Mitochondrial membrane permeabilization (MMP) is the step of commitment to cell death in a broad spectrum of cell injuries. MMP leads to the release of several factors activating the systematic execution of the cells and destroys the mitochondrial capacity to maintain ATP production. MMP is mediated by either pro-apoptotic Bcl-2 family proteins, Bax and Bak or by the permeability transition pore (PTP) that remains to be identified. In recent years, much attention has been focused on Bax that resides largely in the cytoplasm and upon to a variety of stress stimuli, gets inserted and activated in the outer mitochondrial membrane (OMM). By contrast, Bak is constitutively inserted to the OMM in most cell types and can respond to some activators faster than Bax. We and others have recently established that the targeting of Bak to the mitochondria is dependent on the presence of specifically the 2nd isoform of the Voltage Dependent Anion-selective Channel (VDAC2) in the OMM. We have also demonstrated that VDAC2 and the ensuing targeting of Bak to the mitochondria is required for the rapid response to insults that are relayed by Bid to the mitochondria. In our preliminary studies, we have shown that hepatocarcinoma mitochondria are far more sensitive to Bid than normal liver mitochondria and this difference is paralleled by more VDAC2 and Bak being present in the hepatocarcinoma. Our first hypothesis is that the differential sensitivity to Bid-induced cell killing in normal liver and liver tumor results from differential expression of VDAC2 and the ensuing differential recruitment of Bak to the mitochondria, offering a potential target for selective killing of hepatocarcinoma cells. We and others have provided evidence that both (a) Bid-induced mitochondrial membrane permeabilization and (b) IP3 receptor (IP3R)-mediated calcium signaling are effectively sensitized by reactive oxygen species (ROS). We propose that enhanced mitochondrial ROS production locally supports spreading of Bid-induced permeabilization among individual mitochondria and causes local enhancement of IP3R-mediated Ca2+ release and Ca2+ transfer to the mitochondria. The surplus Ca2+ further stimulates ROS production and facilitates opening of the PTP, initiating a vicious cycle that leads to cell death. To test this hypothesis, w have developed a novel molecular toolkit that allows us to directly monitor and perturb [ROS] and [Ca2+] dynamics at the mitochondrial surface or close associations of ER and mitochondria. Completion of this project will not only help to better understand the fundamental control of the mitochondrial membrane integrity but will also shed some light on the presently underappreciated role of Bak in cell killing and might uncover VDAC2 and Bak as candidates for selective targeting of human hepatocarcinomas. Tumors are commonly resistant to cell killing agents, and our work might show an Achilles'heel of hepatocarcinoma.

Public Health Relevance

Mitochondrial membrane permeabilization is central to both physiological and pathophysiological cell death, and is also a potential drug target for death induction in tumors. The proposed work will expose the mechanism of Bak insertion into the mitochondria and its differential regulation and contribution to cell killing in normal and tumor liver. Furthermore, the studies will study a novel local ROS signaling mechanism that takes place at the mitochondrial surface or ER-mitochondrial interface to synergize with Bid and Ca2+ to induce mitochondrial cell death.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Membrane Biology and Protein Processing (MBPP)
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Gindhart, Joseph G
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Thomas Jefferson University
Schools of Medicine
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Naghdi, Shamim; Hajnóczky, György (2016) VDAC2-specific cellular functions and the underlying structure. Biochim Biophys Acta 1863:2503-14
Booth, David M; Enyedi, Balázs; Geiszt, Miklós et al. (2016) Redox Nanodomains Are Induced by and Control Calcium Signaling at the ER-Mitochondrial Interface. Mol Cell 63:240-8
Booth, David M; Joseph, Suresh K; Hajnóczky, György (2016) Subcellular ROS imaging methods: Relevance for the study of calcium signaling. Cell Calcium 60:65-73
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