Molecular Modulation of Actomyosin Mechanics by Cardiac Myosin-Binding Protein C

心肌肌球蛋白结合蛋白 C 对肌动球蛋白力学的分子调节

• 批准号: 8762567
• 负责人: Michael Joseph Previs
• 金额: $ 14.36万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-02 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8762567
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Address; Affect; Affinity; Amino Acids; Australia; Award; Binding; Biochemistry; Biological; Biological Assay; Biophysics; Blood; Calcium; Calcium Binding; Cardiac; Cardiac Myosins; Charge; Clinical; Collaborations; Conflict (Psychology); Consensus; Databases; Development Plans; Disease; Event; Exposure to; Familial Hypertrophic Cardiomyopathy; Fluorescence Microscopy; Future; Gene Mutation; Gene Transfer Techniques; Genes; Goals; Head; Health; Heart; Heart failure; Human; Hybrids; Immunoglobulins; In Vitro; Individual; Institution; International; Knock-out; Knowledge; Label; Lasers; Lead; Light; Link; Literature; Mass Spectrum Analysis; Mechanics; Mentors; Mentorship; Microfilaments; Modeling; Molecular; Molecular Biology; Molecular Models; Molecular Motors; Motor; Mus; Muscle; Mutate; Mutation; Myocardium; Myosin ATPase; Myosin Heavy Chains; N-terminal; Phase; Phosphorylation; Play; Post-Translational Protein Processing; Probability; Protein Isoforms; Proteins; Proteomics; Pump; Regulation; Research; Research Personnel; Resolution; Role; Running; Sampling; Sarcomeres; Science; Scientist; Services; Slide; Solutions; Specific qualifier value; Spectrum Analysis; Staging; Sudden Death; Systole; Thick Filament; Thin Filament; Time; Tissue Banking; Tissue Banks; Training; Transgenic Mice; Universities; Ventricular Myosins; Vertebral column; Work; Yeasts; activated Protein C; base; biophysical techniques; blood pump; career; career development; cell motility; experience; genetic regulatory protein; knowledge base; member; molecular mechanics; molecular modeling; mouse model; mutant; myosin-binding protein C; novel; protein expression; research study; single molecule; skills; success; sudden cardiac death; tool; troponin-tropomyosin complex

DESCRIPTION (provided by applicant): Your heart beats ~70 times per minute, with the ventricles ejecting blood during each beat due to calcium-regulated sliding of actin thin filaments past thick filaments composed of tiny myosin molecular motors. Myosin-binding protein C (MyBP-C) is a 140 kD immunoglobulin protein superfamily member that exists within the myosin thick filament. MyBP-C is a critical modulator of the heart's pumping capacity, which is emphasized by genetic mutations being a leading cause of familial hypertrophic cardiomyopathy and sudden death; most notably in young athletes. Despite its clinical impact, the underlying molecular mechanics by which MyBP-C tunes cardiac contractility in healthy hearts is not well understood. Therefore, understanding such mechanisms under normal conditions is necessary to determine how mutations affect its modulatory capacity. With the support of his mentorship team, Dr. Previs will acquire new technical skills and combine state-of-the-art, single molecule biophysical techniques, in vitro protein expression, and quantitative proteomics to define the molecular basis for MyBP-C's functional impact on calcium-dependent actomyosin interactions using native thick and thin filaments isolated from transgenic mouse and failing human hearts. In an effort to unravel MyBP-C's molecular impact on cardiac contractility, Dr. Previs developed a total internal fluorescence microscopy (TIRFM) assay to visualize single actin filaments sliding over native thick filaments from transgenic mouse hearts, with the guidance of Dr. David Warshaw, an expert in single molecule biophysics. Through a combination of molecular biophysics, mass spectrometry-based proteomics, and analytic modeling, he gathered direct molecular evidence that MyBP-C's N-terminal domains interact with actin and/or the myosin head to slow actin filament sliding only where MyBP-C exists within the thick filament. Thus he demonstrated that MyBP-C acts like a governor in a car engine to limit the heart's pumping power. Both the literature and Dr. Previs' current research (submitted to PNAS) suggests that before applying the molecular brakes, MyBP-C's N-terminal domains rev up the heart during the early stages of contraction by activating calcium-regulated thin filaments at low calcium levels, through an independent molecular mechanism. With additional mentoring from Dr. Warshaw and Drs. Jeffrey Robbins and Kathleen Trybus, having expertise in mouse transgenesis, molecular biology and in vitro protein expression, he is proposing to determine if MyBP-C's activation and inhibition of thin filament sliding involve unique MyBP-C N-terminal domains that specify actin and/or myosin S2 binding. With his mentoring team, he will then develop a novel laser trap-based TIRFM assay to observe the sequence of events by which a single fluorescently-tagged MyBP-C molecule binds to a calcium-regulated thin filament and turns it "on" so that myosin motors will bind under low calcium conditions, where binding is normally inhibited. This assay will have broad implications for investigating thin filament regulation by muscle biologists and for continued use throughout his independent career. Specifically, during the independent phase of the award (R00) and beyond, he will combine his graduate training in quantitative mass spectrometry with the biophysical and molecular biological tools and knowledge gained during the mentored phase of the award (K99), to define MyBP-C's role in altering the contractility of failing human myocardium. These studies will benefit from his direct access to the world's largest human cardiac tissue bank (Sydney Heart Bank, Australia) run by Dr. Cris dos Remedios, an expert in fluoresce spectroscopy of actin binding proteins and the molecular basis for heart failure. The inclusion of Dr. Remedios on Dr. Previs' Mentoring Committee during the K99 phase will provide guidance for scientific and career development, and their independent collaboration (R00) will provide Dr. Previs with human myocardium for his studies and exposure to an international group of scientists who utilize the tissue bank to address similar scientific questions from differing perspectives. The science generated by the proposed studies will advance our understanding of MyBP-C's molecular mechanics, build consensus between conflicting molecular models within the field (i.e. actin and/or myosin binding), and provide a critical translational link to human heart failure, where additional thick and thin filament compensatory and/or decompensatory regulatory mechanisms are at play. This award will provide Dr. Previs with a means to acquire new technical skills necessary to address both his short- and long-term hypotheses, and mentorship in career development to obtain his long-term goal of becoming a successful independent investigator at a prestigious academic institution.

