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首页> 外文学位 >A novel mechanism for human papillomavirus mediated tumorigenesis: Examining a role for HPV E6 protein in CYLD mediated NF-kappaB activation.
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A novel mechanism for human papillomavirus mediated tumorigenesis: Examining a role for HPV E6 protein in CYLD mediated NF-kappaB activation.

机译：人类乳头瘤病毒介导的肿瘤发生的新机制：检查HPV E6蛋白在CYLD介导的NF-κB激活中的作用。

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摘要

Human papillomavirus (HPV) infection of mucosal epithelium by 'high-risk' HPV types has a prominent role in the development of anogenital intraepithelial neoplasias and carcinomas. Human epithelial cells transformed with the HPV E6 oncoprotein survive even under conditions that normally lead to cell apoptosis. This phenomenon has been attributed to HPV E6's ability to promote the degradation of the tumor suppressor protein p53. More recently, it has been demonstrated that HPV E6 contributes to activation of the NF-kappaB pathway. NF-kappaB is a transcription factor involved in the regulation of genes associated with cellular proliferation, apoptosis and inflammatory responses. In addition to p53 suppression, HPV E6 modulation of NF-kappaB activation presents another mechanism for HPV-driven tumorigenesis. However, it was not known how HPV E6 promotes NF-kappaB pathway activation. To address how HPV E6 leads to NF-kappaB activation, we identified an association between HPV E6 and the human cylindromatosis gene product (CYLD). CYLD is an endogenous inhibitor of canonical NF-kappaB activation. We showed that HPV E6 proteins could precipitate CYLD in vitro using a co-immunoprecipitation assay. Demonstrating that HPV E6 and CYLD proteins bind each other raised the possibility that this binding relationship would have a functional effect upon the NF-kappaB pathway by altering CYLD-mediated suppression of NF-kappaB activation. To identify HPV E6 functional relationship with CYLD and to determine how HPV E6 activates the NF-kappaB pathway, we transfected cells with either HPV E6 expression vectors containing the high-risk HPV type 16 E6 or the low-risk HPV type 11 E6 along with a CYLD expression vector. We showed HPV16 E6 expression in 293 cells blocked the ability of CYLD to inhibit CD40 ligand-stimulated NF-kappaB activation. Interestingly, HPV11 E6 was unable to inhibit CYLD mediated suppression of NF-kappaB in our system. CYLD had previously been shown to suppress NF-kappaB activation by removing stimulatory lysine 63-linked ubiquitin chains from TRAF2. We found CYLD expression in 293 cells leads to dose-dependent reduction in TRAF2 levels. This CYLD-mediated loss of TRAF2 is inhibited by co-expression of high-, but not low-risk E6 proteins. It was known that CYLD phosphorylation in vivo suppresses CYLD deubiquitination actions of canonical pathway proteins; we therefore tested the extent of CYLD phosphorylation when co-expressed with HPV E6, and discovered that CYLD