CELL-DEATH ATTENUATION BY USURPIN, A MAMMALIAN DED-CASPASE HOMOLOG THAT PRECLUDES CASPASE-8 RECRUITMENT AND ACTIVATION BY THE CD-95 (FAS, APO-1) RECEPTOR COMPLEX

Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occur s through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous m ammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Gasper, CLARP, FLAME-1, FLIP, I-FL ICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpi n gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. T he Usurpin polypeptide has features in common with pro-caspase-8 and - 10, including tandem 'death effector domains' on the N-terminus of a l arge subunit/small subunit caspase-like domain, but it lacks key resid ues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues th at stabilize the P-1 aspartic acid in substrates. Retro-mutation of th ese residues to functional caspase counterparts failed to restore prot eolytic activity, indicating that other determinants also ensure the a bsence of catalytic potential. Usurpin heterodimerized with pro-caspas e-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin defic it was found in cardiac infarcts where TUNEL-positive myocytes and act ive caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 def icit) occurred in surrounding unaffected cardiac tissue, suggesting re ciprocal regulation of these pro-and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensi tivity in mammalian cells, including the susceptibility of cardiac myo cytes to apoptotic death following ischemia/reperfusion injury.