Specificity of Ubiquitination: the HPVE6/E6AP Paradigm

泛素化的特异性：HPVE6/E6AP 范式

• 批准号: 6541407
• 负责人: JON HUIBREGTSE
• 金额: $ 28.01万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-13 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6541407
• 关键词: cervix neoplasms; enzyme mechanism; human papillomavirus; keratinocyte; ligase; oncoproteins; p53 gene /protein; protein protein interaction; protein structure function; tissue /cell culture; ubiquitin; viral carcinogenesis; virus protein; virus related neoplasm /cancer

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.

描述（由申请人提供）：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，性传播HPV的“高危”亚组的人乳头瘤病毒（HPV）E6和E7蛋白在超过90%的这些癌症中起致病作用。E6蛋白的生物化学活性与致癌作用最明显相关的是它们刺激泛素介导的p53肿瘤抑制蛋白降解的能力。这依赖于细胞泛素连接酶E6 AP。生化分析表明，E6的功能是重定向或重编程E6 AP的底物特异性，使其泛素化p53，一种它通常不识别的蛋白质。虽然许多证据表明p53的靶向降解对HPV在致癌作用中的作用至关重要，但越来越清楚的是，高危HPV E6蛋白具有与细胞永生化相关的额外的、不依赖于p53的功能。对E6突变体的分析表明，这些活性中至少有一些依赖于E6 AP。因此，我们假设高危HPVE 6/E6 AP复合物识别并泛素化一组细胞蛋白，包括但不限于p53，并且这些细胞蛋白的靶向与HPV相关的致癌作用有关。 该提案的第一个目标是表征一组我们已经证明由E6/E6 AP复合物靶向的蛋白质，并确定这些蛋白质的降解是否与细胞永生化相关。这些蛋白质是Scribble、Discs large（DIg）和utrophin。有趣的是，所有这三种蛋白质都与质膜上形成的多蛋白复合物的结构有关，对Scribble和Dlg的研究表明，它们是果蝇中合作的肿瘤抑制因子。虽然对高危HPV E6蛋白的研究导致了E6 AP的发现和HECT E3功能的建立模型，但我们对E6蛋白本身的结构-功能关系的理解仍然非常有限。一些证据表明，这主要是由于表达正确折叠的E6蛋白用于生化分析的困难。这一领域的最新进展构成了本提案第二个目标的基础，即HPV E6蛋白在促进蛋白泛素化中的作用方面的结构-功能关系的表征。该建议的第三个目标是进一步表征E6 AP和HECT泛素连接酶的酶促机制的具体方面。除了宫颈癌之外，大约30种人类HECT E3中的几种在疾病状态中发挥着关键作用，包括利德尔综合征、安吉尔曼综合征和几种类型病毒的生命周期。因此，更深入地了解HECT E3机制将有助于我们理解几个重要的健康相关问题。