Resolving the role of nicotine-mediated phosphorylation on pancreatic fibrosis

描述(申请人提供)：胰腺纤维化的发展是胰腺疾病的一个标志，但纤维化形成和相关的磷酸化途径仍未解决。胰腺星状细胞(PASC)是胰腺纤维化的关键介质。吸烟是胰腺疾病的独立危险因素。此外，香烟烟雾中的主要有毒成分尼古丁与各种细胞类型的纤维化有关。我的目标是调查 尼古丁对PASC磷酸化蛋白改变的影响1)PASC表达的尼古丁受体(NAChR)亚单位并评估其在纤维化中的作用；2)PASC中尼古丁的活性差异；以及3)尼古丁引起的PASC蛋白磷酸化事件的改变。我将测试一种假设，即尼古丁改变PASC中由激酶调节的细胞信号通路，导致形态和功能改变，这可能是胰腺疾病的先兆。在具体目标1中，我将确定参与尼古丁诱导的PASC信号转导的nAChR亚型及其在纤维化中的作用。人类PASC细胞株将在尼古丁和不含尼古丁的情况下孵育。Western blotting将评估PASC激活的蛋白质特征，并识别存在的nAChR亚基。SiRNA将被用来敲除特定的nAChR亚基，并将使用蛋白质印迹和基于四甲基偶氮唑盐的细胞增殖分析进行评估。在特定的目标2中，我将确定尼古丁处理的PASC的激酶谱变化。使用Kinome Profile的Kinase Activity Assay(Kayak)，我将识别和量化暴露于尼古丁时在PASC中表达的激酶。如果需要，蛋白质印迹将被用来根据表达水平来使激酶活性正常化。在具体目标3中，我将确定尼古丁治疗引起的PASC的快速磷酸化改变。利用Gygi实验室开发和建立的全球磷蛋白鉴定策略(即，激酶活性分析、二氧化钛磷酸肽富集法和基于等压串联质量标签(TMT)的定量方法)，我将确定尼古丁处理后PASC蛋白局部磷酸化位点随时间的变化。将使用四甲基偶氮唑盐分析和Western blotting来验证效果。在完成这些目标后，我预计已经确定了1)PASC表达的nAChR亚型，2)参与尼古丁诱导的PASC细胞变化的激酶活性谱，以及3)未经处理或经尼古丁处理的PASC特有的磷酸肽，并绘制了不同细胞条件下的定位磷酸化位点图(即尼古丁，各种激酶抑制剂)。这些数据将使我能够确定可能抵消尼古丁影响的激酶抑制剂。这项工作将对胰腺疾病领域产生影响，并符合国家消化疾病委员会2008年建议的研究目标10.2，旨在调查吸烟和PASC在胰腺纤维化中的作用。本文提出的工作符合我的长期目标，即更清楚地了解胰腺疾病的机制，以开发改进的治疗方法来减缓、阻止或改善慢性胰腺炎和胰腺癌。这项拟议的工作反映了我从研究生院在尼古丁受体方面的工作，到我目前的博士后研究，使用质谱学寻找胰腺生物标记物，再到现在使用最先进的蛋白质组和磷酸蛋白质组技术进行更专注、假设驱动的研究。除了上述研究战略，我还概述了一项职业发展计划，其中包括(在哈佛推广学院)任教，出席研讨会和国内/国际会议并发表演讲，以及(在哈佛医学院和哈佛公共卫生学院)课程作业，以丰富我在细胞信号、胰腺生理学和生物信息学方面的背景。作为一所学术机构，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质谱学家。他在实验室的重点领域包括开发和应用质谱学和蛋白质组学领域的新技术，以及研究对外来细胞扰动的动态反应(例如，磷酸化和其他翻译后修饰)。Gygi博士的实验室通过NIH和产业界获得了充足的资金，他以前的博士后和学生已经在一流大学和生物技术/制药公司获得了职位。作为吉吉博士实验室的一员，我将有机会接触到最先进的质谱仪和专业知识来验证和探索我的数据。吉吉博士参与了实验室的日常运作，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，因此将获得比单一导师更多的支持。综上所述，本文提出的工作不仅将对胰腺疾病领域产生影响，还将让我作为一名独立的科学家成长。我打算利用我提议的工作及其扩展的结果，在我过渡到一个独立的学术职位并在质谱学和胰腺疾病领域创造自己的利基市场时，建立一个坚实的R01拨款申请。

DESCRIPTION (provided by applicant): 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) nicotinc 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 i 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.