A mitochondria-localized protein, p32, was recently identified by our lab as a binding partner for the tumor suppressor protein p14ARF. Preliminary data indicated that p32 is essential for p14ARF, a critical mediator for transducing hyperproliferative, oncogenic stress signals to the Mdm2-p53 tumor suppression pathway, to localize to mitochondria and to induce p53-dependent apoptosis. Importantly, human cancer-derived p14ARF mutations that disrupt p32 binding can impair both of these functions. Recently, our preliminary studies have shown that p32 is in fact essential for apoptosis induced by a broad range of apoptotic stimuli. The overall hypothesis behind the proposed research is that p32 specifies an essential factor for a surveillance system that monitors the integrity of mitochondrial function and promotes apoptosis in response to irreparable mitochondrial damage. The rationale for the hypothesis is based on the following observations. (1) p32 is a mitochondrial protein. (2) Knockdown p32 desensitizes cells to apoptosis induced by a broad range of apoptotic stimuli. (3) Like cytochrome c, p32 accumulates in the cytoplasm during apoptosis. (4) Ectopically expressed, cytoplasm- localized p32 induces apoptosis. (5) p32 is reported to have a role in oxidative phosphorylation. The experimental focus of the proposal is on dynamics and outcome of p32 localization, function and mechanism of p32 in regulating apoptosis, and genetics and biology of p32 in metabolic regulation and tumorigenesis. Based on these observations, the proposed research will focus on characterizing the function and mechanism of p32 in regulating apoptosis by a combination of biochemical, cellular, and genetic approaches. The specific aims are designed to assess p32's role in regulating apoptosis under a broad range of apoptotic conditions and define the dynamics of p32 subcellular localization, to Investigate mechanisms by which p32 promotes apoptotic cell death, and to investigate p32's physiological function using p32 conditional knockout mice. If successful, the proposed study will ascribe new functions to p32. It will also aid in our understanding of apoptotic cell death - a process that is critical during development and in the pathogenesis of diseases such as cancer, rheumatoid arthritis, and neurodegenerative diseases - and may eventually lead to additional drug targets for controlling apoptosis in treatment of these diseases. PUBLIC HEALTH RELEVANCE: The quest to delineate the apoptosis pathway is highly motivated by the possibility of targeting human cancer or other degenerative diseases through manipulating apoptotic signaling. This proposal is built upon our strong preliminary data identifying the mitochondrial protein p32 to be physically and functionally linked to the p14ARF-Mdm2- p53 tumor suppression pathway. The objective of this proposal is test whether and how p32 signals to p14ARF and p53 and regulates apoptosis. The potential high impact of this proposal rests on the promise of establishing p32 as a novel regulator of apoptotic cell death, the aberrant regulation of which is implicated in the development of many diseases including cancer.