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Protein aggregation propensity is a crucial determinant of intracellular inclusion formation and quality control degradation
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.
... 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). ...
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. ...
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]. ...
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.
... Amyloid β (Aβ) peptides are generated by proteolytic processing of a transmembrane amyloid precursor protein, and Aβ42 is a major component of extracellular amyloid plaques in the brains of patients with Alzheimer's disease [27]. Expression of Aβ42-GFP from a galactose-inducible promoter in yeast cells results in the formation of intracellular inclusions [28,29]. Thus, budding yeast has been used as a model to study the aggregation and toxicity of Aβ42 [30,31]. ...
... In addition to mutated Htt proteins, other neurodegenerative disease-associated proteins, such as Aβ42 and α-synuclein, also form IBs in yeast cells [28,30,32,72]. We first confirmed IB formation in yeast cells expressing these proteins tagged with GFP from a galactose-inducible promoter. ...
Protein misfolding, aggregation, and accumulation cause neurodegenerative disorders. One such disorder, Huntington’s disease, is caused by an increased number of glutamine-encoding trinucleotide repeats CAG in the first exon of the huntingtin (HTT) gene. Mutant proteins of Htt exon 1 with polyglutamine expansion are prone to aggregation and form pathological inclusion bodies in neurons. Extensive studies have shown that misfolded proteins are cleared by the ubiquitin-proteasome system or autophagy to alleviate their cytotoxicity. Misfolded proteins can form small soluble aggregates or large insoluble inclusion bodies. Previous works have elucidated the role of autophagy in the clearance of misfolded protein aggregates, but autophagic clearance of inclusion bodies remains poorly characterised. Here we use mutant Htt exon 1 with 103 polyglutamine (Htt103QP) as a model substrate to study the autophagic clearance of inclusion bodies in budding yeast. We found that the core autophagy-related proteins were required for Htt103QP inclusion body autophagy. Moreover, our evidence indicates that the autophagy of Htt103QP inclusion bodies is selective. Interestingly, Cue5/Tollip, a known autophagy receptor for aggrephagy, is dispensable for this inclusion body autophagy. From the known selective autophagy receptors in budding yeast, we identified three that are essential for inclusion body autophagy. Amyloid beta peptide (Aβ42) is a major component of amyloid plaques found in Alzheimer’s disease brains. Interestingly, a similar selective autophagy pathway contributes to the clearance of Aβ42 inclusion bodies in budding yeast. Therefore, our results reveal a novel autophagic pathway specific for inclusion bodies associated with neurodegenerative diseases, which we have termed IBophagy.
... 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. ...
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]. ...
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. ...
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). ...
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). ...
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 ...
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. ...
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.
Scope:
Amyloid-β oligomers (AβO) are causally related to Alzheimer's disease (AD). Dietary natural compounds, especially flavonoids and flavan-3-ols, hold great promise as potential AD-preventive agents but their host and gut microbiota metabolism complicates identification of the most relevant bioactive species. This study aims to investigate the ability of a comprehensive set of phenyl-γ-valerolactones (PVL), the main circulating metabolites of flavan-3-ols and related dietary compounds in humans, to prevent AβO-mediated toxicity.
Methods and results:
The anti-AβO activity of PVLs was examined in different cell model systems using a highly toxic β-oligomer-forming polypeptide (β23) as target toxicant. Multiple PVLs, and particularly the monohydroxylated 5-(4'-hydroxyphenyl)-γ-valerolactone metabolite [(4'-OH)-PVL], relieved β-oligomer-induced cytotoxicity in yeast and mammalian cells. As revealed by atomic force microscopy (AFM) and other in vitro assays, (4'-OH)-PVL interferes with AβO (but not fibril) assembly and actively remodels preformed AβOs into non-toxic amorphous aggregates. In keeping with the latter mode of action, treatment of AβOs with (4'-OH)-PVL prior to brain injection strongly reduced memory deterioration as well as neuroinflammation in a mouse model of AβO-induced memory impairment.
Conclusion:
PVLs, which have been validated as biomarkers of the dietary intake of flavan-3-ols, lend themselves as novel AβO-selective, candidate AD-preventing compounds. This article is protected by copyright. All rights reserved.
