Novel quantitative proteomic approaches to define the altered interplay between OGlcNAcylation and Phosphorylation in myofilament dysfunction of diabetic hearts

新的定量蛋白质组学方法来定义糖尿病心脏肌丝功能障碍中 OGlcNAc 酰化和磷酸化之间相互作用的改变

• 批准号: 9494653
• 负责人: Genaro Antonio Ramirez-Correa
• 金额: $ 12.47万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-09-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9494653
• 关键词: Adrenergic Agents; Adult; Alanine; Anabolism; Aspartic Acid; Basic Science; Biomedical Research; Calcium; Cardiac; Cardiac health; Cardiology; Cardiovascular system; Child; Complication; Core Facility; Coronary Arteriosclerosis; Data; Depressed mood; Diabetes Mellitus; Diagnostic; Disease; Doctor of Philosophy; Echocardiography; Environment; Equilibrium; Etiology; Faculty; Fellowship; Foundations; Frequencies; Functional disorder; Funding; Future; Gene Transfer; Gene Transfer Techniques; Glucose; Goals; Heart; Heart Diseases; Heart failure; Hexosamines; Human; Impairment; In Vitro; Individual; Ions; K-Series Research Career Programs; Knowledge; Label; Lead; Link; Maps; Mass Spectrum Analysis; Measurement; Medical; Medical Students; Mentors; Mexican Americans; Microfilaments; Molecular; Molecular Motors; Muscle; Myocardial; Myocardial dysfunction; Myocardium; National Heart, Lung, and Blood Institute; North America; Pathway interactions; Patients; Pediatric cardiology; Phosphorylation; Phosphorylation Site; Physiological; Play; Post-Translational Protein Processing; Postdoctoral Fellow; Prevalence; Property; Proteins; Proteomics; Reporting; Research; Research Personnel; Research Proposals; Risk; Role; Sarcomeres; Scheme; Scientist; Serine; Site; Techniques; Testing; Threonine; Time; Training; Training Support; Translational Research; Translations; Troponin I; United States National Institutes of Health; Universities; Variant; Work; Workload; adeno-associated viral vector; career; diabetic; diabetic cardiomyopathy; diabetic patient; experience; gene transfer vector; glucose metabolism; heart function; improved; in vivo; inorganic phosphate; interest; medical schools; mitochondrial dysfunction; multiple reaction monitoring; muscle physiology; mutant; novel; novel therapeutics; pandemic disease; pediatric department; professor; response; skills; stoichiometry; success; titanium dioxide; tool

Project Abstract/Summary The candidate I am a Mexican-American MD/PhD who works as a basic scientist. I am also an Assistant Professor in the Johns Hopkins School of Medicine, Division of Pediatric Cardiology where I also completed my post- doctoral fellowship training. My interest in and commitment to a translational and basic research career started as a 4th year medical student. I am convinced that funding through the NIH/NHLBI Mentored Career Development Award to Promote Faculty Diversity in Biomedical Research will be instrumental to achieve my goal, which is to become an independent investigator and a future leader in the field of molecular cardiology and its translation into the advancement of therapies of diabetic cardiomyopathy and heart failure.  Research Proposal: Novel quantitative proteomic approaches to define the altered interplay between O-GlcNAcylation and Phosphorylation in myofilament dysfunction of diabetic hearts In North America, the 2010 prevalence of diabetes was 37.4 million (10.2%) and is on a steady rise16. Diabetic patients are 2 to 4 times more at risk of dying from heart disease than the general population17. Among cardiovascular complications, diabetic cardiomyopathy refers to a progressive diastolic and systolic dysfunction due to a contractile deficit of the cardiac muscle that develops independently from coronary artery disease. While it is present in 60% of diabetic patients, no therapy is currently available to halt or significantly alter the course of diabetic cardiomyopathy18. Post-translational modifications of the sarcomere regulate cardiac function and when dysregulated contribute to cardiac dysfunction. Recent work in our group has focused on the identification, quantification and functional characterization of myofilament O-GlcNAcylation and Phosphorylation1-8. The goal of this proposal is to use state of the art quantitative proteomic approaches to extensively map and perform site-specific quantification of all potentially O-GlcNAcylated and Phosphorylated myofilament proteins of normal and diabetic hearts during baseline cardiac function and during β-adrenergic and force-frequency stimulation. By comparing O-GlcNAc/Phosphate stoichiometry changes between baseline and enhanced workload we will identify key sites for abnormal myofilament function in diabetic cardiomyopathy. By using gene transfer techniques, the present proposal also will perform in vivo and in vitro functional work to define the role of the interplay between O-GlcNAcylation and Phosphorylation and the mechanisms that lead to impaired cardiac contractile reserve in diabetes. Advances in this field can potentially generate early diagnostic tools for diabetic cardiomyopathy and open new therapeutic venues to fix the molecular motors of a failing diabetic heart. The specific aims of this proposal are Aim 1: To perform global myofilament site-specific O- GlcNAcylation and Phosphorylation mapping and quantification in normal and type 2 diabetic hearts. Aim 2: To identify O-GlcNAcylated and Phosphorylated sites with the greatest stoichiometric variation during β-adrenergic and force-frequency stimulation in normal and type 2 diabetic heart myofilaments. Aim 3: To validate the functional impact of altered balance of O-GlcNAcylation and Phosphorylation competing sites on cardiac contractility by manipulating myofilament proteins with gene transfer. The environment The Johns Hopkins School of Medicine possesses an excellent environment to perform basic and translational research. Johns Hopkins University has a strong foundation and facilities in research focused on diseases of adults and children. For example, the Department of Pediatrics presently has 27 million dollars in NIH research dollars. The medical campus will enable the candidate to access numerous state-of-the-art core facilities. The mentors, advisors and collaborators outlined in this application will assist in a successful completion of the candidate career and research goals. We have assembled a superb team of fine scientist and established faculty with many years of experience and great success mentoring young scientists. My main mentor is Dr. Anne M Murphy, co-mentor is Dr. Jennifer Van Eyk. Dr. Gerald W. Hart and Dr. Brian O'Rourke form the advisor committee.

