Ubiquitylation of synphilin-1 and α-synuclein by SIAH and its presence in cellular inclusions and Lewy bodies imply a role in Parkinson's disease

Abstract

Parkinson's disease (PD) is a neurodegenerative disease characterized by Lewy body formation and death of dopaminergic neurons. Mutations in ␣-synuclein and parkin cause familial forms of PD. Synphilin-1 was shown to interact with ␣-synuclein and to promote the formation of cytosolic inclusions. We now report that synphilin-1 interacts with the E3 ubiquitin-ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 both in vitro and in vivo, promoting its degradation by the ubiquitin-proteasome system. Inability of the proteasome to degrade synphilin-1͞SIAH complex leads to a robust formation of ubiquitylated cytosolic inclusions. Ubiquitylation is required for inclusion formation, because a catalytically inactive mutant of SIAH-1, which still binds to synphilin-1, fails to promote inclusions. Like synphilin-1, ␣-synuclein associates with SIAH in intact cells, but the interaction with SIAH-2 was much stronger that with SIAH-1. In vitro experiments show that SIAH-2 monoubiquitylates ␣-synuclein. Further evidence that SIAH proteins may play a role in inclusion formation comes from the demonstration of SIAH immunoreactivity in Lewy bodies of PD patients.␣-synuclein-interacting protein ͉ ubiquitin ͉ inclusion bodies

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

“…Western Blot Analysis-Samples were homogenized as described (8). Blots were probed with antibodies mouse anti-HA (Covance), mouse anti-Myc (Sigma), mouse anti-␣-synuclein (BD Biosciences), rabbit anti-FLAG (Sigma), rabbit anti-Myc, rabbit anti-HA, goat anti-SIAH-1 (N-15), goat anti-SIAH-2 (N-14), goat anti-SIAH-1/2 (H-18), and mouse antiactin (Santa Cruz Biotechnology).…”

“…Inability of the proteasome to degrade polyubiquitylated synphilin-1 leads to robust formation of inclusions (8). Moreover, ubiquitylation of synphilin-1 is essential for its aggregation, as inactive SIAH mutants do not elicit formation of synphilin-1 inclusions (8). Thus, we raised the possibility that ubiquitylation of synphilin-1 and other PD-related proteins might represent an important step for Lewy body formation (13,23,24).…”

“…Synphilin-1 was shown to be ubiquitylated by different E3 ubiquitin-ligases, including Parkin, Dorfin, and SIAH (8, 20 -22). Inability of the proteasome to degrade polyubiquitylated synphilin-1 leads to robust formation of inclusions (8). Moreover, ubiquitylation of synphilin-1 is essential for its aggregation, as inactive SIAH mutants do not elicit formation of synphilin-1 inclusions (8).…”

“…In addition to ubiquitylated synphilin-1, SIAH interacts with and monoubiquitylates ␣-synuclein at lysines found to be ubiquitylated in ␣-synuclein purified from Lewy bodies (8,14). Monoubiquitylation of ␣-synuclein leads to its aggregation and inclusion formation, which are toxic to dopaminergic cells (14).…”

“…Monoubiquitylation of ␣-synuclein leads to its aggregation and inclusion formation, which are toxic to dopaminergic cells (14). The presence of SIAH in Lewy bodies of PD patients (8) indicates that it might represent an additional component of the ubiquitin-proteasome system involved in the disease.…”