title: Monoubiquitylation of alpha-synuclein by seven in absentia homolog (SIAH) promotes its aggregation in dopaminergic cells.
abstract: alpha-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of alpha-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. alpha-Synuclein is ubiquitylated in Lewy bodies, but the role of alpha-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates alpha-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates alpha-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 alpha-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates alpha-synuclein. Moreover, SIAH co-immunoprecipitated with alpha-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 alpha-synuclein by SIAH. Monoubiquitylation increased the aggregation of alpha-synuclein in vitro. At the electron microscopy level, monoubiquitylated alpha-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased alpha-synuclein aggregation in vivo as observed by increased formation of alpha-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 alpha-synuclein aggregation and Lewy body formation.
authors: Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
authors: Ruth
authors: R
authors: Rott
authors: Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
authors: Raymonde
authors: R
authors: Szargel
authors: Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
authors: Joseph
authors: J
authors: Haskin
authors: Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
authors: Vered
authors: V
authors: Shani
authors: Biological Mass Spectrometry Facility, Department of Biological Services, The Weizmann Institute of Science, 76100 Rehovot.
authors: Alla
authors: A
authors: Shainskaya
authors: Pediatric Research and Electron Microscopy Unit, The B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
authors: Irena
authors: I
authors: Manov
authors: Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
authors: Esti
authors: E
authors: Liani
authors: Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
authors: Eyal
authors: E
authors: Avraham
authors: Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096. Electronic address: simone@tx.technion.ac.il.
authors: Simone
authors: S
authors: Engelender
mesh_terms: D045744:Cell Line, Tumor; D004298:Dopamine / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D016631:Lewy Bodies / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000502:physiology*; D051844:alpha-Synuclein / Q000737:chemistry* / Q000378:metabolism; D000098563:Seven in Absentia Proteins
chemical_list: D009687:Nuclear Proteins; D025801:Ubiquitin; D051844:alpha-Synuclein; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D008239:Lysine; D004298:Dopamine