RESUMEN
The nascent polypeptide-associate complex (NAC) is a heterodimeric chaperone complex that binds near the ribosome exit tunnel and is the first point of chaperone contact for newly synthesized proteins. Deletion of the NAC induces embryonic lethality in many multi-cellular organisms. Previous work has shown that the deletion of the NAC rescues cells from prion-induced cytotoxicity. This counterintuitive result led us to hypothesize that NAC disruption would improve viability in cells expressing human misfolding proteins. Here, we show that NAC disruption improves viability in cells expressing expanded polyglutamine and also leads to delayed and reduced aggregation of expanded polyglutamine and changes in polyglutamine aggregate morphology. Moreover, we show that NAC disruption leads to changes in de novo yeast prion induction. These results indicate that the NAC plays a critical role in aggregate organization as a potential therapeutic target in neurodegenerative disorders.
Asunto(s)
Chaperonas Moleculares , Péptidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Péptidos/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Priones/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismoRESUMEN
Dominant missense mutations in DNAJB6, a co-chaperone of HSP70, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests that DNAJB6b is primarily responsible for disease pathogenesis. Knockdown treatment strategies involving both isoforms carry risk, as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform-specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved in vitro in primary mouse myotubes and human LGMDD1 myoblasts, as well as in vivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knockin LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature toward wild type levels. Additional in vivo functional data is required to determine if selective reduction of DNAJB6b is a viable therapeutic target for LGMDD1.
RESUMEN
Molecular chaperones, or heat shock proteins (HSPs), protect against the toxic misfolding and aggregation of proteins. As such, mutations or deficiencies within the chaperone network can lead to disease. Dominant mutations within DNAJB6 (Hsp40)-an Hsp70 co-chaperone-lead to a protein aggregation-linked myopathy termed Limb-Girdle Muscular Dystrophy Type D1 (LGMDD1). Here, we used the yeast prion model client in conjunction with in vitro chaperone activity assays to gain mechanistic insights into the molecular basis of LGMDD1. Here, we show how mutations analogous to those found in LGMDD1 affect Sis1 (a functional homolog of human DNAJB6) function by altering the structure of client protein aggregates, interfering with the Hsp70 ATPase cycle, dimerization and substrate processing; poisoning the function of wild-type protein. These results uncover the mechanisms through which LGMDD1-associated mutations alter chaperone activity, and provide insights relevant to potential therapeutic interventions.
Asunto(s)
Distrofia Muscular de Cinturas , Proteínas de Saccharomyces cerevisiae , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Distrofia Muscular de Cinturas/genética , Mutación , Proteínas del Tejido Nervioso/metabolismo , Nucleótidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The misfolding and aggregation of proteins is often implicated in the development and progression of degenerative diseases. Heat shock proteins (HSPs), such as the ubiquitously expressed Type II Hsp40 molecular chaperone, DNAJB6, assist in protein folding and disaggregation. Historically, mutations within the DNAJB6 G/F domain have been associated with Limb-Girdle Muscular Dystrophy type 1D, now referred to as LGMDD1, a dominantly inherited degenerative disease. Recently, novel mutations within the J domain of DNAJB6 have been reported in patients with LGMDD1. Since novel myopathy-causing mutations in the Hsp40 J domain have yet to be characterized and both the function of DNAJB6 in skeletal muscle and the clients of this chaperone are unknown, we set out to assess the effect of these mutations on chaperone function using the genetically tractable yeast system. The essential yeast Type II Hsp40, Sis1, is homologous to DNAJB6 and is involved in the propagation of yeast prions. Using phenotypic, biochemical, and functional assays we found that homologous mutations in the Sis1 J domain differentially alter the processing of specific yeast prion strains, as well as a non-prion substrate. These data suggest that the newly-identified mutations in the J domain of DNAJB6 cause aberrant chaperone function that leads to the pathogenesis in LGMDD1.
Asunto(s)
Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Enfermedades Musculares/genética , Mutación , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Ratones , Modelos Moleculares , Dominios Proteicos , Replegamiento ProteicoRESUMEN
Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.