Significance
Protein AMPylation in eukaryotes is a comparatively understudied posttranslational modification. With the exception of yeast, all eukaryotes have the enzymatic machinery required to execute this modification. Members of the heat shock protein family in different cellular compartments appear to be preferred targets for AMPylation, but it has proven challenging to adduce its biological function. We show that genetic modifications that affect AMPylation status, through generation of null alleles and a constitutively active version of the AMPylase FIC-1, can have a major impact on the susceptibility of Caenorhabditis elegans to neurodegenerative conditions linked to protein aggregation.
Many complex traits and diseases (e.g., neurodegenerative diseases, cancers) are multifactorial in nature or involve a combination of genomic, epigenomic, interactomics, networks dynamics, and environmental factors. Progress is being made, and complex diseases are beginning to be understood as more likely due to ‘altered states’ of networks, with intrinsic dynamics and interplay, whose properties require comprehensive integrative systems biology approaches. The yeast Saccharomyces cerevisiae, with its main molecular mechanisms, ‘modular’ networks, and subcellular organization essentially similar to those in all eukaryotes, makes an optimal model for systems biology studies. Yeast systems biology experiments also offer the potential to decipher the complexity and interplay of eukaryotic networks and short- and long-term effects of perturbations and dysregulations at the fundamental cellular level, which may illuminate the origin and sequence of events underlying complex diseases. In this chapter, emphasis is focused on the need for careful experimental designs reproducing in vivo perturbations and comprehensive time-course experiments monitored at multiple ‘omics levels, with effective bioinformatics analyses and data integration. The rationale of systems biology studies in yeast models of disease can be reproduced in more complex model systems (e.g., mammalian cells, animal models, and ultimately, human). The greatest progress will come from the integration of knowledge from studies in several disease models, from yeast to human.
Alzheimer's disease (AD), and many neurodegenerative disorders, are multifactorial in nature. They involve a combination of genomic, epigenomic, interactomic and environmental factors. Progress is being made, and these complex diseases are beginning to be understood as having their origin in altered states of biological networks at the cellular level. In the case of AD, genomic susceptibility and mechanisms leading to (or accompanying) the impairment of the central Amyloid Precursor Protein (APP) processing and tau networks are widely accepted as major contributors to the diseased state. The derangement of these networks may result in both the gain and loss of functions, increased generation of toxic species (e.g., toxic soluble oligomers and aggregates) and imbalances, whose effects can propagate to supra-cellular levels. Although well sustained by empirical data and widely accepted, this global perspective often overlooks the essential roles played by the main counteracting homeostatic networks (e.g., protein quality control/proteostasis, unfolded protein response, protein folding chaperone networks, disaggregases, ER-associated degradation/ubiquitin proteasome system, endolysosomal network, autophagy, and other stress-protective and clearance networks), whose relevance to AD is just beginning to be fully realized. In this chapter, an integrative perspective is presented. Alzheimer's disease is characterized to be a result of: (a) intrinsic genomic/epigenomic susceptibility and, (b) a continued dynamic interplay between the deranged networks and the central homeostatic networks of nerve cells. This interplay of networks will underlie both the onset and rate of progression of the disease in each individual. Integrative Systems Biology approaches are required to effect its elucidation. Comprehensive Systems Biology experiments at different 'omics levels in simple model organisms, engineered to recapitulate the basic features of AD may illuminate the onset and sequence of events underlying AD. Indeed, studies of models of AD in simple organisms, differentiated cells in culture and rodents are beginning to offer hope that the onset and progression of AD, if detected at an early stage, may be stopped, delayed, or even reversed, by activating or modulating networks involved in proteostasis and the clearance of toxic species. In practice, the incorporation of next-generation neuroimaging, high-throughput and computational approaches are opening the way towards early diagnosis well before irreversible cell death. Thus, the presence or co-occurrence of: (a) accumulation of toxic Aβ oligomers and tau species; (b) altered splicing and transcriptome patterns; (c) impaired redox, proteostatic, and metabolic networks together with, (d) compromised homeostatic capacities may constitute relevant 'AD hallmarks at the cellular level' towards reliable and early diagnosis. From here, preventive lifestyle changes and tailored therapies may be investigated, such as combined strategies aimed at both lowering the production of toxic species and potentiating homeostatic responses, in order to prevent or delay the onset, and arrest, alleviate, or even reverse the progression of the disease.
Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between side-chains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions. Citation: Fitzpatrick AW, Knowles TPJ, Waudby CA, Vendruscolo M, Dobson CM (2011) Inversion of the Balance between Hydrophobic and Hydrogen Bonding Interactions in Protein Folding and Aggregation. PLoS Comput Biol 7(10): e1002169. Copyright: ß 2011 Fitzpatrick et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from BBSRC (AWF, MV, CMD), the Royal Society (MV) and the Wellcome Trust (CMD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of "soft" intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.
Numerous studies have shown that the ability to form amyloid fibrils is an inherent property of the polypeptide chain. This has lead to the development of several computational approaches to predict amyloidogenicity by amino acid sequences. Here, we discuss the principles governing these methods, and evaluate them using several datasets. They deliver excellent performance in the tests made using short peptides (∼6 residues). However, there is a general tendency towards a high number of false positives when tested against longer sequences. This shortcoming needs to be addressed as these longer sequences are linked to diseases. Recent structural studies have shown that the core element of the majority of disease-related amyloid fibrils is a β-strand-loop-β-strand motif called β-arch. This insight provides an opportunity to substantially improve the prediction of amyloids produced by natural proteins, ushering in an era of personalized medicine based on genome analysis.
The 26S proteasome, in collaboration with ubiquitin, operates the energy-dependent regulated proteolysis process in eukaryotic cells. Over the past 30 years, several studies have comprehensively characterized the structure and molecular/physiological functions of the 26S proteasome. It is a sophisticated 2.5-MDa protein degradation machine comprising a proteolytic core particle (CP) and one or two terminal regulatory particle(s) (RP). The CP consists of two outer α rings and two inner β rings, which are made up of seven structurally similar α and β subunits, respectively. The CP contains catalytic threonine residues (β1, β2, and β5; caspase-like, trypsin-like, and chymotrypsin-like activities, respectively) on the inner surface of the chamber formed by two abutting β rings. Intriguingly, the immunotype proteasomes, named 'immunoproteasome' and 'thymoproteasome', whose catalytic subunits are replaced by the related counterparts, were discovered lately. Both unique isoenzymes essentially contribute to the acquisition of adaptive immunity in vertebrates. The RP, which serves to recognize polyubiquitylated substrate proteins and plays a role in their deubiquitylating, unfolding, and translocation into the interior of the CP for destruction, forms two subcomplexes: the base and the lid. On another front, the PA28 and PA200, alternative CP activator proteins discovered biochemically, both play independent roles in proteolysis of the 26S proteasome. Several studies have highlighted the importance of the proteasome in various intractable diseases that have been increasing in the aged society of the 21st century.
Autophagy is an essential, conserved lysosomal degradation pathway that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles. It begins when double-membraned autophagosomes engulf portions of the cytoplasm, which is followed by fusion of these vesicles with lysosomes and degradation of the autophagic contents. In addition to its vital homeostatic role, this degradation pathway is involved in various human disorders, including metabolic conditions, neurodegenerative diseases, cancers and infectious diseases. This article provides an overview of the mechanisms and regulation of autophagy, the role of this pathway in disease and strategies for therapeutic modulation.
Latrepirdine (Dimebon™), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer's disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model, Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of amyloid-β (Aβ)42 aggregates. Latrepirdine was shown to upregulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro green fluorescent protein (GFP) tagged Aβ yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aβ42. GFP-Aβ42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aβ42 (19 : 34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aβ42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aβ42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aβ42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aβ42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aβ42.
