Biochemical Journal

Research article

The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis

James A. MAHONEY, Joseph A. ODIN, Sarah M. WHITE, David SHAFFER, Andrew KOFF, Livia CASCIOLA-ROSEN, Antony ROSEN

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 [kcat/Km = (4–5)×104 M−1·s−1] at Asp123, whereas caspases 3 and 7 cleave UFD2 approx. 10-fold less efficiently immediately upstream at Asp109. 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.

  • autoantigen
  • granule pathway
  • proteasome
  • U box
  • ubiquitin

Footnotes

  • Abbreviations used: DEVD-CHO, acetyl-Asp-Glu-Val-Asp aldehyde; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin—protein ligase; EST, expressed sequence tag; IVTT, in vitro transcription/translation; LAK, lymphokine-activated killer; PARP, poly(ADP-ribose) polymerase; RT, reverse transcriptase; UFD2, ubiquitin fusion degradation protein-2.