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The focus of our research is to understand the regulatory mechanism of apoptosis in mammalian systems. Many cells in the body die through a process called programmed cell death, or apoptosis. As an essential cellular process to eliminate unwanted cells in development and homeostasis, apoptosis is executed through a precisely orchestrated biochemical pathway. Dysregulation of apoptotic pathways leads to many human diseases including cancer, autoimmune diseases, and neurodegenerative disorders. At the core of apoptotic programs is a group of proteases, named caspases, which are initially synthesized as inactive proenzymes and become activated by proteolytic cleavage only when cells are committed to die through apoptosis. Once activated, caspases further cleave their downstream targets and generate characteristic morphological changes in apoptotic cells. One important group of negative regulators of caspases is a family of proteins called, Inhibitors of Apoptosis (IAP). IAPs prevent apoptosis induced by diverse stimuli by directly binding to and inhibiting activated caspases. Some members of the IAP family of proteins have been shown to highly express in certain human cancers. Therefore, removing IAP inhibition could be critical for sensitizing cancer cells to anticancer drugs. IAP inhibition of apoptosis can be eliminated by Smac protein, an antagonist of IAPs that sensitizes cells to apoptosis. Mature Smac protein bears a stretch of apoptotic peptide at its N-terminus, which binds to and inhibits IAPs and promotes cell death. We are particularly interested in the molecular mechanisms by which the IAP Bruce/Apollon regulates apoptosis and its function in mouse development. Bruce/Apollon is both an IAP protein and a ubiquitin conjugase. Similar to other IAPs, Bruce inhibits apoptosis by binding to caspases through its BIR domain, and its caspase inhibitory activity also requires the C-terminal UBC domain. We have demonstrated in mice that deletion of the C-terminal half of Bruce, including the UBC domain, causes activation of caspases and apoptosis in placenta and yolk sac, leading to embryonic lethality. This apoptosis is associated with upregulation and nuclear localization of the tumor suppressor p53 and activation of mitochondrial apoptosis, which includes upregulation of Bax, Bak and Pidd, translocation of Bax and caspase-2 onto mitochondria, release of cytochrome c and AIF, and activation of caspase-9 and caspase-3. In addition, eliminating p53 by RNAi rescues cell viability induced by Bruce ablation in the human H460 cell line. This viability results from reduced expression of proapoptotic factors Bax, Bak, and Pidd, and from prevention of activation of caspase-2, -9 and -3. Therefore, we have demonstrated a previously unknown pathway of “Bruce-p53 apoptosis,” in which p53 is a downstream effector that mediates the mitochondrial pathway of apoptosis. Activation of p53 usually indicates genotoxic stress. Unicellular organisms respond to the presence of DNA lesions by activating cell cycle checkpoint and repair mechanisms, while multicellular animals have acquired the further option of eliminating damaged cells by triggering apoptosis. The Bruce-p53 apoptosis pathway that we have demonstrated suggests that Bruce may be involved in maintaining genome stability. The intranuclear mechanisms that signal apoptosis after DNA damage overlap with those that initiate cell cycle arrest and DNA repair, and the early events in these pathways are highly conserved. We are currently investigating the possible roles of Bruce in DNA damage response and apoptosis, and investigating whether Bruce plays a role in tipping the balance between cell death and cell survival mediated by DNA repair and genome stability maintenance. We are also interested in how apical caspase-2 is activated in response to DNA damage. It is known that caspase-2 participates in genotoxic stress-induced apoptosis, but the mechanism remains extremely controversial due to the lack of biochemical mechanisms. In particular, how DNA damage signals are received and transmitted to nuclear caspase-2 is completely unknown. We have identified a novel protein complex by immunoprecipitation and mass spectrometry that activates caspase-2 in response to DNA damage. We will decipher how this protein complex activates caspase-2, and how this complex is regulated by DNA damage signals. This study will add new knowledge to the very limited understating of caspase-2 functions. The goal of our research is to utilize protein biochemistry, molecular biology, and mouse genetics to investigate how Bruce and caspase-2 function in apoptosis, DNA damage response, and mouse development. Our studies will identify signaling molecules and new mechanisms that regulate apoptosis in mammalian systems. Academic Appointment: Assistant Professor, Department of Biochemistry & Molecular Biology, The University of Kansas School of Medicine Selected publications Return to Research Team |
Du Lab