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(申请人提供)：你的心脏每分钟跳动70次，由于钙调节的肌动蛋白细丝的滑动，脑室在每一次跳动中射血。 过去由微小的肌球蛋白分子马达组成的粗丝。肌球蛋白结合蛋白C(MyBP-C)是存在于肌球蛋白粗丝中的一个140kD的免疫球蛋白超家族成员。MyBP-C是心脏泵送能力的关键调节因子，基因突变是家族性肥厚型心肌病和猝死的主要原因，这一点在年轻运动员中最为明显。尽管MyBP-C对临床有影响，但MyBP-C调节健康心脏收缩能力的潜在分子机制尚不清楚。因此，了解正常条件下的这种机制对于确定突变如何影响其调节能力是必要的。在他的指导团队的支持下，Previs博士将获得新的技术技能，并结合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，以确定MyBP-C使用从转基因小鼠和衰竭的人类心脏分离出来的天然粗和细丝，对依赖钙的肌球蛋白相互作用的功能影响的分子基础。为了揭示MyBP-C对心肌收缩能力的分子影响，Previs博士在单分子生物物理学专家David Warshaw博士的指导下，开发了一种全内部荧光显微镜(TIRFM)分析方法，可以显示转基因小鼠心脏中单个肌动蛋白细丝穿过天然粗丝的情况。通过结合分子生物物理学、基于质谱学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/或肌球蛋白头部相互作用，以减缓肌动蛋白细丝仅在MyBP-C存在于粗丝内的地方滑动。因此，他证明了MyBP-C的作用就像汽车发动机的调速器一样，限制了心脏的泵送功率。文献和Previs博士目前的研究(提交给PNAS)都表明，在施加分子刹车之前，MyBP-C的N-末端结构域在心脏收缩的早期阶段通过激活低钙水平下受钙调节的细丝，通过一个独立的分子机制使心脏加速。在Warshaw博士、Jeffrey Robbins博士和Kathen Trybus博士的额外指导下，他在小鼠转基因、分子生物学和体外蛋白质表达方面拥有专业知识，他提议确定MyBP-C对细丝滑动的激活和抑制是否涉及指定肌动蛋白和/或肌球蛋白S2结合的独特的MyBP-C N末端结构域。然后，他将与他的指导团队开发一种新型的基于激光陷阱的TIRFM分析方法，以观察单个荧光标记的MyBP-C分子与钙调节的细丝结合并打开它的事件序列，从而使肌球蛋白马达在低钙条件下结合，而低钙条件通常是结合被抑制的。这项测试将对肌肉生物学家研究细丝调节以及在他的独立职业生涯中继续使用具有广泛的意义。具体地说，在奖项的独立阶段(R00)及以后，他将把他在定量质谱学方面的研究生培训与在奖项指导阶段(K99)获得的生物物理和分子生物学工具和知识结合起来，以确定MyBP-C在改变衰竭人类心肌收缩能力方面的作用。这些研究将受益于他直接接触到世界上最大的人类心脏组织库(澳大利亚悉尼心脏银行)，该银行由克里斯·多斯·雷米迪奥斯博士经营，他是肌动蛋白结合蛋白的荧光光谱和心力衰竭的分子基础方面的专家。在K99阶段将雷米迪奥斯博士纳入普雷维斯博士的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(R00)将为普雷维斯博士的研究和与国际科学家小组的接触提供人体心肌，这些科学家利用组织库从不同的角度解决类似的科学问题。拟议中的研究产生的科学将促进我们对MyBP-C分子力学的理解，在该领域内相互冲突的分子模型(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，在那里，额外的粗和细丝代偿和/或失代偿调节机制正在发挥作用。这一奖项将为Previs博士提供一种手段，以获得必要的新技术技能，以解决他的短期和长期假设，并在职业发展方面提供指导，以实现他成为一家著名学术机构的成功独立研究员的长期目标。