Asunto(s)
Mutación con Ganancia de Función , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Fuerza Muscular/genética , Distrofia Muscular de Cinturas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Humanos , Ratones , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Distrofia Muscular de Cinturas/tratamiento farmacológico , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/patología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Saccharomyces cerevisiaeRESUMEN
OBJECTIVE: To understand DNAJB6's function in skeletal muscle and identify therapeutic targets for limb-girdle muscular dystrophy 1D (LGMD1D). METHODS: DNAJB6 knockout (KO) myoblasts were generated with Crispr/cas9 technology, and differentially accumulated proteins were identified using stable isotope labeling, followed by quantitative mass spectrometry. Cultured KO myotubes and mouse muscle from DNAJB6b-WT or DNAJB6b-F93L mice were analyzed using histochemistry, immunohistochemistry, and immunoblot. Mouse functional strength measures included forelimb grip strength and inverted wire hang. RESULTS: DNAJB6 inactivation leads to the accumulation of sarcomeric proteins and hypertrophic myotubes with an enhanced fusion index. The increased fusion in DNAJB6 KO myotubes correlates with diminished glycogen synthase kinase-ß (GSK3ß) activity. In contrast, LGMD1D mutations in DNAJB6 enhance GSK3ß activation and suppress ß-catenin and NFAT3c signaling. GSK3ß inhibition with lithium chloride improves muscle size and strength in an LGMD1D preclinical mouse model. CONCLUSIONS: Our results suggest that DNAJB6 facilitates protein quality control and negatively regulates myogenic signaling. In addition, LGMD1D-associated DNAJB6 mutations inhibit myogenic signaling through augmented GSK3ß activity. GSK3ß inhibition with lithium chloride may be a therapeutic option in LGMD1D.
RESUMEN
The early stages of protein misfolding remain incompletely understood, as most mammalian proteinopathies are only detected after irreversible protein aggregates have formed. Cross-seeding, where one aggregated protein templates the misfolding of a heterologous protein, is one mechanism proposed to stimulate protein aggregation and facilitate disease pathogenesis. Here, we demonstrate the existence of cross-seeding as a crucial step in the formation of the yeast prion [PSI +], formed by the translation termination factor Sup35. We provide evidence for the genetic and physical interaction of the prion protein Rnq1 with Sup35 as a predominant mechanism leading to self-propagating Sup35 aggregation. We identify interacting sites within Rnq1 and Sup35 and determine the effects of breaking and restoring a crucial interaction. Altogether, our results demonstrate that single-residue disruption can drastically reduce the effects of cross-seeding, a finding that has important implications for human protein misfolding disorders.
Asunto(s)
Priones/metabolismo , Agregado de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Modelos Biológicos , Polimerizacion , Unión Proteica , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Molecular chaperones are responsible for managing protein folding from translation through degradation. These crucial machines ensure that protein homeostasis is optimally maintained for cell health. However, 'too much of a good thing' can be deadly, and the excess of chaperones can be toxic under certain cellular conditions. For example, overexpression of Ssa1, a yeast Hsp70, is toxic to cells in folding-challenged states such as [PSI+]. We discovered that overexpression of the nucleotide exchange factor Sse1 can partially alleviate this toxicity. We further argue that the basis of the toxicity is related to the availability of Hsp70 cofactors, such as Hsp40 J-proteins and nucleotide exchange factors. Ultimately, our work informs future studies about functional chaperone balance and cautions against therapeutic chaperone modifications without a thorough examination of cofactor relationships.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Chaperonas Moleculares/metabolismo , Factores de Terminación de Péptidos/genética , Unión Proteica , Pliegue de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.
Asunto(s)
Técnicas Genéticas , Mutación , Proteínas/genética , Poli A/genética , Poli A/metabolismo , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Proteínas/metabolismo , Estabilidad del ARNRESUMEN
The nascent polypeptide-associated complex (NAC) is a highly conserved but poorly characterized triad of proteins that bind near the ribosome exit tunnel. The NAC is the first cotranslational factor to bind to polypeptides and assist with their proper folding. Surprisingly, we found that deletion of NAC subunits in Saccharomyces cerevisiae rescues toxicity associated with the strong [PSI+] prion. This counterintuitive finding can be explained by changes in chaperone balance and distribution whereby the folding of the prion protein is improved and the prion is rendered nontoxic. In particular, the ribosome-associated Hsp70 Ssb is redistributed away from Sup35 prion aggregates to the nascent chains, leading to an array of aggregation phenotypes that can mimic both overexpression and deletion of Ssb. This toxicity rescue demonstrates that chaperone modification can block key steps of the prion life cycle and has exciting implications for potential treatment of many human protein conformational disorders.
Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Chaperonas Moleculares/genética , Factores de Terminación de Péptidos/genética , Priones/genética , Proteínas de Saccharomyces cerevisiae/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Regulación Fúngica de la Expresión Génica , Proteínas HSP70 de Choque Térmico/biosíntesis , Humanos , Chaperonas Moleculares/química , Factores de Terminación de Péptidos/biosíntesis , Péptidos/química , Péptidos/genética , Priones/química , Priones/toxicidad , Agregado de Proteínas/genética , Pliegue de Proteína , Ribosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Eliminación de SecuenciaRESUMEN
Prions are infectious protein polymers that have been found to cause fatal diseases in mammals. Prions have also been identified in fungi (yeast and filamentous fungi), where they behave as cytoplasmic non-Mendelian genetic elements. Fungal prions correspond in most cases to fibrillary ß-sheet-rich protein aggregates termed amyloids. Fungal prion models and, in particular, yeast prions were instrumental in the description of fundamental aspects of prion structure and propagation. These models established the "protein-only" nature of prions, the physical basis of strain variation, and the role of a variety of chaperones in prion propagation and amyloid aggregate handling. Yeast and fungal prions do not necessarily correspond to harmful entities but can have adaptive roles in these organisms.
Asunto(s)
Amiloide/metabolismo , Proteínas Fúngicas/metabolismo , Hongos/metabolismo , Priones/metabolismo , Amiloide/química , Proteínas Fúngicas/química , Priones/químicaRESUMEN
Limb-girdle muscular dystrophy type 1D (LGMD1D) is caused by dominantly inherited missense mutations in DNAJB6, an Hsp40 co-chaperone. LGMD1D muscle has rimmed vacuoles and inclusion bodies containing DNAJB6, Z-disc proteins and TDP-43. DNAJB6 is expressed as two isoforms; DNAJB6a and DNAJB6b. Both isoforms contain LGMD1D mutant residues and are expressed in human muscle. To identify which mutant isoform confers disease pathogenesis and generate a mouse model of LGMD1D, we evaluated DNAJB6 expression and localization in skeletal muscle as well as generating DNAJB6 isoform specific expressing transgenic mice. DNAJB6a localized to myonuclei while DNAJB6b was sarcoplasmic. LGMD1D mutations in DNAJB6a or DNAJB6b did not alter this localization in mouse muscle. Transgenic mice expressing the LGMD1D mutant, F93L, in DNAJB6b under a muscle-specific promoter became weak, had early lethality and developed muscle pathology consistent with myopathy after 2 months; whereas mice expressing the same F93L mutation in DNAJB6a or overexpressing DNAJB6a or DNAJB6b wild-type transgenes remained unaffected after 1 year. DNAJB6b localized to the Z-disc and DNAJB6b-F93L expressing mouse muscle had myofibrillar disorganization and desmin inclusions. Consistent with DNAJB6 dysfunction, keratin 8/18, a DNAJB6 client also accumulated in DNAJB6b-F93L expressing mouse muscle. The RNA-binding proteins hnRNPA1 and hnRNPA2/B1 accumulated and co-localized with DNAJB6 at sarcoplasmic stress granules suggesting that these proteins maybe novel DNAJB6b clients. Similarly, hnRNPA1 and hnRNPA2/B1 formed sarcoplasmic aggregates in patients with LGMD1D. Our data support that LGMD1D mutations in DNAJB6 disrupt its sarcoplasmic function suggesting a role for DNAJB6b in Z-disc organization and stress granule kinetics.
Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Chaperonas Moleculares/metabolismo , Distrofia Muscular de Cinturas/metabolismo , Miofibrillas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSP40/genética , Humanos , Queratinas/metabolismo , Ratones , Ratones Transgénicos , Chaperonas Moleculares/genética , Distrofia Muscular de Cinturas/patología , Mutación , Miofibrillas/patología , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Isoformas de Proteínas/metabolismoRESUMEN
Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1 and its human orthologue Hdj1 had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas del Choque Térmico HSP40/genética , Priones/genética , Levaduras/genética , Evolución Molecular , Proteínas Fúngicas/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Levaduras/metabolismoRESUMEN
The molecular chaperone network protects against the toxic misfolding and aggregation of proteins. Disruption of this network leads to a variety of protein conformational disorders. One such example recently discovered is limb-girdle muscular dystrophy type 1D (LGMD1D), which is caused by mutation of the HSP40 chaperone DNAJB6. All LGMD1D-associated mutations localize to the conserved G/F domain of DNAJB6, but the function of this domain is largely unknown. Here, we exploit the yeast HSP40 Sis1, which has known aggregation-prone client proteins, to gain insight into the role of the G/F domain and its significance in LGMD1D pathogenesis. Strikingly, we demonstrate that LGMD1D mutations in a Sis1-DNAJB6 chimera differentially impair the processing of specific conformers of two yeast prions, [RNQ+] and [PSI+]. Importantly, these differences do not simply correlate to the sensitivity of these prion strains to changes in chaperone levels. Additionally, we analyzed the effect of LGMD1D-associated DNAJB6 mutations on TDP-43, a protein known to form inclusions in LGMD1D. We show that the DNAJB6 G/F domain mutants disrupt the processing of nuclear TDP-43 stress granules in mammalian cells. These data suggest that the G/F domain mediates chaperone-substrate interactions in a manner that extends beyond recognition of a particular client and to a subset of client conformers. We propose that such selective chaperone disruption may lead to the accumulation of toxic aggregate conformers and result in the development of LGMD1D and perhaps other protein conformational disorders.