Structural mechanisms underlying latency and activation of GDF8

生长分化因子8(GDF8/myostatin)是一种强有力的骨骼肌发育负调控因子 是治疗遗传性、急性或慢性肌肉萎缩的重要靶点 疾病。GDF8属于由33个成员组成的转化生长因子β(β)家族，负责调节 胚胎模式，组织和器官发育，以及动态平衡。成员在内部或内部处理 胞外转化为由生长因子(GF)二聚体非共价结合的前复合体 PRODOWS。前体域可以与细胞外基质(ECM)成分相互作用，并靶向前复合体以 用于存储的ECM。分离的一些成员的前复合体，如前GDF8和前转化生长因子-βS 1-3，是 潜伏期。原-GDF8的激活需要Tolloid(TLD)金属蛋白酶介导的原结构域的切割。我们的 先前的研究揭示了潜伏的转化生长因子原-β1的交叉臂构象和相反的开臂构象 非潜伏期前体BMP9的构象。我们假设与ECM组件的相互作用稳定PRO- GDF8为交叉臂构象，而TLD-裂解诱导为开臂构象。我们 建议揭示前GDF8潜伏期和激活的结构机制。目标1决定 负染电子显微镜观察GDF8前体络合物的整体结构并验证我们的假说 这种TLD-裂解导致张开双臂的构象，而相互作用的大分子，如 糖胺多糖稳定了交叉臂构象。目的2探讨PRO-2的构象动力学 GDF8通过氢/氢交换(HDX)。HDX将检验TLD-裂解和TLD-裂解 大分子结合改变裂解或结合部位局部的交换率，定义这些正构体 由于全球构象变化(变构位点)，也改变了远端位点的交换。HDX GDF8前复合体、前结构域和GF之间的比较将有助于确定中介区域 原结构域与GF的相互作用。这些测量将为监管过程提供重要的见解 交叉臂构象或开放臂构象。目的3从结构和生化细节上对原GDF8进行了表征。我们 将解决PRO-GDF8的晶体结构。突变和基于细胞的活性分析将测试 原结构域-GF相互作用揭示的晶体结构对GDF8的潜伏期和四个神秘作用的影响 保守的原结构域半胱氨酸。结合研究将揭示GDF8前体域是否与GFS结合 合作或独立，并检验潜伏期与原结构域-GF亲和力相关的假设。目标 4确定横纹肌肉瘤细胞和肌肉组织中与前GDF8共同作用的大分子。 我们将研究共结合蛋白如何调节整个前GDF8结构、潜伏期和激活。 我们在GDF8上的工作将为理解TLD-裂解和关联提供新的概念性进展 利用ECM组件调节GDF8存储、前复杂构象和延迟，并将产生深远的影响 开发治疗肌肉萎缩疾病的新疗法的意义。

Growth differentiation factor 8 (GDF8/myostatin) is a potent negative regulator of skeletal muscle development and a therapeutically important target for treating muscle wasting associated with inherited, acute, or chronic disease. GDF8 belongs to the 33-member transforming growth factor beta (TGF-β) family that regulates embryonic patterning, tissue and organ development, and homeostasis. Members are processed intra- or extracellularly into a pro-complex consisting of a growth factor (GF) dimer non-covalently associated with two prodomains. Prodomains can interact with extracellular matrix (ECM) components and target pro-complexes to the ECM for storage. Isolated pro-complexes of some members, e.g., pro-GDF8 and pro-TGF-βs 1-3, are latent. Activation of pro-GDF8 requires Tolloid (Tld) metalloprotease-mediated cleavage of the prodomain. Our previous studies revealed a cross-armed conformation for latent pro-TGF-β1 and a contrasting open-armed conformation for non-latent pro-BMP9. We hypothesize that interaction with an ECM component stabilizes pro- GDF8 in a cross-armed conformation, whereas Tld-cleavage induces an open-armed conformation. We propose to uncover the structural mechanisms underlying pro-GDF8 latency and activation. Aim 1 determines overall structures of GDF8 pro-complexes by negative stain electron microscopy (EM) and tests our hypothesis that Tld-cleavage induces an open-armed conformation, whereas interacting macromolecules such as glycosaminoglycans stabilize a cross-armed conformation. Aim 2 probes conformational dynamics of pro- GDF8 by hydrogen/deuterium exchange (HDX). HDX will test the hypothesis that Tld-cleavage and macromolecule binding alters exchange rates local to the cleavage or binding sites, defining these orthosteric sites, and also alters exchange in distal sites due to global conformational change (allosteric sites). HDX comparisons between GDF8 pro-complex, prodomains, and GF will help identify regions that mediate prodomain–GF interactions. These measurements will provide important insights into processes that regulate cross- or open-armed conformations. Aim 3 characterizes pro-GDF8 in structural and biochemical detail. We will solve the crystal structure of pro-GDF8. Mutations and cell-based activity assays will test the importance of prodomain–GF interactions revealed by the crystal structure for GDF8 latency and the role of four mysterious conserved prodomain cysteines. Binding studies will reveal whether GDF8 prodomains bind to GFs cooperatively or independently, and test the hypothesis that latency correlates with prodomain–GF affinity. Aim 4 identifies macromolecules that co-associate with pro-GDF8 in rhabdomyosarcoma cells and muscle tissue. We will investigate how co-associating proteins regulate overall pro-GDF8 structure, latency, and activation. Our work on GDF8 will provide new conceptual advances in understanding how Tld-cleavage and association with ECM components regulate GDF8 storage, pro-complex conformation, and latency, and will have profound implications for developing novel therapies to treat muscle-wasting conditions.