MOLECULAR MECHANISMS OF GENUS BETA E6 AND E7 PROTEINS

人乳头瘤病毒(HPV)是一大类感染粘膜或皮肤上皮的病毒， 引起一系列增生性病变。有越来越多的证据表明，贝塔病毒属 在鳞状细胞皮肤癌(SCSC)的病因学中发挥作用，尽管E6 而E7蛋白可能对癌症的发生起到作用尚不清楚。与高危甲型HPV不同的是 与宫颈癌相关的是，贝塔病毒属并不存在于肿瘤的每一个细胞中，E6/E7也不是如此。 促进P53或Rb的降解。我们的工作模型是E6/E7的表达导致细胞 增殖，从而增加了对紫外线诱变敏感的细胞池的大小。作为对.的回应 紫外线损伤E6钝化修复和凋亡过程，增加带有P53或其他基因的细胞的存活率 突变，这可能会发展为浸润性癌症。为了检验这些假设，我们提出了四个具体目标： 1.研究βE6蛋白对紫外线损伤诱导的细胞凋亡反应的抑制作用。 我们建议：a)确定BAK的降解机理。我们将确定E6AP或 其他一些泛素连接酶，靶向Bak的降解；研究Bcl-xl和Mcl-1在阻断中的作用 BAK降解；并确定介导BAK结合和降解的E6序列(目标2)； 检查内在和外在的凋亡途径的其他方面，以充分描述放松调控的特征 贝塔e6蛋白；c)确定贝塔e6是否通过阻断p300诱导的乙酰化来使p53失活；以及d) 检测人类皮肤癌和癌前病变中E6-HFKs中研究的凋亡标记物。2.识别 βE6蛋白的保守功能和新功能。我们建议：a)确定E6的区域 参与针对Bak的降解；b)确定能够诱导 和绘制关键残基；更广泛地说，c)我们计划寻找新的结合伙伴 使用TAP标记的E6、GST-E6下拉的βE6蛋白，还将重点关注独特的氨基酸和 羧基末端结构域。3.研究βE7蛋白对S期的促进作用 进入和扰乱上皮细胞分化。我们将评估HPV8和38E7的促进能力 在三种生长试验中进入S阶段，我们将检测p130在单层和 器官类型培养。4.建立E6/E7基因表达调控的转基因小鼠模型。我们 建议建立可调节的“Tet-on”双转基因小鼠系统，K14-RTTA/Tetre-HPV8E6/E7，在 在小鼠暴露于多西环素(Dox)之前，E6/E7的表达是关闭的。我们将确定是否 癌前病变一旦启动，在没有HPV表达的情况下可以维持或进展。我们 还将探索低剂量紫外线是否可以刺激HPV-on小鼠的肿瘤形成 与自然发育相比，非转基因小鼠产仔，并加速病变的形成。我们会 确定所需的E6表达的时间和持续时间。组织学和细胞凋亡标记物 在自发发生和紫外线诱导的损伤中进行研究。

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.