are consistent with previous findings in which amyloid was also expressed cytosolically. In yeast, cytosolic expression of wild-type Aβ42-GFP, as well as of a comprehensive set of Aβ peptide variants (fused to GFP), was not associated with any observable cytotoxicity (Morell et al., 2011;Villar-Pique and Ventura, 2013). Caine et al. (2007) reported a minor reduction (5%) in growth of yeast cells (at ∼10 h) expressing cytosolically localized Aβ42-GFP. ...
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Amyloid-beta (Aβ) containing plaques are a major neuropathological feature of Alzheimer's disease (AD). The two major isoforms of Aβ peptide associated with AD are Aβ40 and Aβ42, of which the latter is highly prone to aggregation. Increased presence and aggregation of intracellular Aβ42 peptides is an early event in AD progression. Improved understanding of cellular processes affecting Aβ42 aggregation may have implications for development of therapeutic strategies. Aβ42 fused to green fluorescent protein (Aβ42GFP) was expressed in ∼4600 mutants of a Saccharomyces cerevisiae genome-wide deletion library to identify proteins and cellular processes affecting intracellular Aβ42 aggregation by assessing the fluorescence of Aβ42GFP. This screening identified 110 mutants exhibiting intense Aβ42GFP-associated fluorescence. Four major cellular processes were overrepresented in the data set, including phospholipid homeostasis. Disruption of phosphatidylcholine, phosphatidylserine and/or phosphatidylethanolamine metabolism had a major effect on intracellular Aβ42 aggregation and localisation. Confocal microscopy indicated that Aβ42GFP localisation in the phospholipid mutants was juxtaposed to the nucleus, most likely associated with the endoplasmic reticulum/ER membrane. These data provide a genome-wide indication of cellular processes that affect intracellular Aβ42GFP aggregation and may have important implications for understanding cellular mechanisms affecting intracellular Aβ42 aggregation and AD disease progression.
... Hence, avoiding the use of harsh chaotropic or utilizing mild denaturing conditions for solubilization was the primary goal with the hope to obtain bioactive fusion protein. Studies on the use of non-denaturing agents such as N-lauryl sarcosine, dimethyl sulfoxide (DMSO), 5% n-propanol, mild non-ionic detergents, high pH buffers, and low denaturant concentration that preserve the native-like state of the fusion proteins have been reported [52][53][54][55][56][57]. To solubilize IBs a combination of denaturants such as Sodium do-decyl sulphate (SDS), urea and organic solvents such as 40% (v/v) 1-Propanol and 20% (v/v) 2-Butanol in an acidic pH (range of 2-3) are used [58]. ...
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Expression of affinity-tagged recombinant proteins for crystallography, protein–protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli , thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli , resulting in yields of 10–50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 ( Cr FAP65AH1 and Cr FAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster ® , diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.
... In contrast to pathological amyloids 43,48,51,[68][69][70][71] and similar to functional amyloids, 20,72,73 GAPR-1-GFP expression is not toxic for the cells. These observations support the concept that in vivo amyloid-like oligomerisation of GAPR-1 may have functional relevance. ...
Article
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Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid−liquid phase separation. Here, we study the assembly of human Golgi- associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.
... The inspection of the sequence of the fragment 117-132 highlights the presence of several leucine/isoleucine residues that generally have major impacts on the formation of both α-helix and β-rich structures [56,57]. Of these, some (I122, L125, L129 and L132), separated by two or three residues in the sequence, were selected for mutagenesis analyses (Bruckmann C. et al., in preparation). ...
Article
The ability of many proteins to fold into well-defined structures has been traditionally considered a prerequisite for fulfilling their functions. Protein folding is also regarded as a valuable loophole to escape uncontrolled and harmful aggregations. Here we show that the PBX-regulating protein-1 (PREP1), an important homeodomain transcription factor involved in cell growth and differentiation during embryogenesis, is endowed with an uncommon thermostability. Indeed, circular dichroism analyses indicate that it retains most of its secondary structure at very high temperatures. These findings have important implications for PREP1 functions since it is a stabilizing factor of its partner PBX1. Predictive analyses suggest that the observed PREP1 thermostability could be related to the presence of aggregation-prone regions. Interestingly, synthetic peptides corresponding to these regions exhibit a remarkable propensity to form toxic β-rich amyloid-like aggregates in physiological conditions. On this basis, we suggest that PREP1 stability is an effective way to prevent or limit the formation of harmful aggregates. Notably, one of these PREP1 fragments (residues 117-132) is able to reversibly switch from α-helical to β-rich states depending on the environmental conditions. The chameleon conformational behavior of this peptide makes it an ideal system to study this intriguing and widespread structural transition.
