Survivin Regulation of Apoptosis

Univ Of Massachusetts Med Sch Worcester, Worcester MA

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Abstract

DESCRIPTION (provided by applicant): The mechanism(s) of survivin cytoprotection have not been fully elucidated and the controversy stems from the following: (1) survivin lacks a functional "hook" region that in the crystal structure of other IAP's mediates caspase binding, (2) conflicting data has been reported as to the ability of survivin to actually bind and inhibit caspase function in vitro, (3) it is possible that experiments using recombinant survivin may not be valid because the protein is largely unfolded by 2-D NMR, and (4) we have preliminary data "unexpectedly" implicating the mitochondria in survivin cytoprotection. In the present application we seek to further elucidate the cellular pathways and molecular interactions taking place between survivin and members of the apoptosome both in the cytosol and at the mitochondria. Experiments will be carried out to study the role of mitochondria in the phosphorylation mutant, T34A-mediated apoptosis; the loss of viability; the loss of the mitochondrial membrane potential (Dym); the release from the intramitochondrial space to the cytosol of cytochrome C; and the release from the mitochondrial matrix of malate dehydrogenase (MDH). Secondly, using gel filtration chromatography we wish to characterize, in our models, the formation of the apoptosome and the possibility that survivin might physically associate with it, interacting with caspase-3 and/or -9 within the complex. That the T34A survivin mutant complexes with the apoptosome and inhibits this caspase-3 and -9 dependent apoptosis will also be evaluated.