Resolving the role of nicotine-mediated phosphorylation on pancreatic fibrosis

解决尼古丁介导的磷酸化对胰腺纤维化的作用

• 批准号: 8735012
• 负责人: Joao A Paulo
• 金额: $ 12.78万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8735012
• 关键词: Adenocarcinoma; Affect; Applications Grants; Architecture; Area; Binding; Bioinformatics; Biological Assay; Biological Markers; Biotechnology; Cell Line; Cell Proliferation; Cells; Cellular Morphology; Characteristics; Cigarette; Data; Development; Development Plans; Digestive System Disorders; Disease; Drosophila acetylcholine receptor alpha-subunit; Eating; Educational process of instructing; Event; Exposure to; Extracellular Matrix; Fibrosis; Funding; Gland; Goals; Hospitals; Human; Incubated; Industry; Institution; Intention; International; Intervention; Laboratories; Malignant neoplasm of pancreas; Maps; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Mentors; Methods; Molecular; Morphology; Nicotine; Nicotinic Receptors; Organ; Pancreas; Pancreatic Diseases; Pathway interactions; Pharmacologic Substance; Phosphopeptides; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Play; Positioning Attribute; Post-Translational Protein Processing; Postdoctoral Fellow; Production; Proteins; Proteomics; Public Health Schools; RNA; Recommendation; Research; Risk; Risk Factors; Role; Schools; Scientist; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Smoke; Smoking; Solid; Students; Surface; Technology; Testing; Time; Titania; Titanium; Toxic effect; Toxin; United States National Institutes of Health; Universities; Validation; Western Blotting; Work; base; career development; cell type; chronic pancreatitis; cigarette smoking; cigarette smoking; expectation; extracellular; fibrogenesis; improved; in vitro Assay; inhibitor/antagonist; kinase inhibitor; mass spectrometer; medical schools; member; new technology; operation; professor; public health relevance; receptor; response; stellate cell; symposium; therapy development; titanium dioxide; trafficking

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.

描述（由申请人提供）：胰腺纤维化的发展是胰腺疾病的标志，但纤维发生和相关磷酸化的途径仍未得到解决。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，香烟烟雾的主要毒性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磷蛋白改变的影响，以鉴定1）PaSC表达的尼古丁受体（nAChR）亚基并评估其在纤维化中的作用，2）PaSC尼古丁激酶活性谱的差异，3）尼古丁引起的PaSC蛋白磷酸化事件的改变。我将检验尼古丁改变PaSC中激酶调节的细胞信号传导途径导致形态和功能改变的假设，这可能是胰腺疾病的前兆。 在特定目标1中，我将确定参与PaSC中尼古丁诱导的信号转导的nAChR亚型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。蛋白质印迹法将评估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，并将使用蛋白质印迹和基于MTT的细胞增殖测定进行评估。在具体目标2中，我将确定尼古丁处理的PaSC的激酶谱改变。使用Kinase ActivitY Assay for Kinome profiling（KAYAK），本人将鉴定并定量暴露于尼古丁后PaSC中表达的激酶。如果需要，将使用蛋白质印迹法通过表达水平标准化激酶活性。在具体目标3中，我将确定尼古丁处理导致的PaSC快速磷酸化改变。使用Gygi实验室开发和建立的全局磷蛋白鉴定策略（即，激酶活性测定、二氧化钛磷酸肽富集和基于同量异位串联质量标签（TMT）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试验和蛋白质印迹法对效果进行验证。在完成这些目标后，我期望已经确定了1）PaSC表达的nAChR亚型，2）尼古丁诱导的PaSC细胞改变中涉及的激酶活性谱，和3）未处理或尼古丁处理的PaSC特有的磷酸肽，并在各种细胞条件下定位磷酸化位点（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，旨在研究吸烟和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，以开发改进的疗法来减缓、停止或改善慢性胰腺炎和胰腺癌。拟议的工作反映了从我在研究生院对烟碱受体的研究，到我目前的博士后研究，使用质谱法寻找胰腺生物标志物，再到现在使用最先进的蛋白质组学和磷酸蛋白质组学技术进行更集中的假设驱动研究的进展。 沿着上述研究策略，我已经概述了一个职业发展计划，其中包括教学（在哈佛推广学校），出席研讨会和国家/国际会议并发表演讲，以及课程（在哈佛医学院和哈佛公共卫生学院），以丰富我在细胞信号传导，胰腺生理学和生物信息学方面的背景。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，哈佛医学院的教授史蒂文·P·吉吉博士，是一位世界知名的质谱仪专家。他实验室的重点领域包括开发和应用质谱和蛋白质组学领域的新技术，以及研究动态响应（例如，磷酸化和其他翻译后修饰）对外来细胞扰动的影响。Gygi博士的实验室通过NIH和行业获得了充足的资金，他以前的博士后和学生也在高水平大学和生物技术/制药公司获得了职位。作为Gygi博士实验室的一员，我将有机会使用最先进的质谱仪和专业知识来验证和探索我的数据。Gygi博士参与实验室的日常运作，并随时担任导师。我还选择了顾问和合作者，他们都是各自领域的专家，因此将得到超越单一导师的支持。 总之，本文提出的工作不仅会对胰腺疾病领域产生影响，而且会让我成长为一名独立的科学家。这是我打算使用的结果，从我提出的工作，及其扩展，以建立一个坚实的R 01补助金申请，因为我过渡到一个独立的学术立场，并创建自己的利基在质谱和胰腺疾病领域。