Apoptotic caspase activation in the yeast Saccharomyces cerevisiae
Abstract
The complex patterns of caspase gene expression in most mammalian cells prohibits a direct assessment of individual caspase function. To circumvent these issues, we expressed human caspase-8[Special characters omitted.] , -10, -3, and -6 in the budding yeast, to investigate the autocatalytic and sequential proteolytic processing of these zymogens. In S. cerevisiae , expression of caspase-8[Special characters omitted.] or caspase-10 from the inducible galactose promoter, pGAL1 , caused these procaspases to be efficiently processed suggesting that either is capable of catalyzing its own activation. Nonetheless, the maturation of caspase-8[Special characters omitted.] or -10 was distinguished by their effects on cell lethality as only caspase-8[Special characters omitted.] was cytotoxic. This suggests that critical death substrates for caspase-10 might not be present in yeast. Expression of wild-type caspase-3 or -6, however, did not induce autoactivation and cytotoxicity. When caspase-10 or -8[Special characters omitted.] was co-expressed with caspase-3, processing of the caspase-3 zymogen was detected. Caspase-8[Special characters omitted.] and -10 appeared to be equally proficient in initiating the maturation of caspase-3 supporting the notion that caspase-3 is a physiological substrate of at least caspase-8. Interestingly, however, neither effected the processing of the caspase-6 zymogen. Caspase-8[Special characters omitted.] and/or -10 may lie upstream of caspase-6 in a proteolytic cascade; however, additional or augmented caspase activity are apparently necessary for procaspase-6 processing. In addition, the lack of processing by activated caspase-3 on procaspase-6 and visa versa in yeast cells suggests additional caspase activities are required, even though the cytotoxic effects of the autoprocessing forms of the caspase-3 and -6, caspase-3-rev and -6-rev, respectively can be effectively blocked by co-expressing the baculovirus pan-caspase inhibitor, p35. Taken together, these data distinguish a sequential mode of caspase activation in vivo , whereby caspase-3 and -6 require initiating protease function(s) for processing, while caspase-8[Special characters omitted.] and caspase-10 lie upstream of caspase-3 in the activation cascade. Furthermore, the cytotoxic consequences of the autocatalytic processing of caspase-8[Special characters omitted.] in the presence of active site mutant caspase-8[Special characters omitted.] C345S was also examined. Differences in the substrate specificity of capase-8[Special characters omitted.] , -10, -3, -3-rev, and -6-rev were evident in distinct morphological alterations of cells expressing various combinations of these cytotoxic proteases. (Abstract shortened by UMI.)
Recommended Citation
Jason Jongho Kang,
"Apoptotic caspase activation in the yeast Saccharomyces cerevisiae"
(January 1, 1999).
ETD Collection for Thomas Jefferson University.
Paper AAI9921397.
http://jdc.jefferson.edu/dissertations/AAI9921397
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