Resolving the role of nicotine-mediated phosphorylation on pancreatic fibrosis

摘要 胰腺纤维化的发展是胰腺疾病的标志，但胰腺纤维化的途径是胰腺炎的一个重要因素。 纤维形成和相关的磷酸化仍然没有解决。胰腺星状细胞（PaSC）是关键 胰腺纤维化的介质。吸烟是胰腺疾病的独立危险因素。此外，本发明还提供了一种方法， 尼古丁是香烟烟雾的主要有毒成分，它与各种细胞类型的纤维化有关。 我的目的是研究尼古丁对PaSC磷蛋白改变的影响，以确定1） 烟碱受体（nAChR）亚单位表达的PaSC，并评估其在纤维化中的作用，2）差异， PaSC中尼古丁的激酶活性谱，以及3）由于 尼古丁。我将检验尼古丁改变PaSC中激酶调节的细胞信号通路的假设。 导致形态和功能改变，这可能是胰腺疾病的前兆。 在具体目标1中，我将确定参与尼古丁诱导的信号转导的nAChR亚型， PaSC及其在纤维化中的作用。人PaSC细胞系将在有和没有尼古丁的情况下孵育。西方 印迹法将评估PaSC活化的蛋白质特征并鉴定存在的nAChR亚基。 siRNA将用于敲低特定的nAChR亚基，并使用Western blot进行评估。 印迹和基于MTT的细胞增殖测定。在具体目标2中，我将确定激酶谱 尼古丁处理的PaSC的改变。使用Kinase ActivitY Assay for Kinome profiling（KAYAK）， 鉴定和定量暴露于尼古丁后PaSC中表达的激酶。蛋白质印迹法将 如果需要，用于通过表达水平标准化激酶活性。在具体目标3中，我将确定快速 尼古丁处理导致的PaSC磷酸化改变。使用全局磷蛋白 Gygi实验室开发和建立的鉴定策略（即，激酶活性测定，钛 二氧化物磷酸肽富集和同量异位串联质量标签（TMT）为基础的定量），我将确定 尼古丁处理后PaSC蛋白的局部磷酸化位点的时间依赖性变化。 将使用MTT试验和Western印迹法进行效应验证。在完成这些 目的，我希望确定1）PaSC表达的nAChR亚型，2）激酶活性谱 参与尼古丁诱导的PaSC细胞改变，以及3）未治疗或 尼古丁处理的PaSC并绘制各种细胞条件下的局部磷酸化位点（即，尼古丁， 各种激酶抑制剂）。这些数据将使我能够确定激酶抑制剂， 尼古丁的影响 这项工作将对胰腺疾病领域产生影响，并且符合研究目标 10.2国家消化系统疾病委员会2008年建议， 研究吸烟和PaSC在胰腺纤维化中的作用。本文件所建议的工作符合 我的长期目标是更清楚地了解胰腺疾病的机制， 减缓、停止或改善慢性胰腺炎和胰腺癌的治疗。拟议的工作是 这反映了我从研究生院研究尼古丁受体到现在的博士后研究的进展。 研究使用质谱法寻找胰腺生物标志物，现在更集中，假设- 使用最先进的蛋白质组学和磷酸化蛋白质组学技术推动研究。 沿着上述的研究策略，我还概述了一个职业发展计划， 包括教学（在哈佛推广学校），出席和介绍研讨会和国家/ 国际会议和课程（在哈佛医学院和哈佛公共卫生学院）， 丰富了我在细胞信号传导、胰腺生理学和生物信息学方面的背景知识。作为一个学术机构， 哈佛医学院及其附属医院是科学研究和发现的中心。 我的导师，哈佛医学院教授史蒂文·P·吉吉博士，是一位世界知名的弥撒 光谱测量师他的实验室的重点领域包括开发和应用质量领域的新技术， 光谱分析和蛋白质组学以及研究动态响应（例如，磷酸化和其他后 翻译修饰）对外来细胞扰动的影响。Gygi博士的实验室通过 国家卫生研究院和工业界以及他以前的博士后和学生已经在高级大学获得了职位 生物技术/制药公司。作为吉吉博士实验室的一员， 先进的质谱仪和专业知识来验证和探索我的数据。吉吉博士每天都参与 实验室的操作，并随时可以作为导师。我还选择了顾问和合作者， 他们是各自领域的专家，因此将获得超越单一导师的支持。 总之，本文提出的工作将不仅对胰腺疾病领域产生影响， 也让我成长为一名独立的科学家。我打算利用我提出的工作的结果， 及其扩展，以建立一个坚实的R 01赠款申请，因为我过渡到一个独立的学术 定位并在质谱和胰腺疾病领域创造自己的利基。

ABSTRACT The development of pancreatic fibrosis is a hallmark of pancreatic disease, yet the pathways of fibrogenesis and associated phosphorylation remain unresolved. Pancreatic stellate cells (PaSC) are key mediators of pancreatic fibrosis. Smoking is an independent risk factor for pancreatic disease. In addition, nicotine, the major toxic component of cigarette smoke is implicated in fibrosis in various cell types. I aim to investigate the effects of nicotine on the phosphoprotein alterations of PaSC in efforts to identify 1) nicotinic receptor (nAChR) subunits expressed by PaSC and assess their roles in fibrosis, 2) differences in the kinase activity profiles of PaSC ¿ nicotine, and 3) alterations in protein phosphorylation events in PaSC due to nicotine. I will test the hypothesis that nicotine alters kinase-regulated cellular signaling pathways in PaSC resulting in morphological and functional alterations, which may be precursors of pancreatic disease. In Specific Aim 1, I will determine the nAChR subtypes involved in nicotine-induced signal transduction in PaSC and their roles in fibrosis. A human PaSC cell line will be incubated with and without nicotine. Western blotting will assess proteins characteristic of PaSC activation and identify nAChR subunits that are present. siRNA will be used to knockdown specific nAChR subunits and assessments will be made using western blotting and MTT-based cell proliferation assays. In Specific Aim 2, I will determine the kinase profile alterations of nicotine-treated PaSC. Using the Kinase ActivitY Assay for Kinome profiling (KAYAK), I will identify and quantify kinases that are expressed in PaSC upon exposure to nicotine. Western blotting will be used to normalize kinase activity by expression level, if needed. In Specific Aim 3, I will determine the rapid phosphorylation alterations in PaSC resulting from nicotine