Molecular Mechanism of Troponin Function in Health and Disease

描述（由申请人提供）：我们的长期目标是阐明细胞内Ca 2+和跨桥与横纹肌细肌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理解遗传性疾病（如家族性肥厚型心肌病）和开发药物（如Ca 2+增敏剂）至关重要。横纹肌收缩的Ca 2+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），其由TnI、TnC和TnT组成。细肌丝蛋白之间的Ca 2+依赖性相互作用是横纹肌调节中的关键事件。在低Ca ~（2+）时，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。一个重要的未解问题是：肌动蛋白和肌钙蛋白I如何相互作用以阻止细丝的激活？在高[Ca 2 +]下，Tnl的调节位点与TnC的N-叶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移动到肌动蛋白丝的内部结构域。有证据表明，TnI的调节位点与TnC的N端半叶之间的相互作用应引起比抑制性区域/第二肌动蛋白-Tm位点从肌动蛋白的简单释放更多的作用。有证据表明，在高[Ca 2 +]时，Tnl的抑制区可能与TnC的中心连接区相互作用。最新的结构和生化数据表明，在Tn复合物的连接区的三个Tn组件之间的相互作用，包括TnC的中心连接器，TnI和TnT的抑制区，可能在Ca ~（2+）激活中发挥作用。基于这些数据，我的假设是，能量平衡的Ca 2+依赖的相互作用之间的细丝蛋白质是至关重要的Ca 2+依赖的调节肌丝活性。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，Ca 2+结合测量，acto-S1 ATP酶测定，SPR和FRET测量进行研究。

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.