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+ 和横桥与横纹肌细丝相互作用调节肌丝活性的分子机制。阐明肌肉调节的分子机制对于了解遗传性疾病（如家族性肥厚型心肌病）和开发药物（如 Ca2+ 增敏剂）至关重要。横纹肌收缩的 Ca2+ 依赖性调节需要蛋白质复合物、原肌球蛋白 (Tm) 和肌钙蛋白 (Tn)，其中肌钙蛋白 (Tn) 由 Tn1、TnC 和 TnT 组成。细丝蛋白之间依赖 Ca2+ 的相互作用是横纹肌调节的关键事件。在低 Ca2+ 条件下，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 ATP 酶活性。然而，这种抑制的分子机制仍有待解决。尚未解答的重要问题是：肌动蛋白和 Tnl 如何相互作用以阻止细丝的激活？在高 [Ca2+] 时，Tnl 的调节位点与 TnC N 叶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 移动到肌动蛋白丝的内部结构域。有证据表明，Tnl 的调节位点和 TnC 的 N 叶之间的相互作用不仅仅会导致肌动蛋白的抑制区/第二肌动蛋白-Tm 位点的简单释放。有证据表明 Tnl 的抑制区可能在高 [Ca2+] 时与 TnC 的中央连接区相互作用。最近的结构和生化数据表明，Tn 复合物连接区的三个 Tn 成分之间的相互作用（由 TnC 的中心连接区、Tn1 和 TnT 的抑制区组成）可能在 Ca2+ 激活中发挥作用。基于这些数据，我的假设是，细丝蛋白之间能量平衡的 Ca2+ 依赖性相互作用对于肌丝活性的 Ca2+ 依赖性调节至关重要。我们将使用定点突变、合成肽阵列测定、基于固相的蛋白质结合测定、光交联、生物信息学分析、Ca2+结合测量、acto-S1 ATPase 测定、SPR 和 FRET 测量进行研究。