Molecular Mechanism of Troponin Function in Health and Disease

描述(申请人提供)：我们的长期目标是阐明细胞内钙离子和与横纹肌细丝的跨桥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，如家族性肥厚性心肌病，以及开发药物，如钙增敏剂至关重要。依赖于钙离子的横纹肌收缩调节需要蛋白质复合体、原肌球蛋白(TM)和肌钙蛋白(TN)，TN由TnL、TnC和TnT组成。细丝蛋白之间依赖于钙离子的相互作用是横纹肌调节的关键事件。在低钙时，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ATPase活性。然而，这种抑制的分子机制仍有待解决。一个重要的悬而未决的问题是：肌动蛋白和TNL如何相互作用来防止细丝的激活？在高[Ca~(2+)]时，TnL的调控位点与新暴露的TNC N叶疏水表面相互作用。这种相互作用导致肌动蛋白与肌动蛋白相互作用的部位从肌动蛋白上移开，使肌动蛋白转移到肌动蛋白细丝的内区。有证据表明，TnL的调控位点和TNC的N叶之间的相互作用应该不仅仅是从肌动蛋白中简单地释放抑制区/第二肌动蛋白-TM位点。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。最近的结构和生化数据表明，位于TN复合体连接区的三个TN组分之间的相互作用可能在钙激活中起作用，其中包括TNC的中心连接区、TnL和TNT的抑制区。基于这些数据，我的假设是，细丝蛋白之间能量平衡的钙依赖的相互作用在钙依赖的肌丝活性调节中至关重要。我们将使用定点突变、合成多肽阵列分析、基于固相的蛋白质结合分析、光交联、生物信息学分析、钙结合测定、acto-S1 ATPase测定、SPR和FRET测定来进行研究。

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.