MOLECULAR MECHANISMS OF GENUS BETA E6 AND E7 PROTEINS

β E6 和 E7 属蛋白质的分子机制

• 批准号: 8307528
• 负责人: DENISE A. GALLOWAY
• 金额: $ 37.82万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8307528
• 关键词: Acetylation; Anogenital cancer; Apoptosis; Apoptotic; Automobile Driving; Behavior; Benign; Binding; Biological Assay; Biology; CDKN2A gene; Carcinoma; Cell Differentiation process; Cell Fraction; Cell Proliferation; Cell Size; Cell Survival; Cells; Cutaneous; Development; Dose; Doxycycline; EP300 gene; Epidemiology; Epithelial; Epithelial Cells; Epithelium; Etiology; Exposure to; Family; Genes; Genital system; Growth; Histology; Human; Human Papillomavirus; Learning; Lesion; Life Cycle Stages; Longevity; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Maps; Measures; Mediating; Modeling; Molecular; Mus; Mutation; Pathogenicity; Pathway interactions; Play; Premalignant; Process; Property; Proteins; Role; S Phase; Skin Cancer; Skin Neoplasms; Squamous Cell; System; Telomerase; Testing; Tetanus Helper Peptide; Thymine Dimers; Time; Transgenic Mice; UV Mutagenesis; UV Radiation Exposure; UV induced; Ultraviolet Rays; Viral Genes; Virus; Work; carcinogenesis; clinical epidemiology; clinically significant; high risk; monolayer; mouse model; neoplastic cell; novel; promoter; repaired; response; tumor; tumorigenesis; ubiquitin ligase; ultraviolet damage

Human papillomaviruses (HPVs) are a large family of viruses that infect mucosal or cutaneous epithelium, causing causing a spectrum of proliferative lesions. There is mounting evidence that the genus beta HPVs play a role in the etiology of squamous cell skin cancers (SCSC), though the mechanism by which the E6 and E7 proteins might contribute to carcinogenesis is not known. Unlike the high-risk alpha HPVs that are associated with cervical cancers, the genus beta HPVs do not persist in every cell in the tumor, nor do E6/E7 promote the degradation of p53 or Rb. Our working model is that E6/E7 expression causes cellular proliferation, thereby increasing the pool size of cells that are susceptible to UV mutagenesis. In response to UV damage E6 blunts repair and apoptotic processes, increasing the survival of cells with p53 or other mutations, which can progress to invasive cancer. To test these hypotheses we propose four specific aims: 1. To investigate the ability of genus beta E6proteins to blunt the apoptotic response to UVdamage. We propose to: a) determine the mechanism of Bak degradation. We will determine whether E6AP, or some other ubiquitin ligase, targets Bak for degradation; investigate the role of Bcl-XL and Mcl-1 in blocking Bak degradation; and identify the sequences of E6 that mediate Bak binding and degradation (in aim 2); b) examine other aspects of the intrinsic and extrinsic apoptotic pathways to fully characterize deregulation by beta E6 proteins; c) determine whether beta E6s inactivate p53 by blocking p300-induced acetylation; and d) examine markers of apoptosis studied in E6-HFKs in human skin cancers and pre-cancers. 2. To identify both conserved and novel functions of the beta E6 proteins. We propose to: a) identify regions ofE6 involved in targeting Bak for degradation; b) identify the beta HPV E6s that are capable of inducing telomerase and map the critical residues; and more broadly, c) we plan to identify new binding partners for the beta E6 proteins using TAP-tagged E6, GST- E6 pull downs, and will also focus on the unique amino and carboxy -terminal domains. 3. To investigate the ability of genus beta E7 proteins to promote S phase entry and perturb epithelial cell differentiation. We will assess the ability of HPV 8 and 38 E7 to promote entry into S phase in three growth assays; and we will examine whether p130 is degraded in monolayer and organotypic culture. 4. To develop transgenic mouse models with regulated expression of E6/E7. We propose to develop regulatable "Tet-On" bitransgenic mouse systems, K14-rtTA/TetRE-HPV8E6/E7, in which E6/E7 expression is off until the mice are exposed to doxycycline (Dox). We will determine whether precancerous lesions once initiated can be maintained or progress in the absence of HPV expression. We will also explore whether low doses of UV can stimulate tumorigenesis in HPV- ON mice compared nontransgenic littermates, and accelerate lesion formation compared to spontaneous development. We will determine the timing and duration of E6 expression that is required. Histology and markers of apoptosis will be studied in the spontaneously occurring and UV-induced lesions.

人乳头瘤病毒（HPV）是一个大型病毒家族，感染粘膜或皮肤上皮， 引起一系列增殖病变。有越来越多的证据表明β属HPV属 在鳞状细胞皮肤癌（SCSC）的病因中发挥作用，尽管E6的机制 E7蛋白可能导致致癌作用。与高风险的Alpha HPV不同 与颈癌相关，βHPV属在肿瘤的每个细胞中都不持续，E6/E7也不持续 促进p53或rb的降解。我们的工作模型是E6/E7表达会导致细胞 增殖，从而增加了容易受到紫外线诱变的细胞的池大小。响应 紫外线损害E6钝化修复和凋亡过程，增加了用p53或其他细胞的生存 突变，可以发展为侵入性癌症。为了检验这些假设，我们提出了四个具体目标： 1。研究βe6蛋白属钝化对紫外线反应的能力。 我们建议：a）确定BAK降解的机理。我们将确定是E6AP还是 其他一些泛素连接酶的目标是降解；研究BCL-XL和MCL-1在阻塞中的作用 贝克退化；并确定介导BAK结合和降解的E6序列（在AIM 2中）； b） 检查固有和外在凋亡途径的其他方面，以完全表征通过 βE6蛋白； c）确定βE6是否通过阻断p300诱导的乙酰化来灭活p53；和D） 检查在人类皮肤癌和癌症中的E6-HFK中研究的凋亡的标记。 2。识别 βE6蛋白的保守和新功能。我们建议：a）识别E6的区域 参与靶向Bak进行降解； b）确定能够诱导的βHPVE6S 端粒酶并绘制关键残基；更广泛地，c）我们计划确定新的约束伙伴 使用Taged E6，GST-E6下拉的βE6蛋白，还将集中在独特的氨基上 羧基末端域。 3。研究βE7蛋白属促进S相的能力 进入和敏感性上皮细胞分化。我们将评估HPV 8和38 E7促进的能力 在三个增长测定中进入S阶段；我们将检查P130是否在单层中降解 器官文化。 4。开发具有E6/E7的调节表达的转基因小鼠模型。我们 建议开发可调节的“ Tet-On” Bittransgenic Mouse Systems，K14-RTTA/TETRE-HPV8E6/e7，in e6/e7的表达熄灭，直到小鼠暴露于多西环素（DOX）。我们将确定是否 在没有HPV表达的情况下，可以保持癌前病变，或者可以维持或进展。我们 还将探索低剂量的紫外线是否可以刺激小鼠HPV-的肿瘤发生 与自发发育相比，非转基因同窝窝和加速病变形成。我们将 确定所需的E6表达的时间和持续时间。凋亡的组织学和标记将 在自发发生和紫外线引起的病变中进行研究。