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: 8919943
• 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/8919943
• 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; DNA Sequence Alteration; 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，由于钙调节的肌动蛋白薄丝的滑动，每次拍子在每次节拍过程中射出血液 过去由微小的肌球蛋白分子电机组成的厚细丝。肌球蛋白结合蛋白C（MYBP-C）是一种140 kD免疫球蛋白蛋白超家族成员，存在于肌球蛋白厚细丝内。 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性心肌病和猝死的主要原因。最值得注意的是在年轻运动员中。尽管它具有临床影响，但MyBP-C在健康心脏中心脏收缩力的潜在分子力学尚不清楚。因此，必须在正常条件下理解这种机制，以确定突变如何影响其调节能力。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，以定义MYBP-C对钙依赖性actosyosin对钙依赖性actymyosin相互作用的功能影响的分子基础，利用天然型和薄型跨性别的跨性交小鼠和透射型人的心脏，并将其定义。 为了揭示MyBP-C对心脏收缩性的分子影响，Previs博士开发了总内荧光显微镜（TIRFM）测定法，以可视化单基因小鼠心脏中天然厚细丝的单个肌动蛋白丝，并在单分子生物生物学专家David Warshaw的指导下从转基因小鼠心脏的天然厚细丝上滑落。通过分子生物物理学，基于质谱的蛋白质组学和分析建模的结合，他收集了直接的分子证据，表明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，以减慢肌动蛋白丝的慢速肌动蛋白丝，仅在MyBP-C存在于MyBP-C内的位置。因此，他证明了MYBP-C像汽车发动机中的州长一样，以限制心脏的泵送力量。 文献和Previs博士的当前研究（已提交给PNA）都表明，在施加分子制动器之前，MYBP-C的N末端结构域在收缩的早期阶段通过独立的分子机制激活低钙水平的钙调节的薄细丝，在收缩的早期阶段将心脏升高。在Warshaw博士和Drs博士的额外指导下。 Jeffrey Robbins和Kathleen Trybus在小鼠转基因，分子生物学和体外蛋白表达方面具有专业知识，他提议确定MYBP-C的激活和抑制薄丝滑动是否涉及独特的MyBP-C N末端域，这些MYBP-C N末端域指定了肌动蛋白和/或肌蛋白S2结合。随后，他将借助他的指导团队开发一种新型的激光陷阱TIRFM测定法，以观察事件的序列，通过该测定，单个荧光标记的MYBP-C分子与钙调节的薄丝结合，并将其旋转为“上”，以便在低钙条件下结合肌球蛋白运动，而在较低的钙下结合，在该结合下均受到约束的约束。该测定法对调查肌肉生物学家的细丝调节以及在整个独立职业中的持续使用将具有广泛的影响。具体而言，在奖励的独立阶段（R00）及以后的独立阶段，他将在定量质谱学领域的研究生培训与生物物理和分子生物学工具以及在奖励阶段（K99）中获得的知识相结合，以定义MYBP-C在改变失败人类心肌的收缩性中的作用。这些研究将受益于他直接进入由CRIS DOS REMEDIOS（澳大利亚悉尼心脏银行，澳大利亚悉尼心脏银行）的通道，Cris Dos Remedios是肌动蛋白结合蛋白的荧光光谱专家，也是心力衰竭的分子基础。在K99阶段，将Remedios博士纳入Previs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Previs博士提供人类心肌，以进行研究并接触国际科学家，这些科学家利用组织库从不同的角度来解决类似的科学问题。 拟议的研究产生的科学将提高我们对MYBP-C分子力学的理解，在该领域内（即肌动蛋白和/或肌球蛋白结合）内建立共识，并提供与人体心力衰竭的关键转化联系，在这种情况下，又有较厚的细丝补偿性和/或薄丝补偿性和/或分解的调节性机械机制在效果上。该奖项将为Previs博士提供一种获得所需的新技术技能的方法，以解决他的短期和长期假设以及职业发展的指导，以获得他的长期目标，即成为享有声望的学术机构成功独立研究员的长期目标。