SARCOPENIA AND APOPTOSIS: ROLE OF SERCA AND BCL-2

骨骼肌的生物衰老与逐渐增加的细胞凋亡水平有关， 促进了骨质疏松症的发展。这些凋亡机制似乎涉及到特定的 在ER和/或SR级别启动和执行的路径。不足为奇的是，抗凋亡蛋白 Bcl2定位于内质网，通过轻微(约20%-30%)降低内质网钙水平来抑制细胞凋亡。 然而，这一过程的实际机制才刚刚开始被揭开。一条可能的路线是 内质网钙的降低是一种依赖于Bcl2的肌醇-1，4，5-磷酸受体(IP3R)的兴奋。 导致内质网钙水平降低的第二个潜在机制涉及Bcl2依赖的部分 肌浆网/内质网Ca-ATPase(SERCA)失活。 这项建议的长期目标是界定第二个机制的各个步骤，即 抗凋亡蛋白Bcl2对SERCA的调节作用，并比较SERCA/Bcl2相互作用的影响 Bcl2在IP3R上的表达。这些机制将在四个具体目标中进行研究，这将(1)表征 体内外Bcl2与SERCA复合体的电子显微镜、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Bcl2磷酸化对Bcl2/SERCA复合体形成的影响及其生物学意义 用细胞内钙的时间分辨测量研究Bcl2/SERCA的相互作用，(2)表征 促细胞凋亡蛋白Bak和Bad对SERCA/Bcl2相互作用的影响(3)执行电子 Hsp70与SERCA和SERCA共定位的显微镜和体内及细胞培养功能研究 BCL-2、Bcl2在SERCA中的作用及Hsp70对Bcl2与SERCA相互作用的影响 SERCA，以及用于化学计量分析的化学计量分析的质谱学实验-- 2/SERCA复合体；(4)体外鉴定纯化SR中Bcl2与SERCA的功能相互作用。 在Hsp70、钙网蛋白和磷蛋白存在下的SERCA中，并对化学计量蛋白进行定量 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.