... A comprehensive summary of all the tools and algorithms available for aggregation/solubility prediction is beyond the scope of this review. We therefore focus in the following only on TANGO (Fernandez-Escamilla et al., 2004), AGGRESCAN (Conchillo-Sole et al., 2007) and ZYGGREG-ATOR (Tartaglia and Vendruscolo, 2008), three of the more widely employed publically available web tools (Carija et al., 2016;Gallardo et al., 2016;Villar-Pique and Ventura, 2013). The TANGO tool employs a statistical mechanics algorithm, which identifies β-aggregating regions in a given amino-acid sequence, considering four conformational states (β-turn, α-helix, the folded state, and β-aggregates), while taking into account hydrophobicity, solvation energetics, electrostatic interactions, and hydrogen-bonding (Fernandez-Escamilla et al., 2004). ...
Article
Bacterial inclusion bodies (IBs) consist of unfolded protein aggregates and represent inactive waste products often accumulating during heterologous overexpression of recombinant genes in Escherichia coli. This general misconception has been challenged in recent years by the discovery that IBs, apart from misfolded polypeptides, can also contain substantial amounts of active and thus correctly or native-like folded protein. The corresponding catalytically-active inclusion bodies (CatIBs) can be regarded as a biologically‐active sub-micrometer sized biomaterial or naturally-produced carrier-free protein immobilizate. Fusion of polypeptide (protein) tags can induce CatIB formation paving the way towards the wider application of CatIBs in synthetic chemistry, biocatalysis and biomedicine. In the present review we summarize the history of CatIBs, present the molecular-biological tools that are available to induce CatIB formation, and highlight potential lines of application. In the second part findings regarding the formation, architecture and structure of (Cat)IBs are summarized. Finally, an overview is presented over the available bioinformatic tools that potentially allow for the prediction of aggregation and thus (Cat)IB formation. This review aims at demonstrating the potential of CatIBs for biotechnology and hopefully contributes to a wider acceptance of this promising, yet not widely utilized, protein preparation.
... led these mutations as the highly deleterious mutations. (2) Aggregation propensity: The Aggrescan3D (A3D) tool, used to predict the propensity of tdp43 aggregation based on the solubility, revealed the aggregation prone conformational state of both D169G and K263E mutants than wild type due to the increased hydrophobicity and decreased solubility (Villar-Piqu? & Ventura, 2013). (3) Distortion in the secondary structure: Generally, the exposure of non-polar residues to an aqueous environment describes the gain in hydrophobic nature which is associated either to the par- tial/complete unfolding or the aggregation of a protein. Such structural relaxation is mainly mediated by the distortions at the secondary str ...
Article
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One of the multitasking proteins, transactive response DNA-binding protein 43 (tdp43) plays a key role in RNA regulation and the two pathogenic mutations such as D169G and K263E, located at the RNA Recognition Motif (RRM) of tdp43, are reported to cause neurological disorders such as Amyotrophic Lateral Sclerosis (ALS) and Fronto Temporal Lobar Degeneration (FTLD). As the exploration of the proteinopathy demands both structural and functional characterization of mutants, a comparative analysis on the wild type and mutant tdp43 (D169G and K263E) and their complexes with RNA have been performed using computational approaches. Molecular dynamics simulations revealed comparatively stable mutant structures compared to wild type tdp43. Both mutants show lesser binding affinity towards RNA molecule when compared to the wild type tdp43. Some of the observed features, including the increased solvent accessible surface area, conformational flexibility as well as unfolding of tdp43 and the altered RNA conformation in tp43-RNA complex, reveal the susceptibility of these mutants to induce conformational changes in tdp43 for a possible aggregation in the cytoplasm. Particularly, the enhanced aggregation propensity of both mutants also evidences the higher probability of cytoplasmic aggregation of tdp43 mutants. Hence, the present analysis highlighting the structural and functional aspects of wild and mutant tdp43 will form the basis to gain insight into the proteinopathy of tdp43 and the related structure based drug discovery. Thus, tdp43 can be used as target to develop novel therapeutic approaches or drug designing.
... The inherent aggregation propensity of amyloid proteins often results in their aggregation into insoluble IBs when they are produced in bacteria [29]. In several cases, these intracellular aggregates have been shown to display amyloid-like properties. ...
