Vulnerability of newly synthesized proteins to proteostasis stress

Abstract

The capacity of the cell to produce, fold and degrade proteins relies on components of the proteostasis network. Multiple types of insults can impose a burden on this network, causing protein misfolding. Using thermal stress, a classic example of acute proteostatic stress, we demonstrate that ∼5-10% of the soluble cytosolic and nuclear proteome in human HEK293 cells is vulnerable to misfolding when proteostatic function is overwhelmed. Inhibiting new protein synthesis for 30 min prior to heat-shock dramatically reduced the amount of heat-stress induced polyubiquitylation, and reduced the misfolding of proteins identified as vulnerable to thermal stress. Following prior studies in C. elegans in which mutant huntingtin (Q103) expression was shown to cause the secondary misfolding of cytosolic proteins, we also demonstrate that mutant huntingtin causes similar 'secondary' misfolding in human cells. Similar to thermal stress, inhibiting new protein synthesis reduced the impact of mutant huntingtin on proteostatic function. These findings suggest that newly made proteins are vulnerable to misfolding when proteostasis is disrupted by insults such as thermal stress and mutant protein aggregation.

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in C. elegans

“…Therefore, it is likely that these proteins self-assemble into amyloid structures directly from the unfolded state occurring during or shortly after translation rather than undergoing unfolding from their natively folded state prior to amyloid formation. This is consistent with protein folding intermediates being particularly at risk of aggregating as exemplified by a recent study revealing that newly synthesized proteins constitute the majority of the insoluble fraction prompted by thermal stress (31). Furthermore, artificially increasing ribosome pausing during translation causes widespread protein aggregation (9).…”

NEDDylation promotes nuclear protein aggregation and protects the Ubiquitin Proteasome System upon proteotoxic stress

“…In addition to pre-existing proteins, newly synthesised polypeptides are particularly vulnerable and damaged under proteotoxic stress 33 . Treatment of cells with the translational inhibitor Cycloheximide (CHX) prevented the increase in protein NEDDylation upon HS, indicating that the NEDD8 response to proteotoxic stress depends on protein synthesis (Fig.…”

Functional Modules of the Proteostasis Network

“…Although it is unclear what fraction of the mammalian proteome denatures and misfolds during heat stress (Wallace et al 2015), the level of ubiquitylated proteins rapidly increases in stressed cells (Fujimuro et al 1997), suggesting that degradation of misfolded proteins via the UPS is critical in reestablishing proteome balance (Parag et al 1987). Nascent polypeptides synthesized prior to heat stress, possibly still bound to ribosomes in unfolded states, are thought to represent the major vulnerable fraction of the proteome that is targeted for degradation (Medicherla and Goldberg 2008;Xu et al 2016). A comprehensive genome-wide deletion screen for induction of the HSR in yeast (Brandman et al 2012) under nonstress conditions revealed an enrichment of individual chaperones and members of the UPS, among other inducers, pointing to a strong cross talk between the two PN modules.…”