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一个标志，但胰腺纤维化的途径 纤维发生和相关的磷酸化仍未解决。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， 尼古丁是香烟烟雾的主要有毒成分，与多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，以努力确定 1) PaSC 表达的烟碱受体 (nAChR) 亚基并评估其在纤维化中的作用，2) PaSC 的激酶活性概况 ¿ 尼古丁，以及 3) PaSC 中蛋白质磷酸化事件的改变 尼古丁。我将检验尼古丁改变 PaSC 中激酶调节的细胞信号传导途径的假设 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信号转导的 nAChR 亚型 PaSC 及其在纤维化中的作用。人类 PaSC 细胞系将​​在有或没有尼古丁的情况下进行培养。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 将用于敲低特定的 nAChR 亚基，并使用 Western 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。使用激酶活性分析进行激酶组分析 (KAYAK)，我将 识别并定量暴露于尼古丁后在 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 尼古丁治疗导致 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 蛋白局部磷酸化位点发生时间依赖性变化。 将使用 MTT 测定和蛋白质印迹法验证效果。完成这些后 目标，我希望确定 1) PaSC 表达的 nAChR 亚型，2) 激酶活性概况 参与尼古丁诱导的 PaSC 细胞改变，以及 3) 未经处理或未经处理的磷酸肽所特有的 尼古丁处理的 PaSC 并绘制各种细胞条件下的局部磷酸位点图谱（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的激酶抑制剂 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 国家消化疾病委员会 2008 年建议中的 10.2 条旨在 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 我的长期目标是更清楚地了解胰腺疾病的发展机制 减缓、停止或改善慢性胰腺炎和胰腺癌的疗法。拟议的工作是 反映了我从研究生院研究烟碱受体到目前博士后的进展 使用质谱法寻找胰腺生物标志物的研究，现在更加集中，假设- 使用最先进的蛋白质组学和磷酸化蛋白质组学技术驱动研究。 除了上述研究策略外，我还概述了一份职业发展计划，其中 包括教学（在哈佛延伸学院）、出席研讨会和国家/地区研讨会和进行演讲 国际会议和课程（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。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。 我的导师史蒂文·P·吉吉博士是哈佛医学院的教授，是一位世界著名的大众学家 光谱仪。他实验室的重点领域包括在质量领域开发和应用新技术 光谱测定和蛋白质组学以及研究动态响应（例如磷酸化和其他后处理） 翻译修饰）到无关的细胞扰动。 Gygi 博士的实验室得到了充足的资助 NIH 和工业界以及他以前的博士后和学生都在一流大学获得了职位 和生物技术/制药公司。作为 Gygi 博士实验室的成员，我将能够接触到最 先进的质谱仪和专业知识来验证和探索我的数据。 Gygi 博士参与日常工作 实验室的运作，并且随时可以作为导师。我还选择了顾问和合作者 都是各自领域的专家，因此将获得超越单一导师的支持。 总之，本文提出的工作不仅会对胰腺疾病领域产生影响，而且还会对胰腺疾病领域产生影响。 也让我成长为一名独立科学家。我打算使用我提议的工作的结果， 及其扩展，以在我过渡为独立学者时构建可靠的 R01 资助申请 在质谱和胰腺疾病领域定位并创造自己的利基市场。