Function and Regulation of the BCL-2 Family

项目摘要 凋亡的线粒体途径对癌症生物学的影响是广泛的，因为BCL-2 （B细胞CLL/淋巴瘤2）家族调节肿瘤的发生和维持，并被抗- 癌症治疗因此，对BCL-2家族功能的机制性理解将推进我们的知识 导致癌症的途径，并在临床上有针对性地治愈癌症。线粒体途径 当BCL-2家族协同破坏线粒体外膜时， （OMM）。这一过程被称为线粒体外膜透化（MOMP），允许促细胞增殖。 线粒体内的凋亡因子进入细胞质，这导致半胱天冬酶激活， 快速分解和去除靶细胞。BAX（BCL-2相关X蛋白）是主要的促凋亡蛋白。 BCL-2蛋白通过在OMM中产生蛋白脂质孔来接合MOMP。BAX依赖性MOMP抑制 另一方面，BAX依赖性细胞凋亡是由大多数常规和 靶向化疗药物。为了使BAX获得促凋亡功能，它有两个一般要求： (1)BAX需要与促凋亡BCL-2家族的一个亚群相互作用：“直接激活剂”BH 3-only 蛋白质，例如，BID和BIM;和（2）BAX需要与线粒体脂质的稳定相互作用， 重排导致BAX的插入、寡聚化和孔形成。虽然数十年的研究 由于专注于理解第一个要求，很少有人发现线粒体环境如何 机械地有助于MOMP。多年来，我的实验室显示线粒体环境 直接控制BAX的功能，而脑内产生的16碳鞘脂代谢产物2-反式- 十六烯醛（2-t-hex）是BAX激活和BAX依赖性凋亡所必需的。通过利用小说 线粒体模型系统加上最先进的生化，细胞和结构技术，我们 现在准备确定2-t-hex在BAX激活过程中的机制贡献， 更广泛地，揭示2-t-hex结合如何定义BCL-2家族内的功能分类。 此外，我们产生的证据表明，癌细胞特异性地破坏BAX和2- t-hex导致抗凋亡-并确定了“一流”的小分子来克服这一点 表型我们的广泛目标是建立一个基础的新机制的见解的作用，2-t-hex BAX依赖性细胞凋亡和BCL-2家族，并利用这些信息开发新的治疗方法， 抗癌策略这些目标将通过三个相辅相成的目标来实现：（1）确定 2-t-hex介导BAX激活的分子机制;（2）阐明S- BAX的亚硝基化促进癌症中的细胞凋亡抗性;和（3）鉴定、改进和表征“首次在 将S-亚硝基化的BAXCys 62从耐药的转化为有能力的， 促进化疗成功。

PROJECT SUMMARY The impact of the mitochondrial pathway of apoptosis on cancer biology is broad because the BCL-2 (B-cell CLL/Lymphoma 2) family regulates tumor initiation and maintenance, and is directly targeted by anti- cancer therapies. Therefore, a mechanistic understanding of BCL-2 family function will advance our knowledge of the pathways that cause cancer, and are clinically targeted to cure cancer. The mitochondrial pathway of apoptosis proceeds when the BCL-2 family collaborates to compromise the outer mitochondrial membrane (OMM). This process, referred to as mitochondrial outer membrane permeabilization (MOMP), allows for pro- apoptotic factors within mitochondria to gain access to the cytoplasm, which leads to caspase activation and rapid dismantling and removal of the targeted cell. BAX (BCL-2 associated X protein) is the major pro-apoptotic BCL-2 protein that engages MOMP by creating proteolipid pores in the OMM. BAX-dependent MOMP inhibits tumorigenesis, and on the flipside, BAX-dependent apoptosis is induced by a majority of conventional and targeted chemotherapeutics. In order for BAX to gain pro-apoptotic function, it has two general requirements: (1) BAX needs to interact with a subset of the pro-apoptotic BCL-2 family: the “direct activator” BH3-only proteins, e.g., BID and BIM; and (2) BAX requires stable interactions with mitochondrial lipids to structurally rearrange leading to BAX’s insertion, oligomerization, and pore formation. While decades of research have focused on understanding the first requirement, little has been discovered on how mitochondrial environment mechanistically contributes to MOMP. Over the years, my laboratory showed that mitochondrial environment directly controls BAX function, and the mitochondrial-produced 16-carbon sphingolipid metabolite 2-trans- hexadecenal (2-t-hex) is required for BAX activation and BAX-dependent apoptosis. By utilizing novel mitochondrial model systems coupled with state-of-the-art biochemical, cellular, and structural techniques, we are now ready to determine the mechanistic contributions of 2-t-hex within the BAX activation process, and more broadly, to reveal how 2-t-hex binding defines functional classifications within the BCL-2 family. Furthermore, we generated evidence that cancer cells specifically disrupt the cooperation between BAX and 2- t-hex leading to apoptotic resistance – and identified ‘first-in-class’ small molecules to overcome this phenotype. Our broad objectives are to build a foundation of novel mechanistic insights into the role of 2-t-hex on BAX-dependent apoptosis and the BCL-2 family, and to use this information to develop novel therapeutic strategies against cancer. These objectives will be accomplished in three complementary aims: (1) Define the molecular mechanism of 2-t-hex mediated BAX activation; (2) Elucidate the molecular mechanism by which S- nitrosylation of BAX promotes apoptotic resistance in cancer; and (3) Identify, refine, and characterize “first-in- class” therapeutics that transform S-nitrosylated BAXCys62 from apoptosis-resistant to apoptosis-competent thus promoting chemotherapeutic success.