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: 8635107
• 负责人: Joao A Paulo
• 金额: $ 12.84万
• 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/8635107
• 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

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.

抽象的 胰腺纤维化的发展是胰腺疾病的标志，但是 纤维发生和相关的磷酸化仍未解决。胰腺星细胞（PASC）是关键 胰腺纤维化的介体。吸烟是胰腺疾病的独立危险因素。此外， 尼古丁是香烟烟雾的主要有毒成分，暗示在各种细胞类型的纤维化中。 我的目的是研究尼古丁对PASC磷蛋白改变以识别的影响1） PASC表达并评估其在纤维化中的作用的烟碱受体（NACHR）亚基，2） PASC的激酶活性谱和3）由于PASC蛋白质磷酸化事件的改变 尼古丁。我将检验以下假设，即尼古丁改变了激酶调节的PASC中的细胞信号传导途径 导致形态和功能改变，这可能是胰腺疾病的前体。 在特定目标1中，我将确定参与尼古丁诱导的信号转移的NACHR亚型 PASC及其在纤维化中的作用。人类PASC细胞系将与尼古丁一起孵育。西 印迹将评估PASC激活的蛋白质特征并识别存在的NACHR亚基。 siRNA将用于敲除特定的NACHR亚基，并将使用西方进行评估 印迹和基于MTT的细胞增殖分析。在特定的目标2中，我将确定激酶曲线 尼古丁处理的PASC的改变。使用激酶活性测定进行Kinome分析（皮划艇），我将 识别和量化暴露于尼古丁后在PASC中表达的激酶。蛋白质印迹将是 如果需要，用于按表达水平归一化激酶活性。在特定的目标3中，我将确定快速 尼古丁治疗引起的PASC的磷酸化改变。使用全球磷蛋白 在GYGI实验室制定和建立的识别策略（即激酶活动评估，钛 我将确定二氧化磷酸二肽富集和同烷串联质量标签（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。 效果的验证将使用MTT分析和蛋白质印迹进行。这些完成后 目的，我希望已经确定1）PASC表达的NACHR亚型，2）激酶活性曲线 参与尼古丁诱导的PASC细胞改变，3）未经处理或 尼古丁处理的PASC并在各种细胞条件下绘制局部磷酸化位置（即nicotine， »各种激酶抑制剂）。这些数据将使我能够确定可能抵消的激酶抑制剂 尼古丁的影响。 这项工作将在胰腺疾病领域产生影响，并符合研究目标 10.2国家消化疾病委员会2008年提出的建议 研究吸烟和PASC在胰腺纤维化中的作用。本文提出的工作与 我的长期目标是更清楚地了解胰腺疾病的机制以改善 缓慢，停止或改善慢性胰腺炎和胰腺癌的疗法。拟议的工作是 反映了我从烟碱受体上的研究生院工作到我目前的博士后的发展 研究使用质谱法搜索胰腺生物标志物，现在进行更集中的假设 - 使用最先进的蛋白质组学和磷酸化蛋白质组学技术进行的驱动研究。 除了理性的研究策略外，我概述了一项职业发展计划 包括教学（在哈佛扩展学校），在半岛和国家/国家/地区的出勤和演讲/ 国际会议和课程工作（哈佛医学院和哈佛公共卫生学院） 丰富了我在细胞信号，胰腺生理和生物信息学方面的背景。作为一个学术机构， 哈佛医学院及其相关医院是科学研究与发现的枢纽。 我的心理，哈佛医学院的教授史蒂文·吉吉（Steven P. Gygi）博士是世界知名的群众 光谱学家。他的实验室重点领域包括在群众领域开发和应用新技术 光谱和蛋白质组学以及研究动态反应（例如，磷酸化和其他柱 转化修饰）对外部细胞扰动。 Gygi博士的实验室通过 NIH和工业界以及他以前的毕业后和学生在高级大学获得职位 以及生物技术/制药公司。作为Gygi博士实验室的成员，我将获得最多的访问权限 高级质谱仪和专业知识，以验证和探索我的数据。 Gygi博士参与了每日 实验室的操作，很容易作为心理使用。我还选择了顾问和合作者 是各自领域的专家，因此将获得超越单一问题的支持。 总而言之，本文提出的工作不仅会对胰腺疾病的领域产生影响，而且还会影响 我打算使用我建议的工作的结果， 并在我过渡到独立学术的过程中建立可靠的R01赠款应用程序的扩展 定位并在质谱和胰腺疾病领域中创建自己的利基市场。