项目摘要/摘要 候选人 我是墨西哥裔美国医学博士/博士，他是基础科学家。我也是一名助理教授 约翰·霍普金斯医学院儿科心脏病学系，我还完成了我的邮政 博士奖学金培训。我对转化和基础研究职业的兴趣和承诺开始 作为四年级的医学生。我坚信，通过NIH/NHLBI指导的职业资金 促进生物医学研究教师多样性的发展奖将对我的 目标是成为分子心脏病领域的独立研究者和未来的领导者 它转化为糖尿病心肌病和心力衰竭疗法的发展。 研究建议：定义定义变化相互作用的新型定量蛋白质组学方法 糖尿病心脏的肌丝功能障碍中的O-Glcnacylation和磷酸化 在北美，2010年的糖尿病患病率为3740万（10.2％），稳定上升16。 糖尿病患者的死亡风险比一般人群高2至4倍17。 在心血管并发症中，糖尿病性心肌病是指进行性舒张压和收缩期 由于对心肌的收缩防御而导致的功能障碍，该防御与冠状动脉独立发展 疾病。虽然它存在于60％的糖尿病患者中，但目前尚无治疗可用于停止或明显地 改变糖尿病心肌病的过程。18。 肌节的翻译后修饰调节心脏功能，并且当失调失调时 导致心脏功能障碍。我们小组的最新工作重点是识别，量化和 肌丝O-Glcnacylation和磷酸化的功能表征1-8。该提议的目的是 使用艺术状态定量蛋白质组学方法来广泛绘制和执行特定地点 正常和 基线心脏功能以及β-肾上腺和力频刺激期间的糖尿病心脏。经过 比较基线和增强工作量之间的O-GLCNAC/磷酸盐化学计量法变化，我们将 确定糖尿病心肌病异常肌丝功能的关键部位。通过使用基因转移 技术，本提案还将在体内和体外功能工作中执行以定义的作用 O-Glcnacylation和磷酸化与导致心脏受损的机制之间的相互作用 糖尿病的收缩储备。该领域的进步可能会生成糖尿病的早期诊断工具 心肌病和开放新疗法场所修复 糖尿病心脏失败的分子电动机。具体目的 该提议是 目标1：执行全球肌丝特定于特定于特定位点的O- Glcnacylation和磷酸化映射和定量 正常和2型糖尿病心脏。 目标2：识别具有O-Glcnacylated和磷酸化位点的 β-肾上腺素能和 正常和2型糖尿病心脏的力频率刺激 肌膜。 目标3：验证O-Glcnacylation和磷酸化竞争的改变平衡的功能影响 通过用基因转移操纵肌丝蛋白来进行心脏收缩的位点。 环境 约翰·霍普金斯医学院（Johns Hopkins of Medicine of Medicine of Medicine of School of Schore of Butason）拥有一个极好的环境 翻译研究。约翰·霍普金斯大学（Johns Hopkins University 成人和儿童的疾病。例如，介绍的儿科部有2700万美元 NIH研究美元。医疗校园将使候选人能够获得许多最先进的核心 设施。该应用程序中概述的导师，顾问和合作者将有助于成功 完成候选职业和研究目标。我们组建了一支精湛的科学家团队 并以多年的经验和巨大的成功心态建立了教师。我的主要 导师是Anne M Murphy博士，同事是Jennifer Van Eyk博士。 Gerald W. Hart博士和Brian O'Rourke博士 组成顾问委员会。