treatment. Using global phosphoprotein identification strategies developed and established in the Gygi laboratory (i.e., kinase activity assays, titanium dioxide phosphopeptide enrichment and isobaric tandem mass tag (TMT)-based quantitation), I will determine time-dependent changes in localized phosphorylation sites of PaSC proteins upon nicotine treatment. Validation of the effects will be performed using MTT assays and Western blotting. Upon completion of these aims, I expect to have determined 1) the nAChR subtypes expressed by PaSC, 2) the kinase activity profiles involved in nicotine-induced PaSC cellular alterations, and 3) phosphopeptides unique to either untreated or nicotine-treated PaSC and map the localized phospho-sites under various cellular conditions (i.e., ¿nicotine, ¿various kinase inhibitors). These data will allow me to determine kinase inhibitors that may counteract the effects of nicotine. This work will have an impact in the field of pancreatic disease and is in accordance with Research Goal 10.2 of the 2008 Recommendations of the National Commission on Digestive Diseases, seeking to investigate the role of smoking and PaSC in fibrosis of the pancreas. The work proposed herein is in line with my long-term goal to understand more clearly the mechanisms of pancreatic disease to develop improved therapies to slow, halt, or ameliorate chronic pancreatitis and pancreatic cancer. The proposed work is reflective of the progression from my graduate school work on nicotinic receptors, to my current postdoctoral research searching for pancreatic biomarkers using mass spectrometry, and now to more focused, hypothesis- driven research using state-of-the-art proteomic and phosphoproteomic technologies. Along with the aforementioned research strategy, I have outlined a career development plan which includes teaching (at the Harvard Extension School), attendance and presentation in seminars and national/ international conferences, and coursework (at Harvard Medical School and Harvard School of Public Health) to enrich my background in cell signaling, pancreatic physiology, and bioinformatics. As an academic institution, Harvard Medical School and its associated hospitals is a hub of scientific research and discovery. My mentor, Dr. Steven P. Gygi, a professor at Harvard Medical School, is a world-renowned mass spectrometrist. Areas of focus in his lab include developing and applying new technologies in the fields of mass spectrometry and proteomics and investigating dynamic responses (e.g., phosphorylation and other post translational modifications) to extraneous cellular perturbations. Dr. Gygi's lab has been well funded via the NIH and industry and his former post-docs and students have acquired positions at high-ranking universities and biotechnology/pharmaceutical companies. As a member of Dr. Gygi's lab, I will have access to the most advanced mass spectrometers and expertise to validate and explore my data. Dr. Gygi is involved in the daily operation of the lab and is readily available as a mentor. I have also chosen consultants and collaborators who are experts in their respective fields and as such will have support beyond a single mentor. In summary, the work proposed herein will not only have an impact on the field of pancreatic disease, but also allow me to grow as an independent scientist. It is my intention to use the results from my proposed work, and extensions thereof to build a solid R01 grant application as I transition into an independent academic position and create my own niche in the fields of mass spectrometry and pancreatic disease.