title: The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis.
abstract: In the present study, we demonstrate that a human homologue of Ufd2p (a yeast protein that catalyses the formation of long polyubiquitin chains, and is implicated in responses to environmental stress), UFD2 (ubiquitin fusion degradation protein-2), is cleaved during apoptosis induced by multiple stimuli, including UVB irradiation, Fas ligation, staurosporine treatment and cytotoxic lymphocyte granule-induced death. Caspase 6 and granzyme B efficiently cleave UFD2 [k(cat)/K(m)=(4-5) x 10(4) M(-1) x s(-1)] at Asp(123), whereas caspases 3 and 7 cleave UFD2 approx. 10-fold less efficiently immediately upstream at Asp(109). Thus UFD2 is added to the growing list of proteins with closely spaced caspase and granzyme B cleavage sites, suggesting the presence of a previously unrecognized, conserved motif. Both cleavage sites are contained and conserved within a novel 300-amino-acid N-terminal domain present in apparent UFD2 orthologues in mice and zebrafish, but absent in all UFD2 family members in lower eukaryotes. Full-length recombinant UFD2 exhibited ubiquitin-protein ligase ('E3')-like ubiquitination activity in vitro, but this activity was abolished in recombinant UFD2 truncated at the granzyme B/caspase 6 cleavage site. Cleavage of UFD2 by caspases or granzyme B within this putative regulatory N-terminal domain might have important functional consequences within the apoptotic cascade.
authors.affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, USA. jmahoney@jhmi.edu
authors.forename: James A
authors.initials: JA
authors.lastname: Mahoney
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Odin
authors.forename: Sarah M
authors.initials: SM
authors.lastname: White
authors.forename: David
authors.initials: D
authors.lastname: Shaffer
authors.forename: Andrew
authors.initials: A
authors.lastname: Koff
authors.forename: Livia
authors.initials: L
authors.lastname: Casciola-Rosen
authors.forename: Antony
authors.initials: A
authors.lastname: Rosen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017209:Apoptosis; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D015152:Blotting, Northern; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D053804:Granzymes; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D014018:Tissue Distribution; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D014466:Ultraviolet Rays
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C505514:CASP6 protein, human; D053178:Caspase 6; D020169:Caspases; C114683:UFD2 protein, S cerevisiae; D019311:Staurosporine