title: Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator.
abstract: Inhibitors of Apoptosis Protein (IAPs) are guardian ubiquitin ligases that keep classic proapoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5,000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2-conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates, allowing them to be efficiently purified for LC-MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Zhuang
authors.forename: Shenheng
authors.initials: S
authors.lastname: Guan
authors.forename: Haopeng
authors.initials: H
authors.lastname: Wang
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: James A
authors.initials: JA
authors.lastname: Wells
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D053179:Caspase 7 / Q000378:metabolism; D016384:Consensus Sequence; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D019169:Jurkat Cells; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D000075687:NEDD8 Protein; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; D051034:Inhibitor of Apoptosis Proteins; D024101:Mitochondrial Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D044767:Ubiquitin-Protein Ligases; C529563:PGAM5 protein, human; D010749:Phosphoprotein Phosphatases; C505520:CASP7 protein, human; D053179:Caspase 7