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 specificallypathways initiated and executed at the level of the ER and/or SR. Not surprisingly, the anti-apoptotic proteinBcl-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 tothe 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 partialinactivation 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 effectsof Bcl-2 on the IP3R. These mechanisms will be studied in four Specific Aims, which will (1) characterize thecomplex 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, theeffect of aging and Bcl-2 phosphorylation on Bcl-2/SERCA complex formation, and the biologic significanceof the Bcl-2/SERCA interaction using time-resolved measurements of intracellular calcium, (2) characterizethe effects of the pro-apototic proteins Bak and Bad on the SERCA/Bcl-2 interaction, (3) perform electronmicroscopy and functional studies in vivo and in cell culture on the co-localization of Hsp70 with SERCA andBcl-2 and the functional effect of Bcl-2 on SERCA as well as of Hsp70 on the interaction of Bcl-2 withSERCA, 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 andSERCA in the presence of Hsp70, calreticulin and phospholamban, and quantify the stoichiometry proteinproteininteraction.
Showing the most recent 10 out of 171 publications
