Understanding the Structural Mechanism of Puma-induced Apoptosis

描述（由申请人提供）：该提案阐述了 BH3-only 蛋白 PUMA 在细胞凋亡调节中的作用。 PUMA 在基因毒性应激反应中被 p53 转录激活，并且是 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对于人类主要肿瘤抑制途径至关重要。在 St. Jude，我们与 Doug Green 实验室和 Gerry Zambetti 实验室合作，前者发现了 PUMABCL-xLp53BAX 凋亡调节系统，后者发现 PUMA 作为一种 p53 诱导的促凋亡蛋白。 Our preliminary studies have clarified the mechanism of PUMA-induced apoptosis.我们的结果表明，单独使用 PUMA 不足以直接促进线粒体外膜透化 (MOMP) 和细胞凋亡。 Instead, PUMA is a "de-repressor" BH3-only protein and as such cannot directly initiate BAX and/or BAK activation.相反，PUMA 通过替换 BCL-xL 中的直接激活蛋白（即 BID、BIM 和 p53）来间接发挥作用。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 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BCL-2蛋白家族的相互作用来调节细胞质中的细胞凋亡，充当细胞凋亡的“直接激活剂”。默认情况下，胞质 p53 被 BCL-xL 隔离在无活性的复合物中。然而，当表达时，PUMA 结合 BCL-xL，并通过一种独特且先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，引发涉及 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 是唯一一种仅包含 BH3 去阻遏蛋白的蛋白质，可从 BCL-xL 中释放 p53，从而释放 p53 的全部凋亡潜力。我们使用生物化学、生物物理和结构方法以及功能测定来发现 PUMA 从 BCL-xL 中独特释放 p53 的机制。在拟议的研究中，我们的目标是详细阐述和完善我们对这一关键肿瘤抑制机制的初步理解。具体来说，我们的目标是：1) 阐明 PUMA 诱导的 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 和 BCL-xL 之间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。通过拟议研究获得的知识将定义 PUMA、BCL-xL 和 p53 介导的细胞凋亡信号传导的特异性。公共健康相关性：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 的非转录凋亡作用现已被确定为其响应致癌和基因毒性过程的肿瘤抑制功能的重要组成部分。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.