Alzheimer's disease is the most common neurodegenerative disease, associated with aggregation of Aβ peptides. The exact mechanism of neuronal cell dysfunction in Alzheimer's disease is poorly understood and numerous models have been used to decipher the mechanisms leading to cellular death. Yeast cells might be a good model to understand the intracellular toxicity triggered by Aβ peptides. Indeed, yeast has been used as a model to examine protein functions or cellular pathways that mediate the secretion, aggregation, and subsequent toxicity of proteins associated with human neurodegenerative disorders. In the present study, we use the yeast Saccharomyces cerevisiae as a model system to study the effects of intracellular Aβ in fusion with the green fluorescent protein. We sent this fusion protein into the secretory pathway, and showed that intracellular traffic pathways are necessary to generate toxic species. Yeast PICALM orthologs are involved in cellular toxicity, indicating conservation of the mechanisms of toxicity from mammals to yeast. Finally, our model demonstrates the capacity for intracellular Aβ to cross intracellular membranes and target mitochondrial organelles.
Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.
In order for any biological system to function effectively, it is essential to avoid the inherent tendency of proteins to aggregate and form potentially harmful deposits. In each of the various pathological conditions associated with protein deposition, such as Alzheimer's and Parkinson's diseases, a specific peptide or protein that is normally soluble is deposited as insoluble aggregates generally referred to as amyloid. It is clear that the aggregation process is generally initiated from partially or completely unfolded forms of the peptides and proteins associated with each disease. Here we show that the intrinsic effects of specific mutations on the rates of aggregation of unfolded polypeptide chains can be correlated to a remarkable extent with changes in simple physicochemical properties such as hydrophobicity, secondary structure propensity and charge. This approach allows the pathogenic effects of mutations associated with known familial forms of protein deposition diseases to be rationalized, and more generally enables prediction of the effects of mutations on the aggregation propensity of any polypeptide chain.
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.
Aβ (beta-amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Aβ toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aβ was previously unknown. The factors identified in yeast modified Aβ toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aβ impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aβ, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.
Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between side-chains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions.
Eukaryotic cells must contend with a continuous stream of misfolded proteins that compromise the cellular protein homeostasis balance and jeopardize cell viability. An elaborate network of molecular chaperones and protein degradation factors continually monitor and maintain the integrity of the proteome. Cellular protein quality control relies on three distinct yet interconnected strategies whereby misfolded proteins can either be refolded, degraded, or delivered to distinct quality control compartments that sequester potentially harmful misfolded species. Molecular chaperones play a critical role in determining the fate of misfolded proteins in the cell. Here, we discuss the spatial and temporal organization of cellular quality control strategies and their implications for human diseases linked to protein misfolding and aggregation.
Many human diseases owe their pathology, to some degree, to the erroneous conversion of proteins from their soluble state into fibrillar, β-structured aggregates, often referred to as amyloid fibrils. Neurodegenerative diseases, such as Alzheimer and spongiform encephalopathies, as well as type 2 diabetes and both localized and systemic amyloidosis, are among the conditions that are associated with the formation of amyloid fibrils. Several mathematical tools can rationalize and even predict important parameters of amyloid fibril formation. It is not clear, however, whether such algorithms have predictive powers for in vivo systems, in which protein aggregation is affected by the presence of other biological factors. In this review, we briefly describe the existing algorithms and use them to predict the effects of mutations on the aggregation of specific proteins, for which in vivo experimental data are available. The comparison between the theoretical predictions and the experimental data obtained in vivo is shown for each algorithm and experimental data set, and statistically significant correlations are found in most cases.
The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and prion disease, is thought to occur after failure of the cellular protein quality control mechanisms. Here we examine the formation of misfolded protein inclusions in the eukaryotic cytosol of yeast and mammalian cell culture models. We identify two intracellular compartments for the sequestration of misfolded cytosolic proteins. Partition of quality control substrates to either compartment seems to depend on their ubiquitination status and aggregation state. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Notably, disease-associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Enhancing ubiquitination of a prion protein suffices to promote its delivery to the juxtanuclear inclusion. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.
