RESUMEN
A hallmark of diseases of protein conformation and aging is the appearance of protein aggregates associated with cellular toxicity. We posit that the functional properties of the proteostasis network (PN) protect the proteome from misfolding and combat the proteotoxic events leading to cellular pathology. In this study, we have identified new components of the proteostasis network that can suppress aggregation and proteotoxicity, by performing RNA interference (RNAi) genetic screens for multiple unrelated conformationally challenged cytoplasmic proteins expressed in Caenorhabditis elegans. We identified 88 suppressors of polyglutamine (polyQ) aggregation, of which 63 modifiers also suppressed aggregation of mutant SOD1(G93A). Of these, only 23 gene-modifiers suppressed aggregation and restored animal motility, revealing that aggregation and toxicity can be genetically uncoupled. Nine of these modifiers were shown to be effective in restoring the folding and function of multiple endogenous temperature-sensitive (TS) mutant proteins, of which five improved folding in a HSF-1-dependent manner, by inducing cytoplasmic chaperones. This triage screening strategy also identified a novel set of PN regulatory components that, by altering metabolic and RNA processing functions, establish alternate cellular environments not generally dependent on stress response activation and that are broadly protective against misfolded and aggregation-prone proteins.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Pruebas Genéticas/métodos , Péptidos/genética , Péptidos/metabolismo , Interferencia de ARN , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Respuesta al Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Pliegue de Proteína , Deficiencias en la Proteostasis , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity.