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/总结 候选人 我是一名墨西哥裔美国医学博士/博士，担任基础科学家。我也是一名助理教授 在约翰·霍普金斯大学医学院的儿科心脏病学部，我也在那里完成了我的研究生学业。 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了 作为一名四年级医学生。我确信通过 NIH/NHLBI 指导职业提供资金 促进生物医学研究教师多样性的发展奖将有助于实现我的目标 目标是成为分子心脏病学领域的独立研究者和未来的领导者 及其转化为糖尿病心肌病和心力衰竭治疗的进步。  研究提案：新的定量蛋白质组学方法来定义之间改变的相互作用 糖尿病心脏肌丝功能障碍中的 O-GlcNAc 酰化和磷酸化 在北美，2010 年糖尿病患病率为 3740 万人（10.2%），并且正在稳步上升16。 糖尿病患者死于心脏病的风险是普通人群的 2 至 4 倍17。 在心血管并发症中，糖尿病心肌病是指进行性舒张期和收缩期心肌病。 由于独立于冠状动脉而发生的心肌收缩缺陷导致的功能障碍 疾病。虽然 60% 的糖尿病患者存在这种情况，但目前尚无治疗方法可以阻止或显着 改变糖尿病心肌病的病程18。 肌节的翻译后修饰调节心脏功能以及失调时 导致心脏功能障碍。我们小组最近的工作重点是识别、量化和 肌丝 O-GlcNA 酰化和磷酸化的功能表征1-8。该提案的目标是 使用最先进的定量蛋白质组学方法来广泛绘制图谱并进行位点特异性 对正常和正常肌丝蛋白的所有潜在 O-GlcNAcNA 酰化和磷酸化肌丝蛋白进行定量 糖尿病心脏在基线心脏功能期间以及在β-肾上腺素能和力频率刺激期间。经过 比较基线和增强工作负荷之间的 O-GlcNAc/磷酸盐化学计量变化，我们将 确定糖尿病心肌病中肌丝功能异常的关键位点。通过使用基因转移 技术，本提案还将进行体内和体外功能工作，以确定 O-GlcNAc 酰化和磷酸化之间的相互作用以及导致心脏受损的机制 糖尿病的收缩储备。该领域的进步可能会产生糖尿病的早期诊断工具 心肌病并开辟新的治疗场所来修复 糖尿病心脏衰竭的分子马达。具体目标 该提案是 目标 1：执行全局肌丝位点特异性 O- 糖基化和磷酸化的作图和定量 正常和2型糖尿病心脏。 目标 2：识别 O-GlcNA 酰化和磷酸化位点 β-肾上腺素能期间最大的化学计量变化 正常和 2 型糖尿病心脏的力频刺激 肌丝。 目标 3：验证 O-GlcNAcNA 酰化和磷酸化竞争平衡改变的功能影响 通过基因转移操纵肌丝蛋白来调节心肌收缩力的位点。 环境 约翰霍普金斯大学医学院拥有优越的环境来进行基础和 转化研究。约翰·霍普金斯大学在研究领域拥有雄厚的基础和设施 成人和儿童的疾病。例如，儿科目前拥有 2700 万美元 NIH 研究经费。医学园区将使候选人能够接触到众多最先进的核心技术 设施。本申请中列出的导师、顾问和合作者将协助您成功 完成候选人的职业和研究目标。我们组建了一支优秀的优秀科学家团队 并建立了具有多年经验并在指导年轻科学家方面取得巨大成功的师资队伍。我的主要 导师是 Anne M Murphy 博士，共同导师是 Jennifer Van Eyk 博士。杰拉尔德·W·哈特博士和布赖恩·奥罗克博士 组建顾问委员会。