The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N terminus of substrates. J Biol Chem 2013 Jun 28;288(26):18784-8
Date
05/23/2013Pubmed ID
23696636Pubmed Central ID
PMC3696654DOI
10.1074/jbc.C113.477596Scopus ID
2-s2.0-84879597523 (requires institutional sign-in at Scopus site) 90 CitationsAbstract
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.
Author List
Scaglione KM, Basrur V, Ashraf NS, Konen JR, Elenitoba-Johnson KS, Todi SV, Paulson HLMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAtaxin-3
Catalytic Domain
Chromatography, Liquid
Humans
Lysine
Mass Spectrometry
Molecular Sequence Data
Nerve Tissue Proteins
Nuclear Proteins
Peptides
Protein Binding
Protein Interaction Domains and Motifs
Protein Processing, Post-Translational
Repressor Proteins
Sequence Homology, Amino Acid
Ubiquitin
Ubiquitin-Conjugating Enzymes
tau Proteins
