title: The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N terminus of substrates.
abstract: Attachment of ubiquitin to substrate is typically thought to occur via formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and a lysine residue in the substrate. In vitro, Ube2w is nonreactive with free lysine yet readily ubiquitinates substrate. Ube2w also contains novel residues within its active site that are important for its ability to ubiquitinate substrate. To identify the site of modification, we analyzed ubiquitinated substrates by mass spectrometry and found the N-terminal -NH2 group as the site of conjugation. To confirm N-terminal ubiquitination, we generated lysine-less and N-terminally blocked versions of one substrate, the polyglutamine disease protein ataxin-3, and showed that Ube2w can ubiquitinate a lysine-less, but not N-terminally blocked, ataxin-3. This was confirmed with a second substrate, the neurodegenerative disease protein Tau. Finally, we directly sequenced the N terminus of unmodified and ubiquitinated ataxin-3, demonstrating that Ube2w attaches ubiquitin to the N terminus of its substrates. Together these data demonstrate that Ube2w has novel enzymatic properties that direct ubiquitination of the N terminus of substrates.
authors.affiliation: Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109, USA. kscaglio@umich.edu
authors.forename: Kenneth Matthew
authors.initials: KM
authors.lastname: Scaglione
authors.forename: Venkatesha
authors.initials: V
authors.lastname: Basrur
authors.forename: Naila S
authors.initials: NS
authors.lastname: Ashraf
authors.forename: John R
authors.initials: JR
authors.lastname: Konen
authors.forename: Kojo S J
authors.initials: KS
authors.lastname: Elenitoba-Johnson
authors.forename: Sokol V
authors.initials: SV
authors.lastname: Todi
authors.forename: Henry L
authors.initials: HL
authors.lastname: Paulson
mesh_terms: D000595:Amino Acid Sequence; D000067699:Ataxin-3; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry; D009687:Nuclear Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D012097:Repressor Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000737:chemistry*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D016875:tau Proteins / Q000737:chemistry
chemical_list: D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D012097:Repressor Proteins; D025801:Ubiquitin; D016875:tau Proteins; C510626:UBE2W protein, human; D044763:Ubiquitin-Conjugating Enzymes; C092341:ATXN3 protein, human; D000067699:Ataxin-3; D008239:Lysine
keywords: N-terminal Ubiquitination; Neurodegeneration; Post-translational Modification; Ubiquitin; Ubiquitin-conjugating Enzyme (Ubc); Ubiquitination