phosphorylation was increased in the presence of HPV E6. This compilation of experiments suggests that with HPV16 E6 binding to CYLD, the E6 protein blocks CYLD-mediated TRAF2 loss and thereby TRAF2 is available to activate the canonical NF-kappaB pathway. Blocking HPV E6-mediated NF-kappaB activation may prove beneficial as a means for designing therapies that inhibit HPV-mediated tumorigenesis. The differences we detected between HPV11 E6 and HPV16 E6 are supported by other studies that showed E6 protein variations account for molecular and clinical differences among HPV infection outcomes. Similarly, there exist intratype E6 variations in HPV16. We obtained cervical specimens from patients with cytopathogenic changes consistent with the onset of cervical dysplasia infected with HPV16 E6 and the human immunodeficiency virus. We hypothesized the immunocompromised individual may harbor unique HPV16 E6 variants. Using PCR detection methods to amplify the HPV16 E6 DNA and sequencing technology, we identified that some of the samples indeed had nucleotide polymorphisms, resulting in amino acid sequence changes. However, the HPV E6 variants we detected were previously described, and can be classified into known geographic HPV clades. Some of the HPV E6 variants we observed are suggested to be associated with progression to cervical cancer, but further evaluation is required.
机译：人类乳头瘤病毒（HPV）通过“高风险” HPV类型感染粘膜上皮在肛门生殖器上皮内瘤变和癌的发展中具有重要作用。即使在通常导致细胞凋亡的条件下，用HPV E6癌蛋白转化的人上皮细胞也能存活。该现象归因于HPV E6促进肿瘤抑制蛋白p53降解的能力。最近，已证明HPV E6有助于激活NF-κB途径。 NF-κB是一种转录因子，参与调控与细胞增殖，凋亡和炎症反应相关的基因。除了抑制p53外，NF-κB激活的HPV E6调节还为HPV驱动的肿瘤发生提供了另一种机制。但是，尚不知道HPV E6如何促进NF-κB途径的激活。为了解决HPV E6如何导致NF-κB活化的问题，我们确定了HPV E6与​​人类圆柱状体病基因产物（CYLD）之间的关联。 CYLD是典型的NF-κB激活的内源性抑制剂。我们显示，HPV E6蛋白可以使用共免疫沉淀测定法在体外沉淀CYLD。证明HPV E6和CYLD蛋白相互结合，增加了这种结合关系将通过改变CYLD介导的NF-κB激活抑制而对NF-κB通路产生功能作用的可能性。为了确定HPV E6与​​CYLD的功能关系并确定HPV E6如何激活NF-kappaB途径，我们用含有高风险HPV 16 E6或低风险HPV 11 E6以及两者的HPV E6表达载体转染了细胞。 CYLD表达载体。我们显示HPV16 E6在293细胞中的表达阻止了CYLD抑制CD40配体刺激的NF-κB激活的能力。有趣的是，HPV11 E6在我们的系统中无法抑制CYLD介导的对NF-κB的抑制。 CYLD以前已显示可通过从TRAF2去除刺激性赖氨酸63连接的泛素链来抑制NF-κB活化。我们发现293细胞中CYLD的表达导致TRAF2水平的剂量依赖性降低。 CYLD介导的TRAF2的丢失被高风险（但低风险）的E6蛋白的共表达抑制。众所周知，体内CYLD磷酸化可抑制经典途径蛋白的CYLD去泛素作用。因此，我们测试了与HPV E6共表达时CYLD磷酸化的程度，并发现在HPV E6存在下CYLD磷酸化增加。该实验汇编表明，在HPV16 E6与CYLD结合的情况下，E6蛋白可阻止CYLD介导的TRAF2丢失，因此TRAF2可用于激活经典的NF-κB途径。阻断HPV E6介导的NF-κB活化可能被证明是设计抑制HPV介导的肿瘤发生的治疗方法的有益手段。我们在HPV11 E6和HPV16 E6之间检测到的差异得到其他研究的支持，这些研究表明E6蛋白变异解释了HPV感染结局之间的分子和临床差异。类似地，HPV16中存在类型内E6变异。我们从具有致病性变化的患者中获得了宫颈标本，这些病变与感染了HPV16 E6和人类免疫缺陷病毒的宫颈发育异常的发作一致。我们假设免疫受损的个体可能带有独特的HPV16 E6变体。使用PCR检测方法扩增HPV16 E6 DNA和测序技术，我们鉴定出某些样品确实具有核苷酸多态性，从而导致氨基酸序列变化。但是，我们之前检测到的HPV E6变体已经描述过，可以分为已知的地理HPV进化枝。我们观察到的某些HPV E6变体被认为与宫颈癌的进展有关，但是还需要进一步评估。

著录项

  • 作者

    Shaw, Charlie Vincent, Jr.;

  • 作者单位

    Duke University.;

  • 授予单位 Duke University.;
  • 学科 Biology Microbiology.;Biology Virology.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 153 p.
  • 总页数 153
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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