Action of the SV40 T Antigen Chaperone Machine on Tumor Suppressors

肿瘤抑制因子是一种调节蛋白，它接收和整合不同的信号并发挥作用 对细胞增殖、分化和凋亡等关键细胞过程进行控制。因为 它们活动的丧失或干扰往往会导致癌症或其他疾病，而且由于它们的核心作用 在控制生物体发育和组织动态平衡方面，这些蛋白质是非常有意义的。这个 视网膜母细胞瘤蛋白(PRB)是一种具有良好特性的肿瘤抑制因子，它与两个相关的 蛋白质p130和p107部分地通过调节E2F家族的活性来控制细胞周期的进出。 转录因子。 许多病毒，包括猿猴病毒40(SV40)，编码结合RB家族成员的癌蛋白 并干扰它们调节E2F的能力。SV40编码的大肿瘤抗原(T抗原)结合 通过LXCXE基序作用于pRb、p107和p130，并阻断这些蛋白抑制E2F依赖的能力 转录和诱导生长停滞。视网膜母细胞瘤家族已被深入研究，但很少有人研究。 已知病毒(如SV40)阻止其活动的分子基础。事实上，与T的互动 抗原对每种Rb蛋白有不同的影响。例如，在SV40之后，p130被降解 感染或转化，而pRb水平保持不变。因此，T抗原似乎能够 区分不同的Rb-E2F络合物，但这种区分的基础尚不清楚。 像许多调节蛋白一样，pRb和E2F转录因子并不是孤立存在的。相反，他们 作为大型多蛋白组合的一部分，包括染色质修饰物，基础转录 设备以及其他因素，以及这些复合体的动态组装和拆卸至关重要 遵守他们的规定。T抗原有一个J结构域，已被证明具有DNAJ分子的功能 监护人。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 依赖E2F的转录。此应用程序旨在了解以下各项的机制和结构基础 T抗原识别和破坏Rb-E2F复合体的作用首先，生化研究将 探讨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。第二，J域的定向和灵活性的作用将是 用核磁共振和X射线结晶学相结合的方法进行了研究。最后，一个结合了遗传和 将使用生化方法来确定伴侣反应中的其他蛋白质参与者。这些 研究将加强我们对这些肿瘤抑制因子如何控制细胞增殖和生存的理解， 以及病毒或基因突变对这些机制的颠覆如何导致癌症。

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, p130and 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 p130via 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.