Structural, biochemical and genetic analysis of the newly discovered Apaf-1-binding protein CABY

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Inhibition of apoptosis is recognized as a major cause of tumorigenesis, and identification of further apoptosis-regulating proteins as potential targets for molecular therapy has become an important goal. During activation of the mitochondrial apoptosis pathway, the large cytosolic apoptosome complex is assembled, which consists of the oligomerized adaptor protein Apaf-1, Cytochrome c, dATP/ATP and Caspase-9. Apoptosome formation leads to the further recruitment and activation of the effector Caspase-3 and, subsequently, to cell death. This pathway is activated by a plethora of stimuli, including radiation and chemotherapy during cancer treatment. We recently discovered a novel Apaf-1-binding protein, which we named CABY and which sensitizes cells to death-inducing stimuli activating the mitochondrial apoptosis pathway. In the proposed project we plan to carry out a detailed molecular and structural analysis of CABY. For this purpose, we will combine biochemical approaches with NMR spectroscopy to uncover the precise molecular mechanism through which CABY influences activation of the apoptosome complex. Genetic studies using a conditional CABY knockout mouse model will allow us to investigate the physiological function of CABY. Pro-apoptotic proteins may function as tumor suppressor molecules. Our previous results have shown that CABY is downregulated in gastrointestinal stromal tumors (GISTs), and we will perform expression studies to find out whether CABY activity is diminished in other tumor types. Collectively, the experiments will contribute to an improved understanding of apoptosis regulation and the development of new therapeutic approaches for cancer and cancer resistance.