Programmed cell death (PCD) in metazoans is mediated by proteases of the caspase family. These proteases are synthesized as proproteins that are processed, often by other caspases, to form the mature protease. Mutations in the C. elegans caspase gene
ced-3 prevent normally occurring PCD, suggesting that this gene plays an essential role in this process. To understand how CED-3 promotes cell death we are interested in defining regulators and targets of CED-3 activity. Using sequences from the C. elegans genome project and standard hybridization experiments we identified three C. elegans caspase-related genes in addition to
ced-3 . These genes encode alternate transcripts, suggesting that worms may contain a repertoire of at least seven caspase-related proteins (CSP-1A, CSP-1B, CSP-1C, CSP-2A, CSP-2B, CSP-3, and CED-3). Since mammalian caspases can act in proteolytic cascades, we examined whether worm caspases can process each other in vitro . We found that active CSP-1B cleaved proCSP-1B, proCED-3 and proCSP-2B, and that active CED-3 cleaved proCED-3 and proCSP-2B. These experiments suggest that CSP-1B may regulate proCED-3 activation and that CSP-2B may be a target of CED-3. To further understand the functions of
csp-1 ,
csp-2 , and
csp-3 we are in the process of generating mutations in these genes. We have also been searching for CED-3 regulators and targets using a novel screen employing the yeast S. cerevisiae . We found that CED-3 expression in yeast results in growth arrest that is dependent on CED-3 protease activity and is blocked by known caspase inhibitors. Thus, we have constructed a C. elegans yeast expression library to look for genes that inhibit CED-3-mediated growth arrest of yeast. Such genes could represent inhibitors of CED-3 activity or processing, or targets of CED-3 activity. We have isolated 15 different cDNAs so far. Further characterization of the respective genes is ongoing.