Reconstitution of caspase-mediated cell-death signalling in Schizosaccharomyces pombe.
Two pro-apoptotic proteases, caspase-1 and caspase-3, have been expressed as full-length proteins in the fission yeast Schizosaccharomyces pombe. Both proteins autoprocess to generate the corresponding active enzyme and both are lethal to the yeast cell. Lethality is due to catalytic activity since the expression of the inactive mutant forms of both caspases does not result in an obvious phenotype. Caspase-expressing yeast can be rescued by co-expression of the baculovirus protein p35, a known inhibitor of the caspase family. Co-expression of Bcl-2, another anti-apoptotic protein, does not prevent the cell death induced by either caspase. However, Bcl-2 is itself cleaved by both caspase-1 and caspase-3 at two adjacent recognition sites, YEWD(31')A and DAGD(34')V respectively, immediately downstream from the N-terminal BH4 domain, a region of Bcl-2 which is essential for its anti-apoptotic activity; similar cleavage of Bcl-2 by caspases has been demonstrated in mammalian cells. Hence, key elements of the apoptotic pathway can be reliably reconstituted in fission yeast, opening the way to exploit yeast in order to study the control of apoptosis. Furthermore, the activity of caspase-3, although not caspase-1, can be demonstrated in vitro using chromogenic substrates. This offers the possibility of using caspase-producing strains of yeast to screen for chemical inhibitors either in vivo or in vitro.
Ryser
S
S
Ares-Serono, 14 ch. des Aulx, 1228 Plan-Les-Ouates, CH-1211 Geneva, Switzerland. ryser0@cmu.unige.ch
Vial
E
E
Magnenat
E
E
Schlegel
W
W
Maundrell
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017209:Apoptosis; D001665:Binding Sites; D020170:Caspase 1 / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005804:Genes, Lethal / Q000235:genetics; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010641:Phenotype; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015398:Signal Transduction; D013997:Time Factors; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D061945:Caspase Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D010446:Peptide Fragments; D019253:Proto-Oncogene Proteins c-bcl-2; D014764:Viral Proteins; C086430:inhibitor of apoptosis, Nucleopolyhedrovirus; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; D003545:Cysteine