Specificity of Ubiquitination: the HPVE6/E6AP Paradigm

描述(申请人提供)：宫颈癌是全球女性癌症相关死亡的第二大原因，流行病学和实验室研究表明，人乳头瘤病毒(HPV)E6和E7蛋白是性传播HPV的高危亚群，在这些癌症中，超过90%是由HPV E6和E7蛋白引起的。E6蛋白的生化活性最明显地与致癌有关，是它们能够刺激泛素介导的P53肿瘤抑制蛋白的降解。这依赖于细胞泛素连接酶E6AP。生化分析表明，E6的功能是重定向或重新编程E6AP的底物特异性，使其泛化它通常不识别的蛋白质P53。虽然许多证据表明，p53的靶向降解对于HPV在致癌过程中的作用是至关重要的，但越来越清楚的是，高危HPVE6蛋白具有与细胞永生化相关的额外的、P53不依赖的功能。对E6突变体的分析表明，至少其中一些活性依赖于E6AP。因此，我们假设高危HPVE6/E6AP复合体识别并泛化一组细胞蛋白，其中包括但不限于P53，并且这些细胞蛋白的靶向与HPV相关的癌症发生有关。 这项建议的第一个目标是表征一组我们已经证明是E6/E6AP复合体靶向的蛋白质，并确定这些蛋白质的降解是否与细胞永生化有关。这些蛋白质是Scribble、Discs Large(Dig)和utroin。有趣的是，这三种蛋白质都与质膜上形成的多蛋白复合体的结构有关，对Scribble和DLG的研究表明，这些都是果蝇中协同作用的肿瘤抑制因子。虽然对高危HPVE6蛋白的研究导致了E6AP的发现和Hect E3功能的建立，但我们对E6蛋白本身的结构-功能关系的了解仍然非常有限。一些证据表明，这在很大程度上是由于很难表达正确折叠的E6蛋白进行生化分析。这一领域的最新进展构成了这一建议的第二个目标的基础，即表征HPVE6蛋白在促进蛋白质泛素化中的作用的结构-功能关系。这项建议的第三个目标是进一步表征E6AP和Hect泛素连接酶的酶机制的具体方面。在大约30个人类Hect E3中，有几个在宫颈癌以外的疾病状态中发挥着关键作用，包括利德尔综合征、安杰曼综合征和几种类型病毒的生命周期。因此，对Hect E3机制的更深入了解将有助于我们理解几个与健康相关的重要问题。

DESCRIPTION (provided by applicant): Cervical cancer is the second-leading cause of cancer-related deaths among women worldwide, and epidemiologic and laboratory studies have shown that the human papillomavirus (HPV) E6 and E7 proteins of the "high-risk" subgroup of sexually transmitted HPVs play a causative role in over 90% of these cancers. The biochemical activity of the E6 proteins most clearly related to carcinogenesis is their ability to stimulate the ubiquitin-mediated degradation of the p53 tumor suppressor protein. This is dependent on the cellular ubiquitin ligase, E6AP. Biochemical analyses indicate that E6 functions to redirect or reprogram the substrate specificity of E6AP so that it ubiquitinates p53, a protein that it does not normally recognize. While many lines of evidence indicate that targeted degradation of p53 is critical to the role of HPVs in carcinogenesis, it has become increasingly clear that high-risk HPV E6 proteins have additional, p53-independent functions related to cellular immortalization. Analysis of E6 mutants suggests that at least some of these activities are dependent on E6AP. We therefore hypothesize that the high-risk HPVE6/E6AP complex recognizes and ubiquitinates a set of cellular proteins, which includes but is not limited to p53, and that targeting of these cellular proteins is linked to HPV-associated carcinogenesis. The first goal of this proposal is to characterize a set of proteins that we have shown to be targeted by the E6/E6AP complex and to determine if degradation of these proteins is relevant to cellular immortalization. These proteins are Scribble, Discs large (DIg), and utrophin. Interestingly, all three of these proteins are linked to architecture of multiprotein complexes formed at the plasma membrane, and work on Scribble and Dlg indicates that these are cooperating neoplastic tumor suppressors in Drosophila. While study of high-risk HPV E6 proteins led to discovery of E6AP and an established model for HECT E3 function, our understanding of the structure-function relationships of the E6 proteins, themselves, remains very limited. Several lines of evidence suggest that this is largely due to the difficulty of expressing properly folded E6 proteins for biochemical analyses. Recent advances in this area form the basis for the second goal of this proposal, characterization of the structure-function relationships of the HPV E6 proteins with respect to their role in facilitating protein ubiquitination. The third goal of this proposal is to further characterize specific aspects of the enzymatic mechanism of E6AP and HECT ubiquitin ligases, in general. Several of the approximately 30 human HECT E3s play critical roles in disease states in addition to cervical cancer, including Liddle's syndrome, Angelman's syndrome, and the life cycle of several types of viruses. A more thorough understanding of HECT E3 mechanism will therefore contribute to our understanding of several important health-related problems.