Action of the SV40 T Antigen Chaperone Machine on Tumor Suppressors

描述(申请人提供)：肿瘤抑制因子是一种调节蛋白，它接收和整合不同的信号和功能，对细胞增殖、分化和凋亡等关键细胞过程施加控制。因为它们活性的丧失或干扰通常会导致癌症或其他疾病，而且因为它们在调节生物发育和组织动态平衡方面的核心作用，这些蛋白质引起了人们的极大兴趣。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，它与两种相关蛋白p130和p107共同控制细胞周期的进出，部分是通过调节E2F家族转录因子的活性来实现的。许多病毒，包括猿猴病毒40(SV40)，编码与RB家族成员结合的癌蛋白，并干扰他们调节E2F的能力。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依赖的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SV40等病毒阻止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种Rb蛋白都有不同的后果。例如，在SV40感染或转化后，p130被降解，而pRb水平保持不变。因此，T抗原似乎能够区分不同的Rb-E2F复合体，但这种区分的基础尚不清楚。像许多调节蛋白一样，pRb和E2F转录因子并不是孤立存在的。相反，它们是大型多蛋白组合的一部分，包括染色质修饰物、基本转录装置以及其他因素，这些复合体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依赖的转录。这项应用旨在了解T抗原识别和破坏Rb-E2F复合体的作用的机制和结构基础。首先，生化研究将探索T抗原伴侣机器识别和作用于p130-E2F4-DP1、pRb-E2F4-DP1和pRb-E2F1-DP1复合体的能力。其次，将结合核磁共振和X射线结晶学研究J结构域取向和柔韧性的作用。最后，将使用遗传和生化相结合的方法来确定伴侣反应中的其他蛋白质参与者。这些研究将加深我们对这些肿瘤抑制因子如何控制细胞增殖和存活，以及这些机制如何被病毒或基因突变颠覆而导致癌症的理解。

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.