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J. Biol. Chem., Vol. 283, Issue 6, 3316-3328, February 8, 2008

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Monoubiquitylation of -Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells^*

Ruth Rott¹, Raymonde Szargel¹, Joseph Haskin, Vered Shani, Alla Shainskaya, Irena Manov^¶, Esti Liani, Eyal Avraham, and Simone Engelender²

From the Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096, Biological Mass Spectrometry Facility, Department of Biological Services, The Weizmann Institute of Science, 76100 Rehovot, and ^¶Pediatric Research and Electron Microscopy Unit, The B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel

-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of -synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. -Synuclein is ubiquitylated in Lewy bodies, but the role of -synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates -synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates -synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished -synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates -synuclein. Moreover, SIAH co-immunoprecipitated with -synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of -synuclein by SIAH. Monoubiquitylation increased the aggregation of -synuclein in vitro. At the electron microscopy level, monoubiquitylated -synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased -synuclein aggregation in vivo as observed by increased formation of -synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for -synuclein aggregation and Lewy body formation.

Received for publication, June 11, 2007 , and in revised form, November 29, 2007.

^* This work was supported by Israel Academy of Sciences, Ministry of Health, The B. Rappaport Foundation, Brauda Foundation Parkinson Disease Research Fund, and the Technion Research funds (to S. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Bat-Galim, Haifa 31096, Israel. Tel.: 972-4-829-5416; Fax: 972-4-829-5419; E-mail: simone{at}tx.technion.ac.il.

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