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Distrofia Muscular de Cinturas/metabolismo , Mutación Missense , Proteínas del Tejido Nervioso/metabolismo , Agregación Patológica de Proteínas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas del Choque Térmico HSP40/genética , Células HeLa , Humanos , Chaperonas Moleculares/genética , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/patología , Proteínas del Tejido Nervioso/genética , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Amyloidogenic proteins associated with a variety of unrelated diseases are typically capable of forming several distinct self-templating conformers. In prion diseases, these different structures, called prion strains (or variants), confer dramatic variation in disease pathology and transmission. Aggregate stability has been found to be a key determinant of the diverse pathological consequences of different prion strains. Yet, it remains largely unclear what other factors might account for the widespread phenotypic variation seen with aggregation-prone proteins. Here, we examined a set of yeast prion variants of the [RNQ+] prion that differ in their ability to induce the formation of another yeast prion called [PSI+]. Remarkably, we found that the [RNQ+] variants require different, non-contiguous regions of the Rnq1 protein for both prion propagation and [PSI+] induction. This included regions outside of the canonical prion-forming domain of Rnq1. Remarkably, such differences did not result in variation in aggregate stability. Our analysis also revealed a striking difference in the ability of these [RNQ+] variants to interact with the chaperone Sis1. Thus, our work shows that the differential influence of various amyloidogenic regions and interactions with host cofactors are critical determinants of the phenotypic consequences of distinct aggregate structures. This helps reveal the complex interdependent factors that influence how a particular amyloid structure may dictate disease pathology and progression.
Asunto(s)
Amiloide/metabolismo , Chaperonas Moleculares/metabolismo , Priones/metabolismo , Electroforesis en Gel de Poliacrilamida , Priones/clasificación , Conformación ProteicaRESUMEN
Variation in amyloid structures profoundly influences a wide array of pathological phenotypes in mammalian protein conformation disorders and dominantly inherited phenotypes in yeast. Here, we describe, for the first time, naturally occurring, self-propagating, structural variants of a prion protein isolated from wild strains of the yeast Saccharomyces cerevisiae. Variants of the [RNQâº] prion propagating in a variety of wild yeast differ biochemically, in their intracellular distributions, and in their ability to promote formation of the [PSIâº] prion. [PSIâº] is an epigenetic regulator of cellular phenotype and adaptability. Strikingly, we find that most natural [RNQâº] variants induced [PSIâº] at high frequencies and the majority of [PSIâº] variants elicited strong cellular phenotypes. We hypothesize that the presence of an efficient [RNQâº] template primes the cell for [PSIâº] formation in order to induce [PSIâº] in conditions where it would be advantageous. These studies utilize naturally occurring structural variants to expand our understanding of the consequences of diverse prion conformations on cellular phenotypes.
Asunto(s)
Adaptación Fisiológica/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Genes Reporteros/genética , Fenotipo , Priones/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/aislamiento & purificación , TemperaturaRESUMEN
Amyloidogenic proteins, including prions, assemble into multiple forms of structurally distinct fibres. The [PSI(+)] prion, endogenous to the yeast Saccharomyces cerevisiae, is a dominantly inherited, epigenetic modifier of phenotypes. [PSI(+)] formation relies on the coexistence of another prion, [RNQ(+)]. Here, in order to better define the role of amyloid diversity on cellular phenotypes, we investigated how physiological and environmental changes impact the generation and propagation of diverse protein conformations from a single polypeptide. Utilizing the yeast model system, we defined extracellular factors that influence the formation of a spectrum of alternative self-propagating amyloid structures of the Sup35 protein, called [PSI(+)] variants. Strikingly, exposure to specific stressful environments dramatically altered the variants of [PSI(+)] that formed de novo. Additionally, we found that stress also influenced the association between the [PSI(+)] and [RNQ(+)] prions in a way that it superceded their typical relationship. Furthermore, changing the growth environment modified both the biochemical properties and [PSI(+)]-inducing capabilities of the [RNQ(+)] template. These data suggest that the cellular environment contributes to both the generation and the selective propagation of specific amyloid structures, providing insight into a key feature that impacts phenotypic diversity in yeast and the cross-species transmission barriers characteristic of prion diseases.
Asunto(s)
Amiloide/metabolismo , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Priones/metabolismo , Estrés FisiológicoRESUMEN
Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M) domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain (by analogy to ClpB in bacteria). We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.