UC San Diego

Protein homeostasis in is critical to maintain cell health and viability. Protein homeostasis can be divided into two major categories: protein synthesis, and protein degradation. One of the major pathways of protein degradation is via the Ubiquitin Proteasome System (UPS), which, at its core, involves the proteasome; a multi-subunit cellular machine that unfolds and degrades proteins and breaks them down into small peptides to be utilized by the cell for further protein synthesis. Regulation of this machinery is vital to maintain protein homeostasis. We have discovered a posttranslational modification to the AAA ATPase Rpt6 subunit that modulates proteasome activity. Here, we have made mutants that abrogate or mimic this phosphorylation, in order to allow us to further uncover the regulatory mechanisms that govern protein breakdown and homeostasis in eukaryotic cells.

Chapter II investigates the basic cellular effects in the CNS of eliminating phosphorylation dynamics of Rpt6 in novel mouse models that either block Rpt6 phosphorylation or mimic it. Behavioral, electrophysiological, biochemical, and other techniques are utilized in investigating the effects of Rpt6 S120 phosphorylation in the mammalian brain.

In Chapter III, the loss of cocaine sensitization in Rpt6 S120D mice is explored. We find that treatment of cultured neurons with cocaine causes the phosphorylation and activation of CaMKIIα, and concomitant Rpt6 phosphorylation and subsequent increase in proteasome activity. This process is shown to be activated via monaminergic systems in neurons, specifically the inhibition of dopamine uptake. This process is abrogated when Rpt6 S120 phosphorylation is mimicked when mutated to aspartic acid, particularly in the nucleus accumbens and pre-frontal cortex, providing further insight into the pathogenesis of cocaine sensitization.

Chapter IV presents a study of the dynamics Rpt6 phosphorylation and cellular susceptibility to proteotoxic stress. Rpt6 S120 knock-in mutants of S. Cerevisiae are utilized to investigate how cells respond to various proteotoxic stressors. We find Rpt6 is phosphorylated in response to such stressors, and when phosphorylation is abrogated, cells are not able to respond to such stresses and become more susceptible to protein aggregation and cell death.