Phosphorylation of the 19S regulatory particle ATPase subunit, Rpt6, modifies susceptibility to proteotoxic stress and protein aggregation.

Esther Magdalena Marquez-Lona,Ana Lilia Torres-Machorro,Gentry N Patrick,Frankie R Gonzales,Lorraine Pillus

doi:10.1371/journal.pone.0179893

Esther Magdalena Marquez-Lona, Ana Lilia Torres-Machorro + Show 3 more

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https://doi.org/10.1371/journal.pone.0179893

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Journal: PloS one	Publication Date: Jun 29, 2017
Citations: 16	License type: CC BY 4.0

Affiliation: University of California, San Diego

Abstract

The ubiquitin proteasome system (UPS) is a highly conserved and tightly regulated biochemical pathway that degrades the majority of proteins in eukaryotic cells. Importantly, the UPS is responsible for counteracting altered protein homeostasis induced by a variety of proteotoxic stresses. We previously reported that Rpt6, the ATPase subunit of the 19S regulatory particle (RP) of the 26S proteasome, is phosphorylated in mammalian neurons at serine 120 in response to neuronal activity. Furthermore, we found that Rpt6 S120 phosphorylation, which regulates the activity and distribution of proteasomes in neurons, is relevant for proteasome-dependent synaptic remodeling and function. To better understand the role of proteasome phosphorylation, we have constructed models of altered Rpt6 phosphorylation in S. cerevisiae by introducing chromosomal point mutations that prevent or mimic phosphorylation at the conserved serine (S119). We find that mutants which prevent Rpt6 phosphorylation at this site (rpt6-S119A), had increased susceptibility to proteotoxic stress, displayed abnormal morphology and had reduced proteasome activity. Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington’s disease (HD) related huntingtin (Htt) protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead (rpt6-S119A) and phospho-mimetic (rpt6-S119D) mutants. We showed Htt103Q aggregate size to be significantly larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D strains. Furthermore, we observed that phosphorylation of endogenous Rpt6 at S119 is increased in response to various stress conditions. Together, these data suggest that Rpt6 phosphorylation at S119 may play an important function in proteasome-dependent relief of proteotoxic stress that can be critical in protein aggregation pathologies.

Highlights

The balance between synthesis and degradation is crucial for maintaining protein homeostasis
Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington’s disease (HD) related huntingtin (Htt) protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead and phosphomimetic mutants
It is clear that Rpt6 serine 120 (S120) phosphorylation increases proteasome activity and promotes the redistribution of proteasomes to synapses [22, 23, 45], further mechanistic details remain under study

Summary

Introduction

The balance between synthesis and degradation is crucial for maintaining protein homeostasis. Many studies have shown that altered protein homeostasis occurs during normal aging and age-related disease [1,2,3,4,5,6]. In post-mitotic neurons, genetic alterations together with oxidative stress and other forms of cellular damage are thought to underlie many of the abnormalities that contribute to age-related and neurodegenerative diseases. Whereas ribosomes and chaperones ensure proper synthesis and folding of proteins, the ubiquitin proteasome system (UPS) and autophagosomal/ lysosomal pathways control the majority of cellular protein degradation. The UPS is an evolutionarily conserved and tightly regulated biochemical pathway involved in protein degradation [2, 3, 11]. The RP is necessary for interaction and unfolding of ubiquitinated substrates and gating of the CP to ensure proper degradation [13,14,15]

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