Mechanisms of Thin Filament Regulation by Myosin Binding Protein-C

摘要 心肌肌球蛋白结合蛋白-C(cMyBP-C)是心脏功能的重要调节因子，即 对正常的收缩和舒张期心脏功能以及对变力反应增强的收缩能力是必需的 通过许多信号通路使cMyBP-C磷酸化的激动剂。而磷酸化是 低磷酸化与心脏保护有关，在心力衰竭中一直被发现。基因突变 编码cMyBP-C的MYBPC3基因也是肥厚型心肌病最常见的原因 (HCM)，一种患病率约为1：500的疾病。然而，尽管它对心脏健康和疾病具有重要意义， 关于cMyBP-C如何介导其功能效应或cMyBP-C如何发挥作用，人们仍然知之甚少。 磷酸化可增加心肌对变力刺激的收缩能力。我们3个实验室的工作表明 毫无疑问，cMyBP-C直接激活肌肉肌节中的细丝的方式与 肌球蛋白跨越桥梁。因此，我们的数据显示cMyBP-C作用于增强收缩或延迟松弛 是由于cMyBP-C将原肌球蛋白转移到细丝上的激活状态的直接作用。在这里我们 利用低温电子显微镜的革命来揭示cMyBP-C与Thin相互作用的详细结构 细丝和肌球蛋白-S1也是首次发现。我们将使用冷冻-EM重建来确定关键残留物 它调节与细丝和肌球蛋白-S1的相互作用，并将使用 功能性方法，包括一种由我们的实验室独家开发的新型(“Spy-C”)方法，它使我们能够 在肌瘤中原位替换cMyBP-C的N‘末端结构域。使用我们的结构图，我们演示了 通过确定负责原肌球蛋白cMyBP-C激活的氨基酸来证明原理，并表明 磷酸化导致cMyBP-C结构域在细丝上重新定位。具体目标将建立在这些基础上 发现并结合我们独特的资源以：1)确定单个cMyBP-C结构域如何 当与细丝和肌球蛋白-S1结合时，与相邻结构域进行通信，以及2)定义如何 钙离子和磷酸化调节cMyBP-C与细丝和肌球蛋白的关键调控相互作用 S1。通过鉴定控制cMyBP-C与细丝结合的关键分子相互作用 肌球蛋白-S1这些研究将为未来的靶向方法调节心脏收缩和 放松一下。

ABSTRACT Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of heart function that is necessary for normal systolic and diastolic heart function and for enhanced contractility in response to inotropic agonists that phosphorylate cMyBP-C through numerous signaling pathways. Whereas phosphorylation is associated with cardiac protection, hypo-phosphorylation is consistently found in heart failure. Mutations in MYBPC3, the gene encoding cMyBP-C, are also the most common cause of hypertrophic cardiomyopathy (HCM), a disease with a prevalence of ~1:500. However, despite its significance to cardiac health and disease, there is still remarkably little known regarding how cMyBP-C mediates its functional effects or how cMyBP-C phosphorylation increases cardiac contractility in response to inotropic stimuli. Work from our 3 labs has shown unequivocally that cMyBP-C directly activates the thin filament in muscle sarcomeres in the same way as myosin cross-bridges. Our data thus demonstrate cMyBP-C effects to augment contraction or delay relaxation are due to direct effects of cMyBP-C to shift tropomyosin to an activated state on the thin filament. Here we take advantage of the revolution in cryo-EM to reveal detailed structures of cMyBP-C interactions with the thin filament and also with myosin-S1 for the first time. We will use cryo-EM reconstructions to identify key residues that mediate interactions with thin filaments and myosin-S1 and will test model predictions using an arsenal of functional approaches including a novel (“Spy-C”) method developed exclusively in our labs that allow us to replace the N'-terminal domains of cMyBP-C in sarcomeres in situ. Using our structural maps, we demonstrate proof of principle by identifying amino acids responsible for cMyBP-C activation of tropomyosin and show that phosphorylation causes cMyBP-C domains to reposition on the thin filament. Specific aims will build on these discoveries and combine our unique resources to: 1) Determine how individual cMyBP-C domains communicate with neighboring domains when bound to the thin filament and myosin-S1, and 2) Define how Ca2+ and phosphorylation modulate key regulatory interactions of cMyBP-C with the thin filament and myosin- S1. By identifying the critical molecular interactions that control cMyBP-C binding to the thin filament and myosin-S1 these studies will pave the way for future targeted approaches to modulate cardiac contraction and relaxation.