The effect of HPV on apoptosis and cellular proliferation in laryngeal papillomas

描述(由申请人提供)：这项指导临床科学家研究职业发展奖提案的总体目标是帮助候选人在病毒-宿主细胞相互作用研究方面建立独立的研究生涯，特别是人类乳头瘤病毒(HPV)感染导致反复呼吸道乳头状瘤病(RRP)导致声带功能障碍和上呼吸道阻塞的细胞过程。应聘者是一名获得委员会认证的耳鼻咽喉外科医生，专注于嗓音障碍和困难呼吸道的管理。除了他在耳鼻喉科的临床实习经验外，候选人还接受了关于β-HPV属的分子生物学以及它们在DNA损伤后诱导细胞增殖和阻止细胞凋亡的机制的博士后培训--这些问题类似于本项目研究部分中将研究的问题，重点是RPP相关的α-HPV属。该项目的培训和研究部分由共同导师斯蒂芬·泰林博士和托马斯·阿尔布雷希特博士监督。除了有指导的研究部分外，培训计划还要求教授病毒学、生物统计学、科学写作等领域的课程，以及负责任地开展研究的几个方面，并参加相关的地方和国家会议。本研究旨在阐明RPP相关人乳头状瘤病毒抑制角质形成细胞凋亡和诱导细胞增殖的机制。在高危HPV感染中，E6病毒蛋白针对促凋亡的细胞Bak蛋白(以及P53)进行降解，从而防止基因毒性应激诱导正常的细胞凋亡过程。候选人的初步数据显示，与RRP相关的HPV也保留了靶向Bak进行降解的能力。本研究的目的一是利用免疫印迹和shRNA敲除技术检测HPV6和HPV11E6蛋白抑制细胞凋亡反应的能力，并探讨Bak降解的机制。目的2结合蛋白质下拉试验确定6E6和11E6蛋白的保守结合伙伴(Bak，E6AP)和新的结合伙伴。最后，目标3是使用免疫染色技术来确定在未分化和分化的表达E6的细胞培养中针对诱导外源性和/或内在凋亡性通路的治疗的效果。该项目成功完成后，将对与RPP相关的“低风险”HPV的分子生物学和致病机制有更好的了解；这些信息将对开发这一目前无法治愈的疾病过程的有效、功能保留的治疗方法非常有价值。此外，候选人将获得成功竞争R01资金所需的初步数据，以及实现他的长期目标所需的技能集-作为一名专注于RPP和其他喉部相关疾病有效治疗的成熟的、外部资助的外科科学家。

DESCRIPTION (provided by applicant): The overall goal of this Mentored Clinical Scientist Research Career Development Award proposal is to assist in establishing the independent research career of the candidate in the study of virus-host cell interactions, specifically of the cellular processes contributing to vocal cord dysfunction and upper airway obstruction caused by human papillomavirus (HPV) infections that lead to recurrent respiratory papillomatosis (RRP). The candidate is a board-certified otolaryngology surgeon with a focus on the management of voice disorders and difficult airways. Besides his clinical residency in otolaryngology, the candidate received postdoctoral training in the molecular biology of genus beta HPVs and the mechanisms by which they induce cell proliferation and block apoptosis after DNA damage - issues similar to those to be studied in the research component of this project, focused on the RPP-related genus alpha HPVs. The training and research components of the project are under the supervision of the co-mentors, Dr. Stephen Tyring and Dr. Thomas Albrecht. In addition to the mentored research component, the training plan calls for didactic coursework in such areas as virology, biostatistics, scientific writing, and several aspects of the responsible conduct of research, as well as attending relevant local and national meetings. The research project focuses on elucidating the mechanisms by which RPP-related HPVs block apoptosis and induce cellular proliferation in human keratinocytes. In high-risk HPV infections, the E6 viral protein targets the pro-apoptotic cellular Bak protein (as well as p53) for degradation, and so prevents the normal process of apoptosis from being induced by genotoxic stress. The candidate's preliminary data show that the RRP-related HPVs also retain the ability to target Bak for degradation. Aim 1 of the research project is to use immunoblotting and shRNA knockdown to determine the ability of HPV6 and HPV11 E6 proteins to blunt the apoptotic response, and also to determine the mechanism for Bak degradation. Aim 2 is to use a combination of protein pull down assays to identify conserved (Bak, E6AP) and novel binding partners of the 6E6 and 11E6 proteins. Finally, Aim 3 is to use immunostaining techniques to determine the effects of treatments targeted towards the induction of the extrinsic and/or intrinsic apototis pathways in both undifferentiated and differentiated E6-expressing cell cultures. Upon successful completion of this project, there will be a much better understanding of the molecular biology and pathogenic mechanisms of the "low- risk," RPP-related HPVs; this information will be invaluable in developing effective, function-sparing treatments for this presently incurable disease process. Further, the candidate will have obtained the preliminary data needed to compete successfully for R01 funding, and the skill set necessary for him to achieve his long-range goal - success as an established, extramurally funded surgeon-scientist focused on effective treatments for RPP and other laryngeal-related diseases.