A Genetic Screening Strategy Identifies Novel Regulators of the Proteostasis Network

Abstract

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.

Author Summary

A common characteristic of protein conformational diseases is the appearance of protein aggregates associated with late-onset symptoms. Here, we have taken an unbiased genetic approach to test the hypothesis that protein aggregation and toxicity are co-linked genetic traits that are regulated by a common proteostasis network. To address this, we took advantage of the tractable genetic model Caenorhabditis elegans expressing expanded polyglutamines (polyQ) and performed a genome-wide RNA interference (RNAi) screen to identify genes that altered the proteostasis environment and suppressed aggregation and toxicity. These modifiers were subsequently tested on animals expressing mutant SOD1 and on animals expressing endogenous proteins with temperature-sensitive mutations. This screening triage resulted in the identification of nine genes with effects on protein folding, corresponding to new proteostasis pathways involved in metabolism and RNA processing functions.