We have made a preliminary observation that caspase-3 present in mitochondria from Jurkat cells, and that within ten minutes that Fas ligation, it is processed to a p29 form lacking the pro-domain. Over- expression of Bcl-2 prevents the appearance of caspase-3 in mitochondria and blocks Fas-mediated apoptosis in stably transfected CEM cells. Taken together, these findings suggest that caspase activation may occur within mitochondrial inter-membrane space, rather than in the cytosol. Furthermore, it suggests that signals from plasma membrane Fas are transmitted rapidly to mitochondria to stimulate caspase processing. By preparing mitochondria with fully intact outer membranes and through the use of differential sedimentation methods to isolate highly purified fractions of outer mitochondrial membranes, inter-membrane space components, and inner membrane/matrix components, as well as cytosol, we can test the hypothesis that pro-caspase-3 is processed initially in the mitochondria, and we can examine the role of caspase-8 and -9, Apaf-1, Bcl-2, and Bax in this process. We propose the following specific aims: 1) evaluate caspase-3 import into mitochondria using in vitro transcribed/translated caspase-3 and intact mitochondria from control and Fas-ligated cells; 2) Examine intramitochondrial processing of caspase-3. Delineation of these events is essential to understanding the fundamental mechanisms of apoptosis, which may lead to identification of new therapeutic targets for regulating apoptosis. The heart is an organ that is extremely rich in mitochondria and dependent upon their function; apoptosis has been shown to occur in heart failure as well as in ischemia/reperfusion and other myocardial disease processes; therefore our third specific aim will be to: 3) Evaluate cardiomyocytes for parallel events induced by simulated ischemia and reperfusion. Understanding these fundamental processes in the comparatively simple and easily manipulated system of Jurkat cells and Fas-mediated apoptosis can be readily translated to studies in the heart.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL061518-01
Application #
2737636
Study Section
Special Emphasis Panel (ZHL1-CSR-F (S1))
Project Start
1999-01-01
Project End
2003-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Granville, David J; Tashakkor, Babak; Takeuchi, Cindy et al. (2004) Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors. Proc Natl Acad Sci U S A 101:1321-6
Gottlieb, Roberta A (2003) Mitochondrial signaling in apoptosis: mitochondrial daggers to the breaking heart. Basic Res Cardiol 98:242-9
Gottlieb, Roberta A (2003) Cytochrome P450: major player in reperfusion injury. Arch Biochem Biophys 420:262-7
Granville, David J; Gottlieb, Roberta A (2003) The mitochondrial voltage-dependent anion channel (VDAC) as a therapeutic target for initiating cell death. Curr Med Chem 10:1527-33
Sayen, M R; Gustafsson, Asa B; Sussman, Mark A et al. (2003) Calcineurin transgenic mice have mitochondrial dysfunction and elevated superoxide production. Am J Physiol Cell Physiol 284:C562-70
Williams, Scott D; Gottlieb, Roberta A (2002) Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective. Biochem J 362:23-32
Gottlieb, R A (2001) Mitochondria and apoptosis. Biol Signals Recept 10:147-61
He, H; Chen, M; Scheffler, N K et al. (2001) Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection. Circ Res 89:461-7
Gottlieb, R A (2000) Role of mitochondria in apoptosis. Crit Rev Eukaryot Gene Expr 10:231-9
Mutomba, M C; Yuan, H; Konyavko, M et al. (2000) Regulation of the activity of caspases by L-carnitine and palmitoylcarnitine. FEBS Lett 478:19-25

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