Synphilin-1A Inhibits Seven in Absentia Homolog (SIAH) and Modulates α-Synuclein Monoubiquitylation and Inclusion Formation

Abstract

Parkinson disease (PD) is characterized by the presence of ubiquitylated inclusions and the death of dopaminergic neurons. Seven in absentia homolog (SIAH) is a ubiquitin-ligase that ubiquitylates ␣-synuclein and synphilin-1 and is present in Lewy bodies of PD patients. Understanding the mechanisms that regulate the ubiquitylation of PD-related proteins might shed light on the events involved in the formation of Lewy bodies and death of neurons. We show in this study that the recently described synphilin-1 isoform, synphilin-1A, interacts in vitro and in vivo with the ubiquitin-protein isopeptide ligase SIAH and regulates its activity toward ␣-synuclein and synphilin-1. SIAH promotes limited ubiquitylation of synphilin-1A that does not lead to its degradation by the proteasome. SIAH also increases the formation of synphilin-1A inclusions in the presence of proteasome inhibitors, supporting the participation of ubiquitylated synphilin-1A in the formation of Lewy body-like inclusions. Synphilin-1A/SIAH inclusions recruit PD-related proteins, such as ␣-synuclein, synphilin-1, Parkin, PINK1, and UCH-L1. We found that synphilin-1A robustly increases the steady-state levels of SIAH by decreasing its auto-ubiquitylation and degradation. In addition, synphilin-1A blocks the ubiquitylation and degradation of the SIAH substrates synphilin-1 and deleted in colon cancer protein. Furthermore, synphilin-1A strongly decreases the monoubiquitylation of ␣-synuclein by SIAH and the formation of ␣-synuclein inclusions, supporting a role for monoubiquitylation in ␣-synuclein inclusion formation. Our results suggest a novel function for synphilin-1A as a regulator of SIAH activity and formation of Lewy body-like inclusions.

α-Synuclein fate is determined by USP9X-regulated monoubiquitination

“…SIAH is present in Lewy bodies (9) and monoubiquitinates α-synuclein at the same lysine residues that are monoubiquitinated in α-synuclein immunopurified from Lewy bodies (5,10). Most importantly, we found that in the presence of proteolytic inhibitors, monoubiquitination of α-synuclein promoted by SIAH leads to a marked increase in the aggregation of α-synuclein and formation of inclusions, which are toxic to cells (10)(11)(12). Therefore, our data indicate that monoubiquitination by SIAH might play a primary role in the aggregation and toxicity of α-synuclein and possibly in the formation of Lewy bodies.…”

Nuclear Compartmentalization of Serine Racemase Regulates d-Serine Production

“…Mouse anti-histone3 (1:1000) was obtained from Abcam. Rabbit anti-seven in absentia homologue (Siah) (1:500) was a gift from Dr. Simone Engelender (Technion-Israel Institute of Technology) and has been described in earlier studies (38,39). Rabbit affinity-purified anti-SR antibody (0.2-0.3 g/ml) (18) or guinea pig anti-SR have also been described elsewhere (40).…”

Synphilin-1 inhibits alpha-synuclein degradation by the proteasome

“…While Siah-1, and much more efficiently Siah-2, has been reported to interact with alphasynuclein resulting in its monoubiquitylation [50], under our assays conditions we were not able to detect any alphasynuclein modification following Siah-1 expression. The apparent conflicting results can be explained by the fact that monoubiquitylation of alpha-synuclein is quantitatively a minor modification [50,63] that could not be readily detected by direct immunoblotting of whole cell extracts, and in any case Siah-1 expression did not significantly affect alpha-synuclein degradation by the proteasome pathway in the absence of synphilin-1 (Fig. 8).…”