Action of the SV40 T Antigen Chaperone Machine on Tumor Suppressors

SV40 T 抗原伴侣机对肿瘤抑制剂的作用

• 批准号: 7082721
• 负责人: JAMES M PIPAS
• 金额: $ 36.88万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-14 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082721
• 关键词: antigens; neoplasm /cancer; proteins

DESCRIPTION (provided by applicant): Tumor suppressors are regulatory proteins that receive and integrate diverse signals and function to exert control over key cellular processes such as cell proliferation, differentiation, and apoptosis. Because loss or perturbation of their activity often results in cancer or other diseases, and because of their central role in governing organismal development and tissue homeostasis, these proteins are of great interest. The retinoblastoma protein (pRb) is a well characterized tumor suppressor that, in concert with two related proteins, p130 and p107, control cell cycle entry and exit, in part, by regulating the activity of the E2F family of transcription factors. Many viruses, including Simian virus 40 (SV40) encode oncoproteins that bind Rb-family members and interfere with their ability to regulate E2Fs. The large tumor antigen (T antigen) encoded by SV40 binds to pRb, p107, and p130 via an LXCXE motif and blocks the ability of these proteins to inhibit E2F-dependent transcription and to induce growth arrest. The retinoblastoma family has been studied intensively, yet little is known about the molecular basis by which viruses, such as SV40, block their action. In fact, interaction with T antigen has different consequences for each Rb protein. For example, p130 is degraded following SV40 infection or transformation, while the levels of pRb remain unchanged. Thus, T antigen appears to be able to distinguish different Rb-E2F complexes, but the basis for this discrimination is unknown. Like many regulatory proteins pRb and E2F transcription factors do not exist in isolation. Rather they function as part of large multiprotein assemblages that include chromatin modifiers, the basal transcription apparatus, as well as other factors, and the dynamic assembly and disassembly of these complexes is critical to their regulation. T antigen has a J domain and has been shown to function as a DnaJ molecular chaperone. The J domain is required for a vital DNA replication, transcriptional control, and virion assembly. Importantly, the J domain is required for T antigen to block the function of Rb proteins and thus to activate E2F-dependent transcription. This application seeks to understand the mechanistic and structural basis for the action of T antigen's recognition and disruption of Rb-E2F complexes. First, biochemical studies will explore the ability of the T antigen chaperone machine to distinguish and act upon p130-E2F4-DP1, pRb-E2F4-DP1, and pRb-E2F1-DP1 complexes. Second, the role of J domain orientation and flexibility will be examined using a combination of NMR and X-ray crystallography. Finally, a combined genetic and biochemical approach will be used to identify additional protein participants in the chaperone reaction. These studies will enhance our understanding of how these tumor suppressors govern cell proliferation and survival, and how subversion of these mechanisms by viruses or genetic mutation, contribute to cancer.

描述（由申请人提供）：肿瘤抑制因子是一种调节蛋白，可接收和整合多种信号和功能，控制细胞增殖、分化和凋亡等关键细胞过程。由于其活性的丧失或紊乱经常导致癌症或其他疾病，并且由于其在控制生物体发育和组织稳态中的核心作用，这些蛋白质引起了极大的兴趣。视网膜母细胞瘤蛋白（retinoblastoma protein, pRb）是一种特性良好的肿瘤抑制因子，它与两个相关蛋白p130和p107一起，通过调节转录因子E2F家族的活性，部分地控制细胞周期的进入和退出。许多病毒，包括猿猴病毒40 （SV40）编码结合rb家族成员并干扰其调节E2Fs的能力的癌蛋白。SV40编码的大肿瘤抗原（T抗原）通过LXCXE基序与pRb、p107和p130结合，阻断这些蛋白抑制e2f依赖性转录和诱导生长停滞的能力。视网膜母细胞瘤家族已被深入研究，但对病毒（如SV40）阻断其作用的分子基础知之甚少。事实上，与T抗原的相互作用对每个Rb蛋白有不同的影响。例如，p130在SV40感染或转化后被降解，而pRb的水平保持不变。因此，T抗原似乎能够区分不同的Rb-E2F复合物，但这种区分的基础尚不清楚。像许多调节蛋白一样，pRb和E2F转录因子不是孤立存在的。相反，它们作为包括染色质修饰剂、基础转录装置以及其他因子在内的大型多蛋白组合的一部分发挥作用，这些复合物的动态组装和拆卸对它们的调节至关重要。T抗原具有J结构域，并已被证明具有DnaJ分子伴侣的功能。J结构域是至关重要的DNA复制、转录控制和病毒粒子组装所必需的。重要的是，T抗原需要J结构域来阻断Rb蛋白的功能，从而激活e2f依赖性转录。本应用旨在了解T抗原识别和破坏Rb-E2F复合物的作用的机制和结构基础。首先，生化研究将探索T抗原伴侣机区分和作用于p130-E2F4-DP1、pRb-E2F4-DP1和pRb-E2F1-DP1复合物的能力。其次，J畴取向和灵活性的作用将使用核磁共振和x射线晶体学的组合进行检查。最后，结合遗传和生物化学方法将用于识别伴侣反应中的其他蛋白质参与者。这些研究将增强我们对这些肿瘤抑制因子如何控制细胞增殖和存活的理解，以及病毒或基因突变如何破坏这些机制，从而导致癌症。