Methionine Sulfoxide Reductase and Ubiquitin-dependent Degradation of Alpha-synuclein

Abstract

Alpha-synuclein (αSyn) is one of the key proteins considered to be involved in the pathogenesis of the Parkinson`s disease (PD) and being the main component of Lewy bodies (LBs). Studies on the αSyn gene mutants and overexpression of αSyn in yeast and rodent brains indicate that the level of αSyn in neurons is linked to dopaminergic neurons degeneration, and impairment of αSyn degradation is directly related to its aggregation in PD. However, the exact mechanism by which αSyn dysfunction and its degradation are regulated are not well established. Previous studies have suggested that both the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are involved in the degradation of αSyn, in which the ubiquitin (Ub) system plays an important role. Moreover, αSyn was found to bind Ub and the molecular chaperone 14-3-3 proteins, leading to enhanced αSyn aggregation. Our lab recently reported on elevated dopamine and 14-3-3 levels in the methionine sulfoxide reductase A (MsrA) knockout mouse brain, as well as decreased αSyn degradation in MsrA KO yeast cells. Hence, this study will focus on the interactions among MsrA, αSyn degradation, 14-3-3 proteins, and Ub. Here we show that msrA overexpressing (OP) yeast cells extract promotes αSyn aggregation in a Ub- and ATP-dependent manner. Moreover, ubiquitin could promote clearance of both native and A53T mutant αSyn monomers in the presence of msrA OP extract in comparison to a msrA null mutant yeast extract. Furthermore, MsrA was found to inhibit the auto-ubiquitination of Parkin (a known E3 ubiquitin ligase of αSyn). These data suggest that MsrA plays an important role in ubiquitin-dependent αSyn degradation and may function as an E3 Ub ligase. To explore the interactions between αSyn, MsrA, and Ub-bound proteins, a mammalian ex-vivo system was designed using wild-type (W) and MsrA knockout (M) mouse brain extracts. Following pull-down experiments using His-tagged Ub and the mouse brain extracts, it was determined that 14-3-3 proteins were ubiquitinated with dependency on the presence of MsrA (W brain) and ATP. Immunoprecipitation experiments using anti-Ub antibody revealed that M brain exhibited lower levels of ubiquitinated and degraded band products of both αSyn and 14-3-3 proteins, compared with W brain. Complementary immunoprecipitation experiments using anti-14-3-3 antibody showed an enhanced αSyn degradation and ubiquitination in the presence of ATP in W versus M brain extracts, suggesting that 14-3-3 interacts with MsrA and the Ub-system to enhance αSyn degradation. Last but not least, an ex-vivo study performed in yeast cells (wild-type and msrA null mutant strains overexpressing A53T αSyn) showed that the msrA mutant strain possessed significantly much lower total and αSyn ubiquitination levels compared with the wild-type strain. These observations suggest that MsrA plays an important role in the ubiquitination and degradation of αSyn through the ubiquitin-dependent modification of 14-3-3.