Article
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BACKGROUND: An increasing number of proteins are being shown to assemble into amyloid structures that lead to pathological states. Among them, mammalian prions outstand due to their ability to transmit the pathogenic conformation, becoming thus infectious. The structural conversion of the cellular prion protein (PrP(C)), into its misfolded pathogenic form (PrP(Sc)) is the central event of prion-driven pathologies. The study of the structural properties of intracellular amyloid aggregates in general and of prion-like ones in particular is a challenging task. In this context, the evidence that the inclusion bodies formed by amyloid proteins in bacteria display amyloid-like structural and functional properties make them a privileged system to model intracellular amyloid aggregation. RESULTS: Here we provide the first demonstration that recombinant murine PrP and its C-terminal domain (90-231) attain amyloid conformations inside bacteria. Moreover, the inclusions formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity. CONCLUSIONS: Overall, our results suggest that modelling PrP amyloid formation in microbial cell factories might open an avenue for a better understanding of the structural features modulating the pathogenic impact of this intriguing protein.
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The helix, s Applequist, 1963) in which the Zimm-Bragg parameters u and s are defined respectively as the cooperativity factor for helix initiation, and the equi- librium constant for converting a coil residue to a helical ~~~~
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Several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), or prion diseases, are known for their intimate association with protein misfolding and aggregation. These disorders are characterized by the loss of specific neuronal populations in the brain and are highly associated with aging, suggesting a decline in proteostasis capacity may contribute to pathogenesis. Nevertheless, the precise molecular mechanisms that lead to the selective demise of neurons remain poorly understood. As a consequence, appropriate therapeutic approaches and effective treatments are largely lacking. The development of cellular and animal models that faithfully reproduce central aspects of neurodegeneration have been crucial for advancing our understanding of these diseases. Approaches involving the sequential use of different model systems, starting with simpler cellular models and ending with validation in more complex animal models, resulted in the discovery of promising therapeutic targets and small molecules with therapeutic potential. Within this framework, the simple and well-characterized eukaryote Saccharomyces cerevisiae, also known as budding yeast, is being increasingly used to study the molecular basis of several neurodegenerative disorders. Yeast provides an unprecedented toolbox for the dissection of complex biological processes and pathways. Here, we summarize how yeast models are adding to our current understanding of several neurodegenerative disorders. This article is protected by copyright. All rights reserved.
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Conformational protein diseases of the human central nervous system represent a subject that has crucial theoretical and medical implications. They include several important neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's and Creutzfeldt-Jacob's diseases, amyotrophic lateral sclerosis and the tauopathies. They occur when soluble proteins undergo conformational re-arrangements becoming capable of aggregate into β-sheets conformations leading to the production of insoluble complexes known as amyloid deposits, that accumulate and lead to neurons and glial cells death. Theoretical and experimental evidence indicates that a key role in the conformational changes leading to amyloid formation is played by short sequence stretches within a given protein. Thus, the identification of protein regions potentially involved in aggregate formation and the characterization of their properties are relevant questions in the study of conformational proteins diseases. To address these questions, bioinformatics methods might provide an important contribution, suggesting possible mechanisms of protein aggregation, and focusing and orienting the experimental work. Thus, in the first part of present review bioinformatics methods specifically attempting to predict aggregation-prone regions in proteins will be briefly described. Furthermore, the results provided by the combined use of some of them to analyze a set of particularly important proteins involved in human degenerative diseases will be discussed.
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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.
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In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of β-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation.
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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.
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The budding yeast, Saccharomyces cerevisiae, is the best-studied eukaryotic cell, at both genetic and physiological levels. As a eukaryote, yeast shares highly conserved molecular and cellular mechanisms with human cells. Thus, this simple fungus is an invaluable model to study the fundamental molecular mechanisms involved in several human diseases. In the particular case of neurodegenerative disorders, yeast models have been able to recapitulate several important features of complex and devastating disorders, such as Huntington's and Parkinson's diseases. Once validated, these models have also been used to accelerate the identification of both novel therapeutic targets and compounds with therapeutic potential. Here, we review the recent contributions of this simple, but powerful model organism toward our understanding of neurodegeneration.
