Biological aging of skeletal muscle is associated with progressively increasing levels of apoptosis, contributing to the development of sarcopenia. These apoptotic mechanisms appear to involve specifically pathways initiated and executed at the level of the ER and/or SR. Not surprisingly, the anti-apoptotic protein Bcl-2 localizes to the ER and inhibits apoptosis thorugh a slight (ca. 20-30%) reduction of ER calcium levels. However, the actual mechanisms of this process are just beginning to be uncovered. One possible route to the lowering of ER calcium is a Bcl-2-dependent stimulation of the inositol-1,4,5-phosphate receptor (IP3R). A second potential mechanism affording reduced ER calcium levels involves a Bcl-2-dependent partial inactivation of the sarco/endoplasmic reticulum Ca-ATPase (SERCA). The longterm objective of this proposal is to delineate the individual steps of the second mechanism, i.e. SERCA modulation by the anti-apoptotic protein Bcl-2, and compare the SERCA/Bcl-2 interaction to effects of Bcl-2 on the IP3R. These mechanisms will be studied in four Specific Aims, which will (1) characterize the complex between Bcl-2 and SERCA in vivo and in vitro using electron microscopy, photoaffinity labeling, attenuated total reflectance-Fourier transform IR spectroscopy and phosphorescence spectroscopy, the effect of aging and Bcl-2 phosphorylation on Bcl-2/SERCA complex formation, and the biologic significance of the Bcl-2/SERCA interaction using time-resolved measurements of intracellular calcium, (2) characterize the effects of the pro-apototic proteins Bak and Bad on the SERCA/Bcl-2 interaction, (3) perform electron microscopy and functional studies in vivo and in cell culture on the co-localization of Hsp70 with SERCA and Bcl-2 and the functional effect of Bcl-2 on SERCA as well as of Hsp70 on the interaction of Bcl-2 with SERCA, as well as mass spectrometry experiments for a stoichiometric analysis of the composition of Bcl- 2/SERCA complexes, and (4) characterize in vitro in purified SR the functional interaction of Bcl-2 and SERCA in the presence of Hsp70, calreticulin and phospholamban, and quantify the stoichiometry proteinprotein interaction.
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