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J. Biol. Chem., Vol. 282, Issue 38, 27865-27874, September 21, 2007

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Hip Is a Pro-survival Substrate of Granzyme B^*

Daniel R. Hostetter¹², Carly R. K. Loeb¹³, Feixia Chu^¶4, and Charles S. Craik⁵

From the Departments of Pharmaceutical Chemistry and Biochemistry and Biophysics, Tetrad Graduate Program and the ^¶Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94158-2517

The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates, and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein, Hip. Hsp90, Bag1L, and Hip were validated as GrB substrates in vitro, and mutational analysis confirmed the additional cleavage site in Hip. Because the role of Hip in apoptosis is unknown, its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization; therefore, it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer (NK) cell-mediated death. Hip is cleaved at both GrB cleavage sites during NK-mediated cell death in a caspase-independent manner, and its cleavage is due solely to GrB and not other granule components. Furthermore, Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T-cell line, suggesting that Hip is a substrate unique to GrB. RNA interference-mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis, indicating that proteolysis by GrB of Hip contributes to death induction. The small effect of RNA interference-mediated Hip deficiency on cytotoxicity is in agreement with the inherent redundancy of NK cell-mediated cell death. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contribute to understanding the interplay between stress response and apoptosis.

Received for publication, May 25, 2007 , and in revised form, July 2, 2007.

^* This work was supported by National Institutes of Health Grant NIHCA072006 and in part by National Center for Research Resources Grants 01614, 12961, and 15804 and Cancer Center Support Grant P30 CA82103. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and supplemental Figs. S1–S4.

¹ These authors contributed equally to this work.

² Leukemia & Lymphoma Society Fellow.

³ Supported by a Howard Hughes Medical Institute predoctoral fellowship and the Boyer Fund at University of California San Francisco.

⁴ Rett Syndrome Research Foundation postdoctoral fellow.

⁵ To whom correspondence should be addressed. Fax: 415-502-8298; E-mail: craik{at}cgl.ucsf.edu.

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