APOPTOSIS REGULATION BY VIRAL AND CELLULAR PROTEINS

病毒和细胞抗凋亡蛋白BCL-2家族表现出三种主要活性：抑制凋亡、致癌协同作用和抑制细胞增殖。抗凋亡蛋白的致癌活性似乎与抗凋亡活性相关。为了阐明这些蛋白质如何抑制细胞凋亡和抑制细胞增殖，我们将测试两个主要的假设。(1)我们将检验哺乳动物抗凋亡蛋白促进细胞存活的机制之一可能涉及抑制由一组BCL-2家族促凋亡蛋白如BIK、BNIP 1和BNIP 3启动和扩增的蛋白酶（caspase）级联反应的假设。我们假设BIK、BNIP 1和BNIP 3在表达这些促凋亡蛋白的细胞质区域中募集并激活参与细胞凋亡起始的半胱天冬酶。此外，我们认为BIK也可能在其自身的蛋白水解加工后放大蛋白酶激活。我们将研究BIK、BNIP 1或BNIP 3是否与各种启动子半胱天冬酶物理结合并激活。由于线粒体功能障碍在细胞凋亡的启动中起着重要作用，我们将确定线粒体促凋亡蛋白BNIP 3对线粒体完整性和线粒体释放凋亡因子的影响。（二）、抗凋亡蛋白的增殖抑制活性与其抗凋亡活性是可分离的。我们假设这些蛋白质调节某些细胞周期调控蛋白的活性。我们将确定E1 B-19 K和EBV-BHRF 1蛋白是否与已知的细胞周期蛋白/CDK复合物的活性相关并拮抗其活性。我们还将尝试通过HeLa细胞提取物的生化分级分离来鉴定与抗凋亡蛋白相关的蛋白激酶。拟议的研究应该阐明E1 B-19 K和相关BCL-2家族蛋白控制细胞生命和死亡周期的机制。

The viral and cellular BCL-2 family of anti-apoptosis proteins exhibit three major activities: suppression of apoptosis, oncogenic cooperation and restraining of cell proliferation. The oncogenic activity of the anti-apoptosis proteins appears to be linked to the anti-apoptosis activity. To elucidate how these proteins suppress apoptosis and restrain cell proliferation, we will test two major hypotheses. (1) We will test the hypothesis that one of mechanisms by which the mammalian anti-apoptosis proteins promote cell survival may involve suppression of a protease (caspase) cascade initiated and amplified by a set of BCL-2 family pro-apoptotic protein such as BIK, BNIP1 and BNIP3. We postulate that BIK, BNIP1 and BNIP3 recruit and activate caspases involved in initiation of apoptosis in cytoplasmic locales where these pro-apoptotic proteins are expressed. Additionally, we suggest that BIK may also amplify protease activation after its own proteolytic processing. We will investigate if BIK, BNIP1 or BNIP3 physically associate with and activate various initiator caspases. Since mitochondrial dysfunction plays an important role in initiation of apoptosis, we will determine the effect of the mitochondrial pro-apoptotic protein BNIP3 on mitochondrial integrity and release of apoptotic factors from mitochondria. (2). The proliferation restraining activity of the anti-apoptosis proteins is separable from their anti-apoptosis activity. We hypothesize that these proteins modulate the activity of certain cell cycle regulatory proteins. We will determine if E1B-19K and EBV-BHRF1 proteins associated with and antagonize the activity of known cyclin/CDK complexes. We will also attempt to identify the protein kinases associated with the anti-apoptosis proteins by biochemical fractionation of HeLa cell extracts. The proposed studies should illuminate the mechanisms by which E1B-19K and related BCL-2 family proteins control cellular life and death cycles.