描述（由申请人提供）：您的心脏每分钟跳动约70次，由于肌动蛋白细丝的钙调节滑动，心室在每次跳动期间都会喷出血液 穿过由微小肌球蛋白分子发动机组成的粗丝。肌球蛋白结合蛋白C（MyBP-C）是存在于肌球蛋白粗丝内的140 kD免疫球蛋白超家族成员。MyBP-C是心脏泵送能力的关键调节剂，基因突变是家族性肥厚型心肌病和猝死的主要原因，尤其是在年轻运动员中。尽管其临床影响，MyBP-C调节健康心脏心肌收缩力的潜在分子机制尚未得到很好的理解。因此，在正常条件下理解这种机制是必要的，以确定突变如何影响其调节能力。在他的导师团队的支持下，Previs博士将获得新的技术技能，并结合联合收割机最先进的单分子生物物理技术，体外蛋白质表达和定量蛋白质组学，使用从转基因小鼠和衰竭人类心脏中分离的天然粗丝和细丝，确定MyBP-C对钙依赖性肌动球蛋白相互作用的功能影响的分子基础。 为了阐明MyBP-C对心肌收缩力的分子影响，Previs博士在单分子生物物理学专家大卫沃肖博士的指导下，开发了一种全内部荧光显微镜（TIRFM）测定法，以可视化单个肌动蛋白丝在转基因小鼠心脏的天然粗丝上滑动。通过结合分子生物物理学、基于质谱的蛋白质组学和分析建模，他收集了直接的分子证据，证明MyBP-C的N-末端结构域与肌动蛋白和/或肌球蛋白头相互作用，仅在MyBP-C存在于粗丝内的地方减缓肌动蛋白丝的滑动。因此，他证明了MyBP-C的作用就像汽车发动机中的调速器，可以限制心脏的泵送功率。 文献和Previs博士目前的研究（提交给PNAS）都表明，在应用分子制动器之前，MyBP-C的N端结构域通过在低钙水平下激活钙调节的细丝，在收缩的早期阶段通过独立的分子机制加速心脏。Warshaw博士和Jeffrey Robbins博士以及Kathleen Trybus博士在小鼠转基因、分子生物学和体外蛋白质表达方面具有专业知识，他建议确定MyBP-C激活和抑制细丝滑动是否涉及独特的MyBP-C N-末端结构域，该结构域指定肌动蛋白和/或肌球蛋白S2结合。然后，他将与他的指导团队一起开发一种新型的基于激光陷阱的TIRFM检测方法，以观察单个荧光标记的MyBP-C分子与钙调节的细丝结合并将其“打开”的事件序列，以便肌球蛋白马达在低钙条件下结合，其中结合通常被抑制。这种测定将有广泛的影响，研究细丝调节肌肉生物学家和继续使用整个他的独立职业生涯。具体而言，在该奖项的独立阶段（R 00）及以后，他将结合联合收割机他的定量质谱与生物物理学和分子生物学工具和知识的指导阶段期间获得的奖励（K99），以确定MyBP-C的作用，在改变失败的人类心肌收缩性。这些研究将受益于他直接访问世界上最大的人类心脏组织库（悉尼心脏银行，澳大利亚）由Cris dos Remedios博士管理，他是肌动蛋白结合蛋白荧光光谱学和心力衰竭分子基础的专家。在K99阶段，Remedios博士加入Previs博士指导委员会将为科学和职业发展提供指导，他们的独立合作（R 00）将为Previs博士的研究提供人类心肌，并接触国际科学家小组，他们利用组织库从不同角度解决类似的科学问题。 拟议研究产生的科学将促进我们对MyBP-C分子机制的理解，在该领域内冲突的分子模型（即肌动蛋白和/或肌球蛋白结合）之间建立共识，并提供与人类心力衰竭的关键翻译联系，其中额外的粗和细细丝代偿和/或失代偿调节机制正在发挥作用。该奖项将为Previs博士提供一种手段，以获得解决其短期和长期假设所需的新技术技能，并在职业发展方面提供指导，以实现他成为着名学术机构成功的独立调查员的长期目标。