Poly (ADP-ribose) polymerase (PARP) is catalytically active only when bound to DNA strand breaks or ends, as induced by either chemical or ionizing radiation. The enzyme catalyzes the covalent attachment of poly (ADP-ribose) chains, derived from NAD (and hense ATP, to a variety of nuclear proteins. Large amounts of ATP can thus be consumed by cells to support poly (ADP-ribosylation) reactions in response to substantial DNA damage or degradation. The enzyme activities of certain PARP substrate proteins, such as endonucleases and topoisomerases, are inhibited in the poly (adp-ribosylated) state. A cysteine protease that cleaves PARP during apoptosis has been identified. The human enzyme, which we have recently purified and cloned, and termed """"""""apopain"""""""", is itself generated early in apoptosis from a precursor, CPP-32, that is related to the C. Elegans protein CED-3 and to ICE. Apopain cleaves PARP in the DNA binding domain (Asp 216-Gly 217), thereby inactivating the enzyme. We have developed a potent peptide aldehyde inhibitor of apopain which also inhibits apoptosis. The major objective of this proposal is to determine why PARP is inactivated early in apoptosis. A testable hypothesis is that PARP is inactivated to prevent poly(ADP-ribosyl)action-unduced inhibition of nuclear enzymes required for the DNA degradation that occurs during apoptosis, and also to spare futile degradation of NAD and ATP.
Specific Aim I is to characterize the regulation of endonucleases and topoisomerases I and II by poly (ADP-ribosy)lation at the molecular and cellular levels as well as their roles in apoptosis.
This aim will be accomplished using the Merck Gene Index and requires the cloning of a Ca2+Mg2+ -dependent endonuclease that is modified by PARP, and the generation of antibodies to, and inhibitors of the various target enzymes.
Specific Aim II is to develop and characterize apoptotic events involving NAD/ATP in cells stably transfected with constructs encoding either (I) PARP mutants that are not susceptible to cleavage by apopain or (I) apopain antisense RNA. Also to elucidate the effects of these conditions upon other apopain-clevage proteins. Accordingly, osteosarcoma cells manipulated to express PARP mutants that are resistant to apopain cleveage would constitute a feasible and potentially informative experimental model with which to test various hypotheses concerning the biological rationale for PARP's destruction by apopain and the roles for ATP and NAD during apoptosis.
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