Molecular Mechanism of Troponin Function in Health and Disease

肌钙蛋白在健康和疾病中发挥作用的分子机制

• 批准号: 7486819
• 负责人: TOMOYOSHI KOBAYASHI
• 金额: $ 26.34万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7486819
• 关键词: ATP phosphohydrolase; Actins; Binding; Biochemical; Bioinformatics; Biological Assay; Cardiac; Cardiomyopathies; Classification; Complex; Data; Dilated Cardiomyopathy; Disease; Equilibrium; Event; Familial Hypertrophic Cardiomyopathy; Fluorescence Resonance Energy Transfer; Health; Hereditary Disease; Hot Spot; Hydrophobic Surfaces; Link; Lobe; Measurement; Measures; Microfilaments; Molecular; Muscle; Muscle Contraction; Mutagenesis; Mutation; Myosin ATPase; N-terminal; Pharmaceutical Preparations; Phase; Play; Property; Protein Binding; Proteins; Regulation; Relaxation; Restrictive Cardiomyopathy; Roentgen Rays; Role; Site; Solid; Striated Muscles; Structure; Tail; Takeda brand of pioglitazone hydrochloride; Thin Filament; Tropomyosin; Troponin; base; crosslink; prevent; protein protein interaction; synthetic peptide

DESCRIPTION (provided by applicant): Our long-term objective is to elucidate the molecular mechanisms whereby intracellular Ca2+ and cross- bridge interactions with the thin filaments of striated muscle regulate myofilament activity. Elucidation of the molecular mechanisms of the muscle regulation is critical to understand the genetic disease, such as familial hypertrophic cardiomyopathy, and to develop drugs, such as Ca2+-sensitizers. Ca2+-dependent regulation of striated muscle contraction requires the protein complex, tropomyosin (Tm), and troponin (Tn), which consists of Tnl, TnC, and TnT. Ca2+-dependent interactions among thin filament proteins are the key events in striated muscle regulation. At low Ca2+, Tnl interacts with actin through at least two regions and inhibits actin-activated myosin ATPase activity. The molecular mechanism of this inhibition, however, remains to be solved. The important unanswered question is: How do actin and Tnl interact with each other to prevent the activation of the thin filament? At high [Ca2+], the regulatory site of Tnl interacts with the newly exposed hydrophobic surface of the N-lobe of TnC. This interaction causes the actin-interacting sites of Tnl to move away from actin and allows Tm to move to the inner domain of the actin filament. There is evidence that indicates that the interaction between the regulatory site of Tnl and the N- lobe of TnC should cause more than a simple release of the inhibitory region/second actin-Tm site from actin. There is evidence that the inhibitory region of Tnl may interact with the central linker region of TnC at high [Ca2+]. Recent structural and biochemical data suggest that the interaction between three Tn components at the linker region of Tn complex consists of the central linker of TnC, the inhibitory region of Tnl and TnT, may play a role in Ca2+-activation. My hypothesis, based on these data, is that the energetically balanced Ca2+-dependent interactions between the thin filament proteins are critical in the Ca2+-dependent regulation of myofilament activity. We will investigate using site-directed mutations, synthetic peptide-array assay, a solid phase-based protein binding assay, a photocross-linking, Bioinformatics analysis, Ca2+-binding measurement, acto-S1 ATPase assay, SPR, and FRET measurements.

描述（由申请人提供）：我们的长期目标是阐明细胞内Ca2+和与横纹肌调节肌膜肌膜活性的细丝的跨桥相互作用的分子机制。阐明肌肉调节的分子机制对于理解遗传疾病，例如家族性肥厚心肌病和开发药物，例如Ca2+ - 敏感剂至关重要。 Ca2+依赖性调节条纹肌肉收缩需要蛋白质复合物，肌球蛋白（TM）和肌钙蛋白（TN），该蛋白质由TNL，TNC和TNT组成。薄丝蛋白之间的Ca2+依赖性相互作用是条纹肌肉调节的关键事件。在低Ca2+下，TNL通过至少两个区域与肌动蛋白相互作用，并抑制肌动蛋白激活的肌球蛋白ATPase活性。然而，这种抑制的分子机制仍有待解决。重要的未解决的问题是：肌动蛋白和TNL如何相互作用以防止细丝的激活？在高[Ca2+]中，TNL的调节位点与TNC的N-Lobe的新暴露的疏水表面相互作用。这种相互作用导致TNL的肌动蛋白相互作用位点远离肌动蛋白，并允许TM移至肌动蛋白丝的内域。有证据表明，TNL的调节位点与TNC的N叶之间的相互作用不仅仅是抑制区域/第二个肌动蛋白-TM位点的简单释放。有证据表明，TNL的抑制区域可能在高[Ca2+]下与TNC的中央接头区域相互作用。最近的结构和生化数据表明，TN复合物接头区域的三个TN组件之间的相互作用由TNC的中央接头，TNL和TNT的抑制区域组成，可能在Ca2+激活中起作用。我的假设基于这些数据，是薄丝蛋白之间的能量平衡的Ca2+依赖性相互作用在Ca2+依赖性的肌丝活性调节中至关重要。我们将使用位置定向的突变，合成肽 - 阵列测定，一种固相的蛋白质结合测定，一种光叠链链接，生物信息学位分析，Ca2+结合测量，Acto-S1 ATPase ASSPase分析，SPR和FRET测量值。