Regulation of MyBP-C slow via phosphorylation in skeletal muscles

摘要 肌球蛋白结合蛋白-C（MyBP-C）包括一个粗丝相关蛋白家族， 有助于它们的组装和维护，并调节肌动球蛋白跨桥的形成 在收缩期间。三种不同的亚型已被表征，包括心脏（c），缓慢（s）骨骼肌 和快速（f）骨架。心脏同种型的表达局限于发育和成熟的心脏， 而骨骼同种型可共存于同一肌肉中。MyBP-C的核心结构包括： 七个免疫球蛋白（IG）结构域和三个纤连蛋白-III（Fn-III）结构域，从NH 2-末端开始编号 C1-C10。在过去的四十年里，许多研究都集中在阐明机制， 调节cMyBP-C在肌动球蛋白跨桥形成中的活性。反倒是 骨骼亚型的调节和作用仍然不清楚，主要是由于结构上的差异而推断的。 它们与cMyBP-C具有相似性。我们小组一直在研究MyBP-C的缓慢骨骼形式， 了解它的规则和活动。使用分子工具，我们已经证明MYBPC1基因， 编码sMyBP-C的基因被大量剪接，产生多种变体，这些变体可以在相同的细胞中共表达。 肌肉和肌纤维。这些有着共同的领域，但也因包含或跳过小说而有所不同。 插入位于NH 2-末端、FN-III C7结构域和COOH-末端。NH2和 COOH末端可以保留天然的肌球蛋白和肌动蛋白，并调节肌动蛋白丝滑过 肌球蛋白头，虽然在不同程度上，并在一个变种的具体方式。此外，使用蛋白质组学工具， 我们已经证明sMyBP-C经历PKA和PKC介导的磷酸化。我们尤其 在蛋白质的NH2末端确定了四个磷酸化位点，其中一个位于 唯一的插入只存在于选择的变体中。因此，我们假设sMyBP-C包含多个- 通过复杂磷酸化调节功能的粗丝辅助蛋白质的多面家族 它的NH2末端。我们目前的目标是研究磷酸化是如何影响 不同sMyBP-C变体的生物化学和生物物理特性（目的1），并产生第一个 磷酸化突变体sMyBP-C动物模型，以评估磷酸化在体内的作用（目的2）。拟议 这些研究将极大地促进我们对多方面sMyBP-C亚家族调控的理解， 磷酸化，这是一个突出的生物学问题，在肌肉中具有重要和广泛的意义， 病理生理学

ABSTRACT Myosin Binding Protein-C (MyBP-C) comprises a family of thick filament associated proteins that contributes to their assembly and maintenance, and regulates the formation of actomyosin cross-bridges during contraction. Three distinct isoforms have been characterized, including the cardiac (c), slow (s) skeletal and fast (f) skeletal. The expression of the cardiac isoform is confined in the developing and mature heart, whereas the skeletal isoforms can co-exist in the same muscle. The core structure of MyBP-C consists of seven immunoglobulin (Ig) domains and three fibronectin-III (Fn-III) domains, numbered from the NH2-terminus as C1-C10. During the last forty years, numerous studies have focused on elucidating the mechanisms that modulate the activities of cMyBP-C in the formation of actomyosin cross-bridges. On the contrary, the regulation and roles of the skeletal isoforms have remained obscure, and mainly inferred due to the structural similarity they share with cMyBP-C. Our group has been studying the slow skeletal form of MyBP-C aiming to understand its regulation and activities. Using molecular tools, we have shown that the MYBPC1 gene, encoding sMyBP-C, is heavily spliced giving rise to multiple variants that can be co-expressed in the same muscle and myofiber. These share common domains, but also differ by the inclusion or skipping of novel insertions located in the NH2-terminus, the FN-III C7 domain and the COOH-terminus. Both the NH2 and COOH termini can retain native myosin and actin and modulate the sliding velocity of actin filaments past myosin heads, though to different extents and in a variant-specific manner. Moreover, using proteomic tools, we have demonstrated that sMyBP-C undergoes phosphorylation mediated by PKA and PKC. In particular, we have identified four phosphorylation sites in the NH2-terminus of the protein, with one of them located within a unique insertion present only in select variants. We therefore hypothesize that sMyBP-C comprises a multi- faceted family of thick filament accessory proteins whose functions are regulated via complex phosphorylation of its NH2-terminus. Our goals in the current proposal are to examine how phosphorylation affects the biochemical and biophysical properties of the different sMyBP-C variants (Aim 1), and to generate the first phospho-mutant sMyBP-C animal model to assess the role of phosphorylation in vivo (Aim 2). The proposed studies will greatly advance our understanding on the regulation of the multifaceted sMyBP-C subfamily via phosphorylation, which is an outstanding biological question with important and broad implications in muscle pathophysiology.