Author Summary
Protein aggregation is a process by which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are associated with several pathological conditions in humans, including Alzheimer disease, Parkinson disease, and diabetes type II. Furthermore, protein aggregation is a major concern in the biotechnological production of recombinant proteins and the storage of proteins, and is a central mechanism of protein folding. In general, two classes of protein aggregates are classified: first, highly ordered aggregates can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or reorganized into β-sheet–rich aggregates, termed amyloid-like fibrils; and second, amorphous aggregates that lack any long-range order. Here, we demonstrate that bacterial inclusion bodies, which have been believed to be made up of amorphous aggregates, are in fact amyloid-like, comprising cross-β structure that is dependent on amino-acid sequence. These findings suggest that inclusion bodies are structured, that amyloid formation is a process present in both eukaryotes and prokaryotes, that amino acid sequences can evolve to avoid the amyloid conformation, and that there might be no amorphous state of a protein aggregate.
DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation,
both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional
units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange.
The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 Å resolution.
The structure consists of a β-sandwich subdomain followed by α-helical segments. The peptide is bound to DnaK in an extended
conformation through a channel defined by loops from the β sandwich. An α-helical domain stabilizes the complex, but does
not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model
of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.
Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between side-chains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions.
Aβ (beta-amyloid peptide) is an important contributor to Alzheimer’s disease (AD). We modeled Aβ toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aβ was previously unknown. The factors identified in yeast modified Aβ toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aβ impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aβ, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.
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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Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated alpha-synuclein. The molecular mechanism of clearance of alpha-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type alpha-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The alpha-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Deltaatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Deltaatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for alpha-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of alpha-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.
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.
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.
Most proteins in the human body are difficult targets for small-molecule
drugs. This problem may have been overcome with the discovery of
molecules that induce protein degradation, suggesting fresh, modular
approaches to drug discovery.
Misfolded proteins are continuously produced in the cell and present an escalating detriment to cellular physiology if not managed effectively. As such, all organisms have evolved mechanisms to address misfolded proteins. One primary way eukaryotic cells handle the complication of misfolded proteins is by destroying them through the ubiquitin-proteasome system. To do this, eukaryotes possess specialized ubiquitin-protein ligases that have the capacity to recognize misfolded proteins over normally folded proteins. The strategies used by these Protein Quality Control (PQC) ligases to target the wide variety of misfolded proteins in the cell will likely be different than those used by ubiquitin-protein ligases that function in regulated degradation to target normally folded proteins. In this review, we highlight what is known about how misfolded proteins are recognized by PQC ubiquitin-protein ligases.
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.
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.
Protein quality control (PQC) degradation protects the cell by preventing the toxic accumulation of misfolded proteins. In eukaryotes, PQC degradation is primarily achieved by ubiquitin ligases that attach ubiquitin to misfolded proteins for proteasome degradation. To function effectively, PQC ubiquitin ligases must distinguish misfolded proteins from their normal counterparts by recognizing an attribute of structural abnormality commonly shared among misfolded proteins. However, the nature of the structurally abnormal feature recognized by most PQC ubiquitin ligases is unknown. Here we demonstrate that the yeast nuclear PQC ubiquitin ligase San1 recognizes exposed hydrophobicity in its substrates. San1 recognition is triggered by exposure of as few as five contiguous hydrophobic residues, which defines the minimum window of hydrophobicity required for San1 targeting. We also find that the exposed hydrophobicity recognized by San1 can cause aggregation and cellular toxicity, underscoring the fundamental protective role for San1-mediated PQC degradation of misfolded nuclear proteins.
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.
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 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.
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.
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/.
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.
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.
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.
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.
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.
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.
Autophagic protein degradation includes bulk protein turnover with dynamic membrane reorganization, in which formation of novel organelles autophagosomes play key roles. We have shown that Saccharomyces cerevisiae performs the autophagy in the vacuole, a lytic compartment of yeast, in response to various kinds of nutrient starvation. Here we show that the APG1 gene, involved in the autophagic process in yeast, encodes a novel type of Ser/Thr protein kinase. Our results provide direct evidence for involvement of protein phosphorylation in regulation of the autophagic process. We found overall homology of Apglp with C. elegans Unc-51 protein, suggesting that homologous molecular mechanisms, conserved from unicellular to multicellular organisms, are involved in dynamic membrane flow.
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.
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.
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.
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.
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.
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.
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.