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In ageing populations, neurodegenerative diseases increase in prevalence, exacting an enormous toll on individuals and their communities. Multiple complementary experimental approaches are needed to elucidate the mechanisms underlying these complex diseases and to develop novel therapeutics. Here, we describe why the budding yeast Saccharomyces cerevisiae has a unique role in the neurodegeneration armamentarium. As the best-understood and most readily analysed eukaryotic organism, S. cerevisiae is delivering mechanistic insights into cell-autonomous mechanisms of neurodegeneration at an interactome-wide scale.
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Amyloidogenic regions in polypeptide chains are very important because such regions are responsible for amyloid formation and aggregation. It is useful to be able to predict positions of amyloidogenic regions in protein chains. Two characteristics (expected probability of hydrogen bonds formation and expected packing density of residues) have been introduced by us to detect amyloidogenic regions in a protein sequence. We demonstrate that regions with high expected probability of the formation of backbone-backbone hydrogen bonds as well as regions with high expected packing density are mostly responsible for the formation of amyloid fibrils. Our method (FoldAmyloid) has been tested on a dataset of 407 peptides (144 amyloidogenic and 263 non-amyloidogenic peptides) and has shown good performance in predicting a peptide status: amyloidogenic or non-amyloidogenic. The prediction based on the expected packing density classified correctly 75% of amyloidogenic peptides and 74% of non-amyloidogenic ones. Two variants (averaging by donors and by acceptors) of prediction based on the probability of formation of backbone-backbone hydrogen bonds gave a comparable efficiency. With a hybrid-scale constructed by merging the above three scales, our method is correct for 80% of amyloidogenic peptides and for 72% of non-amyloidogenic ones. Prediction of amyloidogenic regions in proteins where positions of amyloidogenic regions are known from experimental data has also been done. In the proteins, our method correctly finds 10 out of 11 amyloidogenic regions. The FoldAmyloid server is available at http://antares.protres.ru/fold-amyloid/.
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Protein aggregation is related to many human disorders and constitutes a major bottleneck in protein production. However, little is known about the conformational properties of in vivo formed aggregates and how they relate to the specific polypeptides embedded in them. Here, we show that the kinetic and thermodynamic stability of the inclusion bodies formed by the Abeta42 Alzheimer peptide and its Asp19 alloform differ significantly and correlate with their amyloidogenic propensity and solubility inside the cell. Our results indicate that the nature of the polypeptide chain determines the specific conformational properties of intracellular aggregates. This implies that different protein inclusions impose dissimilar challenges to the cellular quality-control machinery.
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This chapter describes the conditions and procedures that affect the levels and activities of the vacuolar proteases and present both genetic and biochemical assaying methods for coping with protease problems. Protease B activity in colonies can be assayed using an overlay test. It is based on the principle that states that protease B, which is freed from cells by lysis and from its inhibitor by the SDS present in the overlay, solubilizes the particles of Hide Powder Azure in the overlay, uncovering the colony and surrounding it with a clear halo. Mutant colonies remain covered. Carboxypeptidase Y activity in colonies can be assessed by using an overlay test that relies on the esterolytic activity of the enzyme. The substrate is N-acetyl-DL-phenylalanine β-naphthyl ester, cleavage of which, in colonies anyway, is catalyzed only by CpY. In biochemical analyses, commonly used assay for protease A activity measures the release of tyrosine-containing acid-soluble peptides from aciddenatured hemoglobin. Protease A is apparently the only protease to catalyze the reaction at acid pH. It is reported the use of a protease-deficient strain can ease protease problems. Strains bearing the pleiotropic pep4-3 mutation have greatly reduced, but not zero, levels of PrA, PrB CpY, and ApI. Strains such as EJ101 that carry prb1-1122 have also been used successfully in some purifications. Use of a double mutant is recommended that carries mutations both in the PEP4 gene and the PRB1 gene.
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pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.
