SARCOPENIA AND APOPTOSIS: ROLE OF SERCA AND BCL-2

骨骼肌的生物老化与细胞凋亡水平的逐渐增加有关， 导致肌肉减少症的发展。这些凋亡机制似乎特别涉及 在ER和/或SR水平启动和执行的途径。 Bcl-2定位于ER，并通过轻微的（约100 μ g/ml）抑制细胞凋亡。ER钙水平降低20-30%。 然而，这一过程的实际机制才刚刚开始被发现。一个可能的途径， ER钙的降低是对肌醇-1，4，5-磷酸受体（IP 3R）的Bcl-2依赖性刺激。 第二种可能的机制是降低ER钙水平，涉及Bcl-2依赖性部分凋亡。 肌浆网/内质网Ca-ATP酶（SERCA）失活。 本提案的长期目标是界定第二种机制的各个步骤，即： 抗凋亡蛋白Bcl-2对SERCA的调节作用，并比较SERCA/Bcl-2相互作用与 Bcl-2的表达这些机制将在四个具体目标中进行研究，这些目标将（1）表征 Bcl-2和SERCA之间的复合物在体内和体外使用电子显微镜，光亲和标记， 衰减全反射-傅里叶变换红外光谱和磷光光谱， 衰老和Bcl-2磷酸化对Bcl-2/SERCA复合物形成的影响及其生物学意义 使用细胞内钙的时间分辨测量的Bcl-2/SERCA相互作用，（2）表征 促凋亡蛋白巴克和Bad对SERCA/Bcl-2相互作用的影响 在体内和细胞培养中对Hsp 70与SERCA的共定位进行显微镜和功能研究， Bcl-2和Bcl-2对SERCA的功能作用以及Hsp 70对Bcl-2与 SERCA，以及用于Bcl-2的组成的化学计量分析的质谱实验。 2/SERCA复合物，以及（4）在体外表征纯化SR中Bcl-2和 在热休克蛋白70、钙网蛋白和受磷蛋白存在下的SERCA，并定量化学计量蛋白质蛋白质 互动

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.