Structural, biochemical and genetic analysis of the newly discovered Apaf-1-binding protein CABY
Zusammenfassung der Projektergebnisse
For further analysis of the FAM96A/APAF1 interaction, we mapped five potential peptide regions within APAF1 which might be involved in the binding to FAM96A. Gel filtration experiments suggested that FAM96A is part of the apoptosome complex formed during mitochondrial apoptosis. We found no evidence that FAM96A competes with the antiapoptotic proteins AVEN and HSP70/90 for their binding to APAF1, and we could not detect endogenous ATPase activity for FAM96A. In general, our in vitro experiments to elucidate the precise molecular mechanism of how FAM96A influences apoptosis by binding to APAF1 were severely hampered by the fact that it was impossible to produce larger quantities of purified tag-free recombinant FAM96A. However, we were able to obtain sufficient recombinant protein to determine the structure of an N-terminally deleted FAM96A using standard multidimensional NMR experiments. The structure consists of a central β-sheet with three β-strands showing both parallel and anti-parallel orientations. In addition, six helices of different length complete the structure. Experiments to determine the structure of FAM96A bound to another binding partner, CIAO-1 (a protein essential for cytosolic iron sulfur cluster assembly), are currently being performed. Unfortunately, EUCOMM was only recently able to provide us with conditional FAM96A knockout mice, and the analysis of the animals is ongoing. We are expecting valuable insights into the physiological function of FAM96A from this mouse model which can simultaneously be used to monitor endogenous FAM96A promoter activity. We further investigated the role of the pro-apoptotic FAM96A protein as a tumor suppressor and evaluated a larger cohort of gastrointestinal stroma tumors (GISTs) by immunohistochemistry (IHC). We confirmed the absence of FAM96A expression in this tumor entity and showed in parallel that the cells-of-origin for GISTs, the interstitial cells of Cajal (ICCs), co-express FAM96A with c-KIT. In collaboration with Dr. Tamas Ordog (Mayo Clinic, Rochester, Minnesota, USA) we demonstrated that murine ICC stem cells lose FAM96A during the process of GIST tumorigenesis. At the same time, re-expression of FAM96A in the human GIST882 cell line increases their apoptosis sensitivity in cell culture and abrogates their tumorigenicity in nude mice, confirming the tumor suppressor function of FAM96A in GISTs. IHC studies using multi tumor arrays indicated a similar downregulation of FAM96A in bladder and renal cell carcinoma. In collaboration with Prof. Arndt Hartmann and Dr. Anja Rogler (Pathology Erlangen) we could show that in a cohort of more than 300 bladder carcinoma relapses following chemotherapy, the overall and tumor-specific survival of the patients inversely correlated with the level of FAM96A expression in the tumors. These results suggest a broader role of FAM96A as a general pro-apoptotic tumor suppressor.