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The helix, β-sheet, and coil conformational parameters, Pα, Pβ, and Pc, for the 20 naturally occurring amino acids have been computed from the frequency of occurrence of each amino acid residue in the α, β, and coil conformations in 15 proteins, whose structure has been determined by X-ray crystallography. These values have been utilized to provide a simple procedure, devoid of complex computer calculations, to predict the secondary structure of proteins from their known amino acid sequences. The computed Pα values are within 10% of the experimental Zimm-Bragg helix growth parameters, s, evaluated from poly(α-amino acids). The environmental effects on the s values of polypepljdes and proteins are discussed, showing that Pα values may be more reliable in predicting protein conformation. A detailed analysis of the helix and β-sheet boundary residues in proteins provide amino acid frequencies at the N- and C-terminal ends which are used to delineate helical and β regions. Charged residues are found with the greatest frequency at both helical ends, but are mostly absent in β-sheet regions. The frequencies at the helical ends may also be correlated to the experimental Zimm-Bragg helix initiation parameter, σ, evaluated from poly(α-ammo acids). A mechanism of protein folding is proposed, whereby helix nucleation starts at the centers of the helix (where the Pα values are highest) and propagates in both directions, until strong helix breakers (where Pα values are lowest) terminate the growth at both ends. Similarly, residues with the highest Pβ values will initiate β regions and residues with the lowest Pβ values will terminate β regions. The helical region with the highest α potential (i.e., largest 〈Pα〉) is proposed as the site of the first fold during protein renaturation. The mechanism of folding of myoglobin is discussed. Thus, the protein conformational parameters and the conformational boundary frequencies determined for the first time in their hierarchical order in this paper will enable accurate prediction of protein secondary structure as well as providing insights into tertiary folding.
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Octanol-to-water solvation free energies of acetyl amino amides (Ac-X-amides) [Fauchère, J.L., & Pliska, V. (1983) Eur. J. Med. Chem. --Chim. Ther. 18,369] form the basis for computational comparisons of protein stabilities by means of the atomic solvation parameter formalism of Eisenberg and McLachlan [(1986) Nature 319, 199]. In order to explore this approach for more complex systems, we have determined by octanol-to-water partitioning the solvation energies of (1) the guest (X) side chains in the host-guest pentapeptides AcWL-X-LL, (2) the carboxy terminus of the pentapeptides, and (3) the peptide bonds of the homologous series of peptides AcWLm (m = 1-6). Solvation parameters were derived from the solvation energies using estimates of the solvent-accessible surface areas (ASA) obtained from hard-sphere Monte Carlo simulations. The measurements lead to a side chain solvation-energy scale for the pentapeptides and suggest the need for modifying the Asp, Glu, and Cys values of the "Fauchère-Pliska" solvation-energy scale fro the Ac-X-amides. We find that the unfavorable solvation energy of nonpolar residues can be calculated accurately by a solvation parameter of 22.8 +/- 0.8 cal/mol/A2, which agrees satisfactorily with the AC-X-amide data and thereby validates the Monte Carlo ASA results. Unlike the Ac-X-amide data, the apparent solvation energies of the uncharged polar residues are also largely unfavorable. This unexpected finding probably results, primarily, from differences in conformation and hydrogen bonding in octanol and buffer but may also be due to the additional flaking peptide bonds of the pentapeptides. The atomic solvation parameter (ASP) for the peptide bond is comparable to the ASP of the charged carboxy terminus which is an order of magnitude larger than the ASP of the uncharged polar side chains of the Ac-X-amides. The very large peptide bond ASP, -96 +/- 6 cal/mol/A2, profoundly affects the results of computational comparisons of protein stability which use ASPs derived from octanol-water partitioning data.
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The partitioning of membrane-active oligopeptides into membrane interfaces promotes the formation of secondary structure. A quantitative description of the coupling of structure formation to partitioning, which may provide a basis for understanding membrane protein folding and insertion, requires an appropriate free energy scale for partitioning. A complete interfacial hydrophobicity scale that includes the contribution of the peptide bond was therefore determined from the partitioning of two series of small model peptides into the interfaces of neutral (zwitterionic) phospholipid membranes. Aromatic residues are found to be especially favoured at the interface while charged residues, and the peptide bond, are disfavoured about equally. Reduction of the high cost of partitioning the peptide bond through hydrogen bonding may be important in the promotion of structure formation in the membrane interface.
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Proteasomes are major sites for protein degradation in eukaryotic cells. The recent identification of selective proteasome inhibitors has allowed a definition of the roles of the ubiquitin-proteasome pathway in various cellular processes, such as antigen presentation and the degradation of regulatory or membrane proteins. This review describes the actions of these inhibitors, how they can be used to investigate cellular responses, the functions of the proteasome demonstrated by such studies and their potential applications in the future.
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Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein. A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons. Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear. To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein. The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation. Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation. Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation. However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments. More dramatically, the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.
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Intracellular and extracellular accumulation of aggregated protein are linked to many diseases, including ageing-related neurodegeneration and systemic amyloidosis. Cells avoid accumulating potentially toxic aggregates by mechanisms including the suppression of aggregate formation by molecular chaperones and the degradation of misfolded proteins by proteasomes. Once formed, aggregates tend to be refractory to proteolysis and to accumulate in inclusion bodies. This accumulation has been assumed to be a diffusion-limited process, but recent studies suggest that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules. This microtubule-dependent inclusion body is called an aggresome.
