Understanding the Structural Mechanism of Puma-induced Apoptosis

描述（由申请人提供）：本提案涉及BH3-only蛋白PUMA在细胞凋亡调控中的作用。PUMA在基因毒性应激反应中被p53转录激活，是p53依赖性细胞凋亡的主要介质。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通路的核心。在圣裘德，我们与Doug Green的实验室合作，他们发现了PUMABCL-xLp53BAX细胞凋亡调节系统，以及Gerry Zambetti的实验室合作，他们发现PUMA是一种p53诱导的促凋亡蛋白。我们的初步研究已经阐明了puma诱导细胞凋亡的机制。我们的研究结果表明，单独的PUMA不足以直接促进线粒体外膜通透性（MOMP）和细胞凋亡。相反，PUMA是一种仅限bh3的“去抑制”蛋白，因此不能直接启动BAX和/或BAK激活。相反，PUMA通过取代BCL-xL中的直接激活蛋白（即BID、BIM和p53）间接起作用。重要的是，这些结果表明PUMA和BCL-xL之间的相互作用是PUMA诱导的MOMP和细胞凋亡机制的核心。p53在细胞核中除了作为转录因子外，还通过与BCL-2蛋白家族的相互作用调控细胞质中的凋亡，是细胞凋亡的“直接激活因子”。默认情况下，细胞质p53被BCL-xL隔离在无活性复合物中。然而，当表达时，PUMA结合BCL-xL，并通过一种独特的、以前未知的机制释放被隔离的p53。而释放的p53则直接与BAX相互作用，触发BAX寡聚化、MOMP、细胞色素c释放以及最终凋亡的级联反应。重要的是，PUMA是唯一从BCL-xL中释放p53的BH3-only去抑制蛋白，释放p53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独特地从BCL-xL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肿瘤抑制机制的初步理解。具体而言，我们的目标是：1)阐明PUMA诱导BCL-xL结构重排的分子细节，并充分验证该机制在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结合BCL-xL如何破坏这些相互作用。通过所提出的研究获得的知识将定义PUMA、BCL-xL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的非转录性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。我们提出的PUMA研究将是理解p53的转录和非转录凋亡作用协同预防和维持人类无肿瘤状态的机制的关键。

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.