Degradation of Toll-Like Receptor 8 by RNF216 in response to plasma MicroRNAâs â A Novel Mechanism Regulating Inflammation in Acute Lung Injury

本申请表是K08临床科学家指导研究职业发展奖的申请书，名为 RNF216降解Toll样受体8对血浆microRNA的响应--一种新机制 调节急性肺损伤的炎症反应“。我是一名肺部和重症监护医学的内科医生 匹兹堡大学。我正在申请这个奖项，以获得细胞生物学、翻译方面的高级培训 研究方法和生物信息学，以发展我的职业生涯，作为一名内科科学家，专注于 急性呼吸窘迫综合征(ARDS)。我的提案的主要目的是确定一部小说如何 单核细胞Toll样受体8(TLR8)降解途径调节严重肺损伤。TLR8是一种模式 感知免疫原性RNA的识别受体，包括一些宿主来源的血浆microRNA。TLR8 激活启动信号，导致细胞因子的分泌，导致过度炎症，即 以严重的急性呼吸窘迫综合征为特征。我的初步数据表明，TLR8在单核细胞中被降解的机制 依赖于泛素化的翻译后修饰。此外，我已经确定了一种候选泛素- 转移被称为RNF216的E3连接酶负责靶向TLR8的降解，我观察到 ARDS患者RNF216基因表达降低。最后，我已经确定了循环的一个子集 ARDS患者血浆microRNA可能作为新的TLR8配体发挥作用。这项研究的目的是：i) 确定单核细胞中泛素/蛋白酶体系统调节TLR8蛋白水平的机制II) 确定RNF216是否通过引导TLR8降解来调节炎症信号，并定义RNF216 在ARDS中的表达，以及iii)检测ARDS患者血浆中作为TLR8配体的miRNA。这些研究将 洞察一种新的病理生物学模型，在该模型中，TLR8降解由RNF216介导 蛋白质泛素化，在ARDS中控制炎症反应宿主来源的血浆microRNA。电浆 MiRNA诱导的TLR8激活，被RNF216介导的泛素化和降解所增强 TLR8，可能会导致过度炎症。因此，调制TLR8降解可能是一种新的减少 急性呼吸窘迫综合征的过度炎症反应。这个项目将提供我在细胞生物学和高级技能 分析分子数据集的生物信息学方法。我将接受翻译研究方法方面的培训 以加强我作为一名独立调查员的发展。我已承诺从我们部门进行指导 主任，Rama Mallampalli博士和Bill Chen博士的博士讲解员。另外，我的指导委员会 包括国际先天免疫专家Robert Lafyatis博士、 翻译miRNA生物学，以及布莱恩·麦克弗里博士-一位杰出的翻译科学家和急性核糖核酸学会成员 肺损伤卓越中心负责监督临床急性肺损伤计划。我的工作就会完成 在匹兹堡大学肺、过敏和重症监护医学部，这是 致力于内科科学家的发展。

This application is for a K08 Mentored Clinical Scientist Research Career Development Award entitled “Degradation of Toll-Like Receptor 8 by RNF216 in response to plasma MicroRNA’s – A Novel Mechanism Regulating Inflammation in Acute Lung Injury”. I am a physician in pulmonary and critical care medicine at the University of Pittsburgh. I am applying for this award to acquire advanced training in cell biology, translational research methods, and bioinformatics to develop my career as a physician scientist focused on the study of acute respiratory distress syndrome (ARDS). The main objective of my proposal is to determine how a novel Toll-Like Receptor 8 (TLR8) degradation pathway in monocytes regulates severe lung injury. TLR8 is a pattern recognition receptor that senses immunogenic RNA, including some host-derived plasma microRNA’s. TLR8 activation initiates signaling leading to the secretion of cytokines, contributing to excessive inflammation that is characteristic of severe ARDS. My preliminary data indicate that TLR8 is degraded in monocytes in a mechanism dependent on the post-translational modification of ubiquitination. Further, I have identified a candidate ubiquitin- transferring E3 ligase termed RNF216 responsible for targeting TLR8 for degradation, and I observe that RNF216 mRNA expression is decreased in patients with ARDS. Lastly, I have identified a subset of circulating plasma microRNA’s in ARDS subjects that may function as novel TLR8 ligands. The aims of this study are: i) to define the mechanism regulating TLR8 protein levels by the ubiquitin/proteasome system in monocytes ii) to determine if RNF216 modulates inflammatory signaling by directing TLR8 degradation and define RNF216 expression in ARDS, and iii) to examine plasma miRNA’s in ARDS subjects as TLR8 ligands. These studies will provide insight into a novel pathobiologic model whereby TLR8 degradation, regulated by RNF216 mediated protein ubiquitination, controls inflammation in response to host-derived plasma microRNA’s in ARDS. Plasma miRNA-induced TLR8 activation, augmented by reduced RNF216 mediated ubiquitination and degradation of TLR8, may drive excessive inflammation. Thus, modulating TLR8 degradation may be a novel strategy to reduce excessive inflammatory responses in ARDS. This project will provide me advanced skills in cell biology and bioinformatic approaches to analyze molecular datasets. I will be trained in translational research methodologies to strengthen my development into an independent investigator. I have committed mentoring from our Division Chief, Dr. Rama Mallampalli and a PhD comentor in Dr. Bill Chen. Additionally, my mentoring committee includes Dr. Robert Lafyatis – an international expert in innate immunity, Dr. Stephen Chan – an authority in translational miRNA biology, and Dr. Bryan McVerry – a superb translational scientist and member of the Acute Lung Injury Center of Excellence overseeing the clinical Acute Lung Injury program. My work will be completed within the Division of Pulmonary, Allergy, and Critical Care Medicine at the University of Pittsburgh, which is committed to the development of physician scientists.