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Biochem. J. (2002) 361 (587–595) (Printed in Great Britain)

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

James A. MAHONEY*¹, Joseph A. ODIN*, Sarah M. WHITE*, David SHAFFER†‡, Andrew KOFF†, Livia CASCIOLA-ROSEN§ and Antony ROSEN*

*Department of Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, U.S.A., †Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, U.S.A., ‡Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, U.S.A., §Department of Dermatology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, U.S.A., and Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, U.S.A.

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)×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.

Key words: autoantigen, granule pathway, proteasome, U box, ubiquitin.

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.

¹To whom correspondence should be addressed (e-mail jmahoney@jhmi.edu).

Received 4 September 2001/20 October 2001; accepted 13 November 2001

The Biochemical Society, London © 2002