The Ubiquitin-conjugating Enzyme (E2) Ube2w Ubiquitinates the N Terminus of Substrates
| Autor: | John R. Konen, Kojo S.J. Elenitoba-Johnson, Henry L. Paulson, Kenneth Matthew Scaglione, Venkatesha Basrur, Sokol V. Todi, Naila S. Ashraf |
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| Rok vydání: | 2013 |
| Předmět: |
Molecular Sequence Data
Lysine Nerve Tissue Proteins tau Proteins Plasma protein binding Ubiquitin-conjugating enzyme Biochemistry Mass Spectrometry Ubiquitin Catalytic Domain Humans Protein Interaction Domains and Motifs Amino Acid Sequence Ataxin-3 Molecular Biology Peptide sequence chemistry.chemical_classification Isopeptide bond Sequence Homology Amino Acid biology Nuclear Proteins Active site Cell Biology Ubiquitin ligase Repressor Proteins chemistry Ubiquitin-Conjugating Enzymes biology.protein Peptides Protein Processing Post-Translational Reports Chromatography Liquid Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 288:18784-18788 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.c113.477596 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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