Understanding the Structural Mechanism of Puma-induced Apoptosis

描述(由申请人提供)：这项提案涉及BH3-Only蛋白PUMA在细胞凋亡调控中的作用。PUMA在基因毒性应激下被P53转录激活，是P53依赖的细胞凋亡的主要介导者。因此，对PUMA促凋亡功能的分子基础的研究是人类主要肿瘤抑制途径的核心。在圣裘德，我们与Doug Green的实验室和Gerry Zambetti的实验室合作，前者发现了PUMABCL-xLp53BAX凋亡调控系统，后者发现PUMA是一种由p53诱导的促凋亡蛋白。我们的初步研究阐明了PUMA诱导细胞凋亡的机制。我们的结果表明，PUMA本身不足以直接促进线粒体外膜通透性(MOMP)和细胞凋亡。相反，PUMA只是一种BH3去抑制蛋白，因此不能直接启动BAX和/或BAK的激活。相反，PUMA通过取代bcl-xl中的直接激活蛋白(即Bid、BIM和P53)间接发挥作用。重要的是，这些结果表明，PUMA和bclxl之间的相互作用是PUMA诱导MOMP和细胞凋亡机制的核心。除了作为转录因子的核作用外，P53还通过与bcl2蛋白家族的相互作用来调节细胞质中的细胞凋亡，作为细胞凋亡的“直接激活物”。缺省情况下，胞质中的P53被bclxl隔离在非活性复合体中。然而，当PUMA表达时，它与bclxl结合，并通过一种独特的、以前未知的机制释放隔离的p53。释放的P53，反过来，直接与Bax相互作用，触发一系列涉及Bax寡聚、MOMP、细胞色素c的释放，最终导致细胞凋亡。重要的是，PUMA是唯一的BH3-唯一的去抑制蛋白，可以从bclxl中释放P53，释放P53‘S的全部凋亡潜能。我们使用生化、生物物理和结构方法以及功能分析来发现PUMA从bclxl中独特地释放p53的机制。在拟议的研究中，我们的目标是阐述和完善我们对这一关键的肿瘤抑制机制的初步理解。具体地说，我们的目标是：1)阐明PUMA诱导bCL-xL结构重排的分子细节，并充分验证这一机制在P53介导的细胞凋亡中的生物学作用；2)阐明P53与bCL-xL相互作用的物理基础，以及PUMA与bCL-xL结合如何破坏这些相互作用。通过拟议的研究获得的知识将确定PUMA、bclxl和p53介导的凋亡信号的特异性。公共卫生相关性：了解p53在抑制肿瘤中的作用对于成功治疗癌症患者至关重要。P53的非转录促凋亡作用现已被确定为其肿瘤抑制功能的重要组成部分，以响应致癌和遗传毒性过程。我们对PUMA的研究将是理解PUMA机制的关键(S)，通过该机制，P53的转录和非转录凋亡基因作用共同预防和维持人类的无肿瘤状态。

DESCRIPTION (provided by applicant): This proposal addresses the role of the BH3-only protein, PUMA, in the regulation of apoptosis. PUMA is transcriptionally activated by p53 in response to genotoxic stress and is the primary mediator of p53-dependent apoptosis. Thus, the proposed studies into molecular basis of PUMA's pro-apoptotic function are central to the major tumor suppressor pathway in humans. At St. Jude, we collaborate with Doug Green's lab, which discovered the PUMABCL-xLp53BAX apoptosis regulatory system, and Gerry Zambetti's lab, which discovered PUMA as a p53-induced, pro-apoptotic protein. Our preliminary studies have clarified the mechanism of PUMA-induced apoptosis. Our results show that PUMA alone is insufficient to directly promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Instead, PUMA is a "de-repressor" BH3-only protein and as such cannot directly initiate BAX and/or BAK activation. Instead, PUMA acts indirectly by displacing direct activator proteins (i.e., BID, BIM, and p53) from BCL-xL. Importantly, these results demonstrate that interactions between PUMA and BCL-xL are central to the mechanism of PUMA-induced MOMP and apoptosis. Apart from its nuclear role as a transcription factor, p53 regulates apoptosis in the cytoplasm through interactions with the BCL-2 family of proteins, serving as a "direct activator" of apoptosis. By default, cytosolic p53 is sequestered in inactive complexes by BCL-xL. However, when expressed, PUMA binds BCL-xL and, through a unique and previously unknown mechanism, releases sequestered p53. Released p53, in turn, directly interacts with BAX, triggering a cascade involving BAX oligomerization, MOMP, release of cytochrome c, and, ultimately, apoptosis. Importantly, PUMA is the only de-repressor BH3-only protein that releases p53 from BCL-xL, unleashing p53's full apoptotic potential. We used biochemical, biophysical and structural methods, as well as functional assays, to discover the mechanism by which PUMA uniquely releases p53 from BCL-xL. In the proposed studies, we aim to elaborate and refine our preliminary understanding of this critical tumor suppressor mechanism. Specifically, our aims are: 1) To elucidate the molecular details of PUMA-induced structural rearrangement of BCL-xL and to fully validate the biological role of this mechanism in p53-mediated apoptosis, and 2) To elucidate the physical basis for interactions between p53 and BCL-xL and how these interactions are disrupted by PUMA binding to BCL-xL. The knowledge gained through the proposed studies will define the specificity of apoptotic signaling mediated by PUMA, BCL-xL and p53. PUBLIC HEALTH RELEVANCE: Understanding the roles of p53 in suppressing tumors is essential for successfully treating cancer patients. The non-transcriptional apoptogenic role of p53 has now been established as an essential component of its tumor suppressor function in response to oncogenic and genotoxic processes. Our proposed studies of PUMA will be key in understanding the mechanism(s) through which both the transcriptional and non-transcriptional apoptogenic roles of p53 collaborate in preventing and maintaining a tumor-free state in humans.