The release of cytochrome c from mitochondria is often recognized as the commitment step of apoptosis. The mitochondrial apoptosis-induced channel (MAC) forms early in apoptosis in hematopoietic cells, concurrent with Bax translocation into mitochondria. Our data show MAC could provide a pathway for cytochrome c to exit mitochondria and over-expression of anti-apoptotic Bcl-2 suppresses this channel activity. There is a gap in knowledge defining the relationship between BCL-2 family proteins and MAC, e.g. are Bak and Bax components of MAC? The hypothesis of this proposal is that MAC provides a pathway for cytochrome c, and perhaps other proteins in the mitochondrial intermembrane space, to exit the mitochondria during apoptosis and MAC is a critical target for regulation of the mitochondrial apoptotic response by members of the BCL-2 family of proteins. This proposal is aimed at testing this hypothesis using a combination of electrophysiological, molecular biological and microscopic techniques.
Aim 1 is to systematically characterize the channel properties of MAC and its permeability to cytochrome c, thereby testing whether MAC can permit the release of cytochrome c from mitochondria early in apoptosis. We plan to determine if cytochrome c transports through MAC using electrophysiology and ELISA.
Aim 2 is to determine if pro-apoptotic Bax and Bak are integral, structural components of MAC, and will be tested, in part, by assessing MAC activity in mitochondria of cells that are knockouts for Bax and/or Bak, which will also determine if there is more than one kind of MAC activity.
Aim 3 is to determine the mechanism(s) by which pro-apoptotic BH3-only and anti-apoptotic Bcl-2/Bcl-xL proteins regulate MAC formation. We hypothesize that pro- and anti-apoptotic proteins act by antagonistically regulating formation of MAC. We plan to determine if BH3-only proteins promote the formation of MAC and if anti-apoptotic BCL2 family members can inhibit the formation or activity of MAC. One approach will be to assess the development of MAC activity while patch-clamping mitochondria in the presence of Bcl-2/Bcl-xL and/or representative BH3-only proteins. These experiments will provide fundamental insights into the nature of mitochondrial involvement in apoptosis and the regulation by BCL-2 family proteins. These experiments may identify novel therapeutic targets, e.g., MAC, to modulate apoptosis in cancer and other pathologies, e.g., myocardial infarction.
| Peixoto, Pablo M; Dejean, Laurent M; Kinnally, Kathleen W (2012) The therapeutic potential of mitochondrial channels in cancer, ischemia-reperfusion injury, and neurodegeneration. Mitochondrion 12:14-23 |
| Kinnally, Kathleen W; Peixoto, Pablo M; Ryu, Shin-Young et al. (2011) Is mPTP the gatekeeper for necrosis, apoptosis, or both? Biochim Biophys Acta 1813:616-22 |
| Ryu, Shin-Young; Beutner, Gisela; Kinnally, Kathleen W et al. (2011) Single channel characterization of the mitochondrial ryanodine receptor in heart mitoplasts. J Biol Chem 286:21324-9 |
| Peixoto, Pablo M; Lue, Jennifer K; Ryu, Shin-Young et al. (2011) Mitochondrial apoptosis-induced channel (MAC) function triggers a Bax/Bak-dependent bystander effect. Am J Pathol 178:48-54 |
| Ryu, Shin-Young; Peixoto, Pablo M; Teijido, Oscar et al. (2010) Role of mitochondrial ion channels in cell death. Biofactors 36:255-63 |
| Peixoto, Pablo M; Ryu, Shin-Young; Kinnally, Kathleen W (2010) Mitochondrial ion channels as therapeutic targets. FEBS Lett 584:2142-52 |
| Ryu, Shin-Young; Beutner, Gisela; Dirksen, Robert T et al. (2010) Mitochondrial ryanodine receptors and other mitochondrial Ca2+ permeable channels. FEBS Lett 584:1948-55 |
| Ryu, Shin-Young; Peixoto, Pablo M; Won, Jong Hak et al. (2010) Extracellular ATP and P2Y2 receptors mediate intercellular Ca(2+) waves induced by mechanical stimulation in submandibular gland cells: Role of mitochondrial regulation of store operated Ca(2+) entry. Cell Calcium 47:65-76 |
| Dejean, Laurent M; Ryu, Shin-Young; Martinez-Caballero, Sonia et al. (2010) MAC and Bcl-2 family proteins conspire in a deadly plot. Biochim Biophys Acta 1797:1231-8 |
| Peixoto, Pablo M; Ryu, Shin-Young; Pruzansky, Dawn Pietkiewicz et al. (2009) Mitochondrial apoptosis is amplified through gap junctions. Biochem Biophys Res Commun 390:38-43 |
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