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

描述（由申请人提供）：本指导临床科学家研究职业发展奖提案的总体目标是协助候选人在病毒-宿主细胞相互作用的研究中建立独立的研究生涯，特别是由人乳头瘤病毒（HPV）感染引起的声带功能障碍和上呼吸道阻塞的细胞过程，导致复发性呼吸道乳头瘤病（RRP）。候选人是一名委员会认证的耳鼻喉外科医生，专注于声音障碍和气道困难的管理。除了耳鼻喉科的临床实习外，该候选人还接受了β hpv属的分子生物学及其诱导细胞增殖和DNA损伤后阻止细胞凋亡的机制的博士后培训，这些问题与本项目的研究组成部分类似，重点研究与rpp相关的α hpv属。该项目的培训和研究部分由共同导师Stephen Tyring博士和Thomas Albrecht博士监督。除了受指导的研究部分外，该培训计划还要求在病毒学、生物统计学、科学写作和负责任的研究行为的几个方面进行教学课程，并参加相关的地方和国家会议。本研究的重点是阐明与rpp相关的人乳头状瘤病毒阻断人角化细胞凋亡和诱导细胞增殖的机制。在高危HPV感染中，E6病毒蛋白靶向促凋亡细胞Bak蛋白（以及p53）降解，从而阻止基因毒性应激诱导的正常凋亡过程。候选人的初步数据表明，rrp相关的hpv也保留了靶向Bak降解的能力。本课题的目的1是通过免疫印迹和shRNA敲低检测HPV6和hpv11e6蛋白抑制凋亡反应的能力，并确定Bak降解的机制。目的2是使用蛋白质下拉试验的组合来鉴定6E6和11E6蛋白的保守（Bak, E6AP）和新的结合伙伴。最后，目的3是使用免疫染色技术来确定针对诱导未分化和分化表达e6的细胞培养中外源性和/或内在凋亡途径的治疗效果。在这个项目成功完成后，人们将对“低风险”rpp相关的人乳头状瘤病毒的分子生物学和致病机制有更好的了解；这一信息对于开发有效的、保留功能的治疗目前无法治愈的疾病过程将是无价的。此外，候选人将获得成功竞争R01基金所需的初步数据，以及实现其长期目标所需的技能集-成功成为一名建立的，外部资助的外科科学家，专注于RPP和其他喉部相关疾病的有效治疗。

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.