Monoubiquitylation of alpha-synuclein by seven in absentia homolog (SIAH) promotes its aggregation in dopaminergic cells.
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.
Rott
Ruth
R
Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
Szargel
Raymonde
R
Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
Haskin
Joseph
J
Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
Shani
Vered
V
Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
Shainskaya
Alla
A
Biological Mass Spectrometry Facility, Department of Biological Services, The Weizmann Institute of Science, 76100 Rehovot.
Manov
Irena
I
Pediatric Research and Electron Microscopy Unit, The B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Liani
Esti
E
Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
Avraham
Eyal
E
Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096.
Engelender
Simone
S
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.
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
D009687:Nuclear Proteins; D025801:Ubiquitin; D051844:alpha-Synuclein; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D008239:Lysine; D004298:Dopamine