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The ability of proteins to fold to their functional states following synthesis in the intracellular environment is one of the most remarkable features of biology. Substantial progress has recently been made towards understanding the fundamental nature of the mechanism of the folding process. This understanding has been achieved through the development and concerted application of a variety of novel experimental and theoretical approaches to this complex problem. The emerging view of folding is that it is a stochastic process, but one biased by the fact that native-like interactions between residues are on average more stable than non-native ones. The sequences of natural proteins have emerged through evolutionary processes such that their unique native states can be found very efficiently even in the complex environment inside a living cell. But under some conditions proteins fail to fold correctly, or to remain correctly folded, in living systems, and this failure can result in a wide range of diseases. One group of diseases, known as amyloidoses, which includes Alzheimer's and the transmissible spongiform encephalopathies, involves deposition of aggregated proteins in a variety of tissues. These diseases are particularly intriguing because evidence is accumulating that the formation of the highly organized amyloid aggregates is a generic property of polypeptides, and not simply a feature of the few proteins associated with recognized pathological conditions. That such aggregates are not normally found in properly functional biological systems is again a testament to evolution, in this case of a variety of mechanisms inhibiting their formation. Understanding the nature of such protective mechanisms is a crucial step in the development of strategies to prevent and treat these debilitating diseases.
Article
The ability of proteins to fold to their functional states following synthesis in the intracellular environment is one of the most remarkable features of biology. Substantial progress has recently been made towards understanding the fundamental nature of the mechanism of the folding process. This understanding has been achieved through the development and concerted application of a variety of novel experimental and theoretical approaches to this complex problem. The emerging view of folding is that it is a stochastic process, but one biased by the fact that native-like interactions between residues are, on average, more stable than non-native ones. The sequences of natural proteins have emerged through evolutionary processes such that their unique native states can be found very efficiently even in the complex environment inside a living cell. But under some conditions proteins fail to fold correctly, or to remain correctly folded, in living systems, and this failure can result in a wide range of diseases. One group of diseases, known as amyloidoses, which includes Alzheimer's disease and the transmissible spongiform encephalopathies, involves deposition of aggregated proteins in a variety of tissues. These diseases are particularly intriguing because evidence is accumulating that the formation of the highly organized amyloid aggregates is a generic property of polypeptides, and not simply a feature of the few proteins associated with recognized pathological conditions. That such aggregates are not normally found in properly functional biological systems is again a testament to evolution, in this case of a variety of mechanisms inhibiting their formation. Understanding the nature of such protective mechanisms is a crucial step in the development of strategies to prevent and treat these debilitating diseases.
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Diagnosis and circumvention of multi-drug resistance requires an understanding of the underlying cellular mechanisms. In the model organism Saccharomyces cerevisiae, deletions of PDR5 or ERG6 increase sensitivity to many small lipophilic drugs. Pdr5p is a plasma membrane ATP-binding cassette transporter that actively exports drugs, thereby lowering their intracellular levels. The mechanism by which ERG6, an enzyme in sterol biosynthesis, affects drug accumulation is less clear. We show here that ERG6 limits the rate of passive drug diffusion across the membrane, without affecting Pdr5p-mediated drug export. Consistent with their action by distinct mechanisms, PDR5 and ERG6 effects on drug accumulation are additive.
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This chapter discusses the vacuolar proteases and proteolytic artifacts in Saccharomyces cerevisiae (S.cerevisiae). The yeast S.cerevisiae contains a large number of proteases that are located in various compartments and membranes of the cell. These include endoproteinases, carboxypeptidases, aminopeptidases, and dipeptidylaminopeptidases. Some of the proteases pose significant impediments to the analysis of biochemical processes and/or purification of proteins and can generate artifacts concerning the activity, structure, and intracellular location of proteins. Of the many cellular proteases, lumenal vacuolar proteases probably comprise the major source of problems. Found soluble within the vacuole are endoproteinases A and B, carboxypeptidases Y and S, aminopeptidase I, and aminopeptidase ApY. An additional source of protease problems is Zymolyase because commercial preparations of Zymolyase contain substantial amounts of a protease that may catalyze hydrolysis of azocoll. The chapter describes conditions and procedures that affect the levels and activities of the vacuolar proteases and presents both genetic and biochemical methods for coping with protease problems.