MOLECULAR MECHANISMS OF GENUS BETA E6 AND E7 PROTEINS

人乳头瘤病毒（HPV）是感染粘膜或皮肤上皮的病毒的大家族， 导致一系列增生性病变。越来越多的证据表明β型HPV 在鳞状细胞皮肤癌（SCSC）的病因学中发挥作用，尽管E6 和E7蛋白可能有助于致癌作用是未知的。不像高危的α型HPV 与宫颈癌相关，β HPV属并不存在于肿瘤的每个细胞中，E6/E7也不存在。 促进p53或Rb的降解。我们的工作模型是E6/E7表达导致细胞凋亡， 增殖，从而增加对UV诱变敏感的细胞的池大小。响应于 紫外线损伤E6钝化修复和凋亡过程，增加具有p53或其他蛋白的细胞的存活。 突变，这可能会发展为侵袭性癌症。为了验证这些假设，我们提出了四个具体目标： 1.研究β E6蛋白对紫外线损伤引起的细胞凋亡的影响。 我们建议：a）确定巴克降解的机制。我们将确定E6 AP或 一些其他的泛素连接酶，靶向巴克降解;研究Bcl-XL和Mcl-1在阻断Bcl-XL和Mcl-1表达中的作用。 巴克降解;并鉴定介导巴克结合和降解的E6序列（目的2）; B） 检查内源性和外源性凋亡途径的其他方面，以充分表征 β E6蛋白; c）确定β E6是否通过阻断p300诱导的乙酰化而抑制p53;和d） 检测在人皮肤癌和癌前病变中E6-HFKs中研究的细胞凋亡标志物。2.以识别 β E6蛋白的保守和新功能。我们建议：a）确定E6的区域 参与靶向巴克降解; B）鉴定能够诱导 端粒酶和地图的关键残基;更广泛地说，c）我们计划确定新的结合伙伴， 使用TAP标记的E6，GST-E6唐斯的β E6蛋白，也将集中在独特的氨基和 羧基末端结构域。3.研究β E7蛋白促进S期的能力， 进入并扰乱上皮细胞分化。我们将评估HPV 8和38 E7促进 在三种生长试验中进入S期;我们将检查p130是否在单层中降解， 器官型培养4.建立E6/E7基因表达调控的转基因小鼠模型。我们 建议开发可调控的“Tet-On”双转基因小鼠系统，K14-rtTA/TetRE-HPV 8 E6/E7， 其中E6/E7表达关闭，直到小鼠暴露于多西环素（Dox）。我们将决定 癌前病变一旦开始，可以在HPV表达缺失的情况下维持或发展。我们 我们还将探索低剂量的紫外线是否能刺激HPV- ON小鼠的肿瘤发生， 非转基因同窝仔，并加速病变的形成相比，自发发展。我们将 确定所需的E6表达的时间和持续时间。组织学和凋亡标志物将 在自发发生和UV诱导的病变中进行研究。

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.