Structural mechanisms underlying latency and activation of GDF8

GDF8 潜伏期和激活的结构机制

• 批准号: 9175103
• 负责人: TIMOTHY A SPRINGER
• 金额: $ 41.38万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9175103
• 关键词: Acute; Affect; Affinity; Allosteric Site; BMP7 gene; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cachexia; Cell physiology; Cells; Chronic Disease; Chronic Obstructive Airway Disease; Complement; Complex; Crystallography; Cysteine; Deuterium; Diabetes Mellitus; Disease; Dissociation; Distal; Electron Microscopy; Embryo; Extracellular Matrix; Extracellular Matrix Proteins; Family; Family member; GDF8 gene; Glycosaminoglycans; Grant; Growth; Growth Factor Interaction; Homeostasis; Hydrogen; Immune response; Inherited; Ligands; Light; Mass Spectrum Analysis; Measurement; Mediating; Metalloproteases; Molecular Conformation; Muscle; Muscle Development; Muscular Atrophy; Muscular Dystrophies; Mutation; N-terminal; Negative Staining; Pattern; Polysaccharides; Process; Proteins; Resolution; Rhabdomyosarcoma; Roentgen Rays; Role; Site; Skeletal Muscle; Structure; Testing; Tissues; Transforming Growth Factor beta; Trauma; Work; Wound Healing; X-Ray Crystallography; arm; base; bone; conformational conversion; crosslink; dimer; flexibility; growth-differentiation factor 8; in vivo; inhibitor/antagonist; insight; macromolecule; member; novel; novel therapeutics; organ growth; reconstruction; sarcopenia; skeletal muscle growth; therapeutic development; therapeutic target

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.

生长分化因子 8（GDF8/肌生长抑制素）是骨骼肌发育的有效负调节因子 以及治疗与遗传性、急性或慢性相关的肌肉萎缩的重要治疗目标 疾病。 GDF8 属于 33 个成员的转化生长因子 β (TGF-β) 家族，负责调节 胚胎模式、组织和器官发育以及体内平衡。成员在内部或内部处理 细胞外形成由生长因子 (GF) 二聚体组成的前复合物，该二聚体与两个非共价连接 前域。前结构域可以与细胞外基质 (ECM) 成分相互作用并靶向前复合物 用于存储的ECM。一些成员的分离前复合物，例如 pro-GDF8 和 pro-TGF-βs 1-3，是 潜。 pro-GDF8 的激活需要 Tolloid (Tld) 金属蛋白酶介导的前结构域裂解。我们的 之前的研究揭示了潜在的 pro-TGF-β1 的交叉臂构象和对比的开臂构象 非潜伏 BMP9 前体的构象。我们假设与 ECM 成分的相互作用可以稳定亲 GDF8 呈交叉臂构象，而 Tld 切割则诱导开臂构象。我们 提议揭示 pro-GDF8 潜伏和激活的结构机制。目标 1 决定 通过负染色电子显微镜 (EM) 观察 GDF8 前复合物的整体结构并检验我们的假设 Tld 裂解诱导开臂构象，而相互作用的大分子，例如 糖胺聚糖稳定交叉臂构象。目标 2 探测亲的构象动力学 GDF8 通过氢/氘交换 (HDX)。 HDX 将检验 Tld 裂解和 大分子结合改变了裂解或结合位点的局部交换率，从而定义了这些正构 位点，并且由于整体构象变化（变构位点）也改变了远端位点的交换。 HDX GDF8 前复合物、前结构域和 GF 之间的比较将有助于识别介导的区域 前结构域-GF 相互作用。这些测量将为调节过程提供重要的见解 交叉或开臂构象。目标 3 描述了 pro-GDF8 的结构和生化细节。我们 将解析pro-GDF8的晶体结构。突变和基于细胞的活性测定将测试的重要性 GDF8潜伏期的晶体结构揭示了前结构域-GF相互作用以及四个神秘的作用 保守的前结构域半胱氨酸。结合研究将揭示 GDF8 前结构域是否与 GF 结合 合作或独立，并检验潜伏期与前结构域-GF 亲和力相关的假设。目的 图4鉴定了与横纹肌肉瘤细胞和肌肉组织中的GDF8原共关联的大分子。 我们将研究共缔蛋白如何调节 GDF8 前体的整体结构、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进展 与 ECM 组件一起调节 GDF8 存储、亲复杂构象和延迟，并将产生深远的影响 对开发治疗肌肉萎缩疾病的新疗法的影响。