α-Synuclein and Parkin Contribute to the Assembly of Ubiquitin Lysine 63-linked Multiubiquitin Chains

Ellen W Doss-Pepe,Li Chen,Kiran Madura

doi:10.1074/jbc.m413591200

Abstract

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.

Highlights

The abbreviations used areUbiquitin; E1, Ub-activating enzyme; E2, Ub-carrier/conjugating protein; E3, Ub-protein isopeptide ligase; Ub Cterminal hydrolase (UCH), Ub C-terminal hydrolase; UBC, Ub-conjugating enzymes; UEV, Ub-E2 variant; multi-Ub, multiubiquitin; GST, glutathione Stransferase; PD, Parkinson disease; hDAT, human dopamine transporter; ␣-Syn, ␣-synuclein
The covalent linkage of ubiquitin (Ub)1 to proteins is used by eukaryotic cells to signal different cellular processes
The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes

Summary

The abbreviations used are

Ubiquitin; E1, Ub-activating enzyme; E2, Ub-carrier/conjugating protein; E3, Ub-protein isopeptide ligase; UCH, Ub C-terminal hydrolase; UBC, Ub-conjugating enzymes; UEV, Ub-E2 variant; multi-Ub, multiubiquitin; GST, glutathione Stransferase; PD, Parkinson disease; hDAT, human dopamine transporter; ␣-Syn, ␣-synuclein. The only E2 enzyme that has been reported to assemble UbLys-63 chains is unusual because it is a heterodimer composed of UbcH13 and Uev1a [12, 13] Both subunits resemble E2 enzymes, only UbcH13 is catalytically active in forming a thioester bond with Ub. Ub-E2 variant (UEV) proteins lack the active site cysteine residue, indicating that they perform a different role than that usually performed by E2 enzymes [14, 15]. The turnover of hDAT from the synaptic terminal, following signal transmission, could represent a crucial aspect of dopaminergic signaling in PD We report that both ␣-Syn and Parkin stimulate the formation of Ub-Lys-63 chains. We propose that an altered assembly or recognition of Lys-63 chains could result in errors in the turnover of neuronal cell surface proteins through the endocytic pathway and could underlie an important biochemical defect in PD

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION