Giant Proteins, Slender Filaments And Elastic Cytoskeleton

巨型蛋白质、细长丝和弹性细胞骨架

• 批准号: 7964904
• 负责人: KUAN WANG
• 金额: $ 65.89万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964904
• 关键词: ATP phosphohydrolase; Actins; Address; Adult; Affinity; Affinity Chromatography; Antibodies; Atomic Force Microscopy; Behavior; Binding; Binding Proteins; Binding Sites; Biochemistry; Biocompatible Materials; Biophysics; Biopolymers; C-terminal; Calcium; Calmodulin; Cardiac; Cells; Cessation of life; Collaborations; Coupling; Cytoskeleton; Data; Disease; Elasticity; Engineering; Event; Exons; F-Actin; Family; Family member; Filament; Gel; Genes; Goals; Human; Kinetics; Laboratories; Lasers; Lead; Length; Ligand Binding; Link; Maintenance; Measurement; Measures; Mechanical Stress; Mechanics; Mediator of activation protein; Methodology; Methods; Microfilaments; Molecular; Molecular Conformation; Molecular Mechanisms of Action; Molecular Models; Molecular and Cellular Biology; Motor; Motor Activity; Muscle; Muscle Cells; Muscle Development; Muscle Fibers; Muscle Weakness; Muscle function; Mutation; Myocardium; Myofibrillogenesis; Myopathy; Myosin ATPase; N-terminal; Names; Nanotechnology; Neoplasm Metastasis; Phase; Phosphotransferases; Physiological; Physiology; Play; Process; Proline; Property; Protein Isoforms; Proteins; RNA Splicing; Regulation; Research; Resistance; Resolution; Role; S100 Proteins; Sarcomeres; Signal Pathway; Signal Transduction; Signaling Protein; Site; Skeletal Muscle; Slide; Sodium Chloride; Solutions; Sorting - Cell Movement; Stretching; Striated Muscles; Structural Protein; Structure; Surface; System; Techniques; Technology; Testing; Thin Filament; Tissue Engineering; Tropomyosin; Troponin; United States National Institutes of Health; Universities; Work; X-Ray Crystallography; base; cell assembly; cell motility; connectin; design; genetic regulatory protein; human EMS1 protein; insight; instrument; interdisciplinary approach; interest; member; molecular modeling; monolayer; nanomechanics; nanopattern; nebulin; prevent; protein protein interaction; sensor; simulation; single molecule; skeletal; synthetic peptide; theories; titin kinase

Titin and nebulin are unprecedented giant proteins discovered and named in this laboratory. Our recent work has focused on the roles of titin and nebulin in the structure, regulation, mechanics and assembly of sarcomeres in striated muscles. We have employed a multidisciplinary approach that draws on methodologies and concepts of biochemistry, biophysics, cell and molecular biology, and physiology. (A) Titin: A primary theme of our research is the understanding of the structural and physiological roles of titin, a family of giant structural proteins that constitute an elastic matrix in the striated muscle sarcomeres and some nonmuscle cells. Titin and the elastic matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability, the assembly of nascent sarcomeres in developing muscle cells, and the assembly of myosin in the microfilaments in nonmuscle cells. The following questions of central importance are being addressed: 1. How do titin filaments respond to stretch and release? 2. What is the molecular basis of titin elasticity? 3. Do thin filaments modulate titin elasticity? 4. Does titin participate in the regulation of actin-myosin interactions? 5. How do the signaling capacity of titin and titin kinase unction in muscle? We are testing the hypothesis that reversible unfolding of ordered domains such as titin kinase and reversible unraveling of intrinsically disordered segments are two distinct and complementary mechanisms of titin elasticity and function. The elasticity of isolated full length titin and engineered fragments and analogous elastic molecules from skeletal and cardiac and flight muscles are being measured directly, as single molecules and as gel networks, with laser trap techniques, atomic force microscopy (AFM) and rheometric techniques. The conformation of titin domains and synthetic peptides are being studied by high resolution NMR and X-ray crystallography. Titin elasticity is being explained with theories of biopolymers combined with realistic and experimental structural features of titin domains. In the past two years, we have succeeded in revealing that the elastic PEVK segment of titin is modular in construction, display repeating proline II helix /coil conformational motifs and most importantly, interact with thin filaments in a calcium/S100 sensitive manner. Our observations established the conformational basis of titin PEVK elasticity and identified PEVK as a site of interfilament interaction and that S100 or calcium sensor proteins regulate interaction and titin elasticity. Moreover, we discovered that the titin PEVK segment is a giant force sensing molecule of the SH3 signaling pathways. High resolution structures of a homopolymer of a PEVK module in solution and in gel phase has been determined by NMR techniques in collaboration with Prof. Wittebort at University of Louisville. The extensive differential splicing of titin PEVK exons in various human heart muscles was also elucidated. The PEVK domain is an intrinsically disordered protein with short-range structure. AFM measurements on PEVK suggested that its elasticity was driven in part by salt bridges. NMR data demonstrated the existence of specific salt bridges. Using the NIH Biowulf cluster, we applied molecular modeling techniques to understand dynamic interactions at the atomic level. TheMD and SMD simulations have shown further that the manifold of ionic interactions constantly evolves throughout the stretching. These new insights into the behavior of salt bridges in titin PEVK may provide insights in designing elastic polyampholytes as biomaterials for nanotechnology and tissue engineering. These simulations also help us to understand how ligand binding can be regulated by force and only bind when the protein is stretched to unmask SH3 binding sites. We are also designing a new type of instrrument to measure ligand binding and protein-protein interactions as proteins are being stretched. This new instrument will allow us to investigate directly how signaling protein interactions respond to mechanical stress. (B) Nebulin: Nebulin is the second largest protein ( 0.7 MDa) in the skeletal muscle sarcomere. It acts as a ruler for thin filament length and may act as a calcium-linked regulatory protein that is distinct from the classic troponin/tropomyosin system. In muscle development, nebulin is thought to expedite the maturation process of the thin filament assembly. Mutation in the nebulin gene leads to the disease nemalin myopathy, which causes muscle weakness and early death. The following questions are being addressed: 1. How does nebulin regulate the length of thin filaments? 2. How does nebulin regulate actin-myosin interaction? 3. What role does the nanomechanics of nebulin play in muscle function The length, mass and sequence of nebulin size isoforms have been compared with thin filament lengths of a wide range of skeletal muscles in developing and adult muscles. Together with the effect of over expression of nebulin domains on myofibrillogenesis in cultured skeletal muscle cells, we concluded that both the number of nebulin repeats and the specific N-terminal and C-terminal termination sequences are necessary to give rise to uniform thin filament length distribution within each sarcomere. In the past two years, we have successfully purified native full length nebulin from skeletal muscles and tested its effect on the length distribution of polymerizing or performed actin filaments. The measurement of actin filment length unfortunately was complicated by the gelling of actin filaments by the multivalent actin binding sites on each nebulin molecule. To alleviate this roblem, we are designing a new nanotechnology-based platform to measure the effect of nebulin on actin filament length distribution on a nanopatterned substrate that allows parallel actin filaments to elongate in the same direction and polarity without gelation. The kinetics of nebulin on ATPase activities is being studied by stop flow methods to define plausible molecular mechanisms of action. The influence of nebulin and calcium/calmodulin on the sliding velocity of actin over myosin is being investigated to reveal the effect of nebulin on mechanical coupling of myosin motors. We are searching for other potential calcium mediators by affinity chromatography and blotting. We are particularly interested in the possible involvement of S100 proteins as well as the kinase/phosphotase that phosphorylate nebulin as physiological effectors of actin/myosin interaction. We measured the nanomechncial properties of full-length native nebulin with the AFM. These required the implementation of the use of high affinity sitespecific antibodies pairwise to stretch nebulin between the nebulin; the use nof protein resistant self-assembled monolayers to prevent non-specific tip-surface interaction, a new method of sorting out single vs. multiple molecular stretching and the adaption of a powerful empirical mode decomposition (HHT) method to identify force-generating events. The most unique and unexpected finding is that nebulin must be stretched long enough to wrap around and match the peroriodicity of F-actin. We believe that these studies of full length nebulin and nebulin fragments will lead to a deeper understanding of how nebulin interacts with actin, myosin and other cellular components in the multiple roles it plays in muscle function. These results and the technology platform can also be applied to other members of the nebulin family, such as NRAP and cortactin which plays a role in muscle assemblyin muscle assemblyin muscle assembly, cell motility and hin muscle assembly, cell motility and has been implicated in muscle diseases and metastasis of cancer.

肌联蛋白和星云蛋白是该实验室发现并命名的前所未有的巨型蛋白质。我们最近的工作重点是肌联蛋白和星云蛋白在横纹肌肌节的结构、调节、力学和组装中的作用。我们采用了多学科方法，借鉴了生物化学、生物物理学、细胞和分子生物学以及生理学的方法和概念。 (A) 肌动蛋白： 我们研究的一个主要主题是了解肌联蛋白的结构和生理作用，肌联蛋白是一个巨大的结构蛋白家族，构成横纹肌肌节和一些非肌肉细胞的弹性基质。肌联蛋白和弹性基质可能发挥重要的生理作用，包括长程弹性的发生、肌节稳定性的维持、发育中的肌肉细胞中新生肌节的组装以及非肌肉细胞中微丝中肌球蛋白的组装。 正在解决以下至关重要的问题： 1.肌动蛋白丝如何响应拉伸和释放？ 2. 肌动蛋白弹性的分子基础是什么？ 3. 细丝是否调节肌动蛋白弹性？ 4.肌动蛋白是否参与肌动蛋白-肌球蛋白相互作用的调节？ 5.肌联蛋白和肌联蛋白激酶的信号传导能力如何在肌肉中发挥作用？ 我们正在测试这样的假设：肌动蛋白激酶等有序结构域的可逆展开和本质上无序片段的可逆解开是肌动蛋白弹性和功能的两种不同且互补的机制。 使用激光陷阱技术、原子力显微镜 (AFM) 和流变技术，可以直接测量分离的全长肌动蛋白和工程片段以及来自骨骼肌、心肌和飞行肌的类似弹性分子的弹性，作为单分子和凝胶网络。肌联蛋白结构域和合成肽的构象正在通过高分辨率 NMR 和 X 射线晶体学进行研究。肌联弹性可以通过生物聚合物理论与肌联结构域的实际和实验结构特征相结合来解释。 在过去的两年里，我们成功地揭示了肌联蛋白的弹性PEVK片段在结构上是模块化的，显示出重复的脯氨酸II螺旋/螺旋构象基序，最重要的是，它以钙/S100敏感的方式与细丝相互作用。我们的观察建立了肌动蛋白PEVK弹性的构象基础，并确定PEVK是丝间相互作用的位点，并且S100或钙传感器蛋白调节相互作用和肌动蛋白弹性。此外，我们发现titin PEVK片段是SH3信号通路的一个巨大的力传感分子。与路易斯维尔大学的 Wittebort 教授合作，通过 NMR 技术确定了溶液和凝胶相中 PEVK 模块均聚物的高分辨率结构。还阐明了各种人类心肌中肌动蛋白 PEVK 外显子的广泛差异剪接。 PEVK 结构域是一种本质上无序的蛋白质，具有短程结构。对 PEVK 的 AFM 测量表明，其弹性部分是由盐桥驱动的。 NMR 数据证明了特定盐桥的存在。使用 NIH Biowulf 集群，我们应用分子建模技术来了解原子水平的动态相互作用。 MD 和 SMD 模拟进一步表明，离子相互作用的多样性在整个拉伸过程中不断演变。这些对 titin PEVK 中盐桥行为的新见解可能为设计弹性聚两性电解质作为纳米技术和组织工程的生物材料提供见解。这些模拟还帮助我们了解如何通过力调节配体结合，并且仅当蛋白质被拉伸以暴露 SH3 结合位点时才结合。 我们还设计了一种新型仪器来测量蛋白质被拉伸时的配体结合和蛋白质-蛋白质相互作用。这种新仪器将使我们能够直接研究信号蛋白相互作用如何响应机械应力。 (B) 星布林： Nebulin 是骨骼肌肌节中第二大蛋白质 (0.7 MDa)。它充当细丝长度的标尺，并且可能充当与经典肌钙蛋白/原肌球蛋白系统不同的钙连接调节蛋白。在肌肉发育中，星云蛋白被认为可以加速细丝组件的成熟过程。 nemalin 基因突变会导致 nemalin 肌病，导致肌肉无力和过早死亡。正在解决以下问题： 1. 星云蛋白如何调节细丝的长度？ 2. nebulin 如何调节肌动蛋白-肌球蛋白相互作用？ 3. 星云蛋白的纳米力学在肌肉功能中发挥什么作用 星云蛋白大小异构体的长度、质量和序列已与发育中和成年肌肉中各种骨骼肌的细丝长度进行了比较。结合星云蛋白结构域的过度表达对培养的骨骼肌细胞中肌原纤维形成的影响，我们得出结论，星云蛋白重复的数量以及特定的 N 端和 C 端终止序列对于在每个肌节内产生均匀的细丝长度分布是必要的。 在过去的两年中，我们成功地从骨骼肌中纯化了天然全长星云蛋白，并测试了其对聚合或预加工肌动蛋白丝长度分布的影响。不幸的是，肌动蛋白膜长度的测量由于每个星云蛋白分子上的多价肌动蛋白结合位点使肌动蛋白丝胶凝而变得复杂。 为了缓解这个问题，我们正在设计一种基于纳米技术的新平台，以测量星云蛋白对纳米图案基底上肌动蛋白丝长度分布的影响，该基底允许平行肌动蛋白丝以相同方向和极性伸长而不发生凝胶化。 正在通过停流方法研究星云蛋白对 ATP 酶活性的动力学，以确定合理的分子作用机制。正在研究星云蛋白和钙/钙调蛋白对肌动蛋白在肌球蛋白上滑动速度的影响，以揭示星云蛋白对肌球蛋白马达机械耦合的影响。 我们正在通过亲和层析和印迹寻找其他潜在的钙介体。我们特别感兴趣的是 S100 蛋白以及磷酸化星云蛋白的激酶/磷酸酶作为肌动蛋白/肌球蛋白相互作用的生理效应器的可能参与。 我们用 AFM 测量了全长天然星云的纳米机械特性。这些需要实现使用高亲和力位点特异性抗体成对地在星云蛋白之间拉伸星云蛋白；使用 nof 蛋白抗性自组装单层来防止非特异性尖端-表面相互作用，一种区分单分子与多分子拉伸的新方法，以及采用强大的经验模式分解 (HHT) 方法来识别力生成事件。最独特和意想不到的发现是，星云蛋白必须被拉伸得足够长，才能包裹并匹配 F-肌动蛋白的周期性。 我们相信，这些对全长星云蛋白和星云蛋白片段的研究将有助于更深入地了解星云蛋白如何与肌动蛋白、肌球蛋白和其他细胞成分相互作用，从而发挥其在肌肉功能中的多种作用。这些结果和技术平台也可以应用于nebulin家族的其他成员，例如NRAP和cortactin，它们在肌肉组装、细胞运动和肌肉组装、细胞运动中发挥作用，并与肌肉疾病和癌症转移有关。