A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B
FUNCTIONAL RELATIONSHIPS ESTABLISHED FOR KEY MEDIATORS OF APOPTOSIS*
- Nancy A. Thornberry‡§,
- Thomas A. Rano¶,
- Erin P. Peterson‡,
- Dita M. Rasper‖,
- Tracy Timkey¶,
- Margarita Garcia-Calvo‡,
- Vicky M. Houtzager‖,
- Penny A. Nordstrom‖,
- Sophie Roy‖,
- John P. Vaillancourt‖,
- Kevin T. Chapman¶ and
- Donald W. Nicholson‖
- From the Departments of ‡Enzymology and¶Molecular Design and Diversity, Merck Research Laboratories, Rahway, New Jersey 07065 and the ‖Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire, Dorval, Quebec H9R 4P8, Canada
Abstract
There is compelling evidence that members of the caspase (interleukin-1β converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 andCaenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Dept. of Enzymology, Merck Research Laboratories, R80W-250, P. O. Box 2000, Rahway, NJ 07065. E-mail: Nancy_Thornberry{at}merck.com.
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↵1 The abbreviations used are: ICE, interleukin-1β converting enzyme; PS-SCL, positional scanning synthetic combinatorial library; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; AMC, aminomethylcoumarin.
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- Received March 6, 1997.
- Revision received May 1, 1997.
