The broad goal of this proposal is to characterize novel protein interactions involving the inositol trisphosphate receptor (InsP3R) Ca2+ release channel. The InsP3R participates in generation of complex Ca2+ signals that regulate many physiological processes. Interactions between the InsP3R and cellular components could provide effective means to couple Ca2+ release to specific targets, or to regulate Ca2+ release properties of the channel. Recently, we identified a novel mechanism that directly links the InsP3R to programmed cell death. We identified BC!-XL, a pro-survival member of the Bcl-2 family of pro- and anti-apoptotic proteins, as an interactor with the InsP3R. Bel-XL binding sensitizes channel gating to extremely low InsP3 concentrations that exist in unstimulated cells, reducing Ca2+ in the endoplasmic reticulum (ER) and enhancing Ca2+ signaling. The interaction profoundly enhances the ability of cells to withstand apoptotic insults, thereby identifying a molecular mechanism that links the ER, InsP3R and Bcl-2 proteins to cell survival in a novel paradigm. Because apoptosis is orchestrated by Bcl-2 proteins in normal physiology and pathophysiology, our identification of a novel physiological target of Bcl-2 proteins may provide new opportunities for interventions in human diseases involving apoptosis, including cancer, neurodegeneration and cardiovascular disease. We propose three aims to characterize the mechanisms and function of the interaction between the InsP3R and Bcl-2 proteins. First, we will define the mechanisms of Bcl-Xt regulation of the channel using a combination of biochemistry, cell imaging, single channel recording of recombinant InsP3R in native ER membranes, and physiological assays. Second, we will determine the structural bases for the interaction, using mutagenesis, peptide competition and biophysical approaches. We will also examine directly the solution structure of the InsP3R/Bcl-2 complex by NMR spectroscopy. Third, we will determine the physiological relevance of the interaction of pro-survival Bcl-2 proteins with the InsP3R. First, we will identify the mechanisms whereby this interaction confers apoptosis resistance, by testing the hypothesis that mitochondrial bioenergetics is a primary sensor. Second, we will determine if disruption of the interaction affects apoptosis resistance in vivo. The results of these studies should provide important insights into the molecular mechanisms that regulate apoptosis, and into the development of novel targets for therapeutic interventions in human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056328-12
Application #
7795101
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Rivera-Rentas, Alberto L
Project Start
1999-02-01
Project End
2011-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
12
Fiscal Year
2010
Total Cost
$392,967
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Payne, Riley; Hoff, Henry; Roskowski, Anne et al. (2017) MICU2 Restricts Spatial Crosstalk between InsP3R and MCU Channels by Regulating Threshold and Gain of MICU1-Mediated Inhibition and Activation of MCU. Cell Rep 21:3141-3154
Vais, Horia; Mallilankaraman, Karthik; Mak, Don-On Daniel et al. (2016) EMRE Is a Matrix Ca(2+) Sensor that Governs Gatekeeping of the Mitochondrial Ca(2+) Uniporter. Cell Rep 14:403-410
Cárdenas, César; Müller, Marioly; McNeal, Andrew et al. (2016) Selective Vulnerability of Cancer Cells by Inhibition of Ca(2+) Transfer from Endoplasmic Reticulum to Mitochondria. Cell Rep 14:2313-24
Yang, Jun; Vais, Horia; Gu, Wenen et al. (2016) Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability. Proc Natl Acad Sci U S A 113:E1953-62
Vais, Horia; Tanis, Jessica E; Müller, Marioly et al. (2015) MCUR1, CCDC90A, Is a Regulator of the Mitochondrial Calcium Uniporter. Cell Metab 22:533-5
Foskett, J Kevin; Philipson, Benjamin (2015) The mitochondrial Ca(2+) uniporter complex. J Mol Cell Cardiol 78:3-8
Foskett, J Kevin; Madesh, Muniswamy (2014) Regulation of the mitochondrial Ca(2+) uniporter by MICU1 and MICU2. Biochem Biophys Res Commun 449:377-83
Joseph, J Donald; Peng, Yi; Mak, Don-On Daniel et al. (2014) General anesthetic isoflurane modulates inositol 1,4,5-trisphosphate receptor calcium channel opening. Anesthesiology 121:528-37
Millholland, Melanie G; Mishra, Satish; Dupont, Christopher D et al. (2013) A host GPCR signaling network required for the cytolysis of infected cells facilitates release of apicomplexan parasites. Cell Host Microbe 13:15-28
Mak, Don-On Daniel; Vais, Horia; Cheung, King-Ho et al. (2013) Nuclear patch-clamp electrophysiology of Ca2+ channels. Cold Spring Harb Protoc 2013:885-91

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