Structural mechanisms underlying latency and activation of GDF8

GDF8 潜伏期和激活的结构机制

基本信息

批准号：
9302311
负责人：
TIMOTHY A SPRINGER
金额：
$ 39.46万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
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/9302311
关键词：
Acute Disease 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 Crystallization Crystallography Cysteine Deuterium Diabetes Mellitus Dimerization Disease Dissociation Distal Electron Microscopy Embryo Extracellular Matrix Extracellular Matrix Proteins Family Family member GDF8 gene Glycosaminoglycans Grant Growth Growth Factor 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 Therapeutic Tissues Transforming Growth Factor beta Trauma Work Wound Healing X-Ray Crystallography base bone conformational conversion crosslink dimer extracellular 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

项目摘要

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/Myostatin）是骨骼肌发育的潜在负调节剂以及治疗与遗传，急性或慢性相关的肌肉浪费的治疗上重要的靶标疾病。 GDF8属于调节的33名成员转化生长因子β（TGF-β）家族胚胎图案，组织和器官发育以及稳态。成员在内部或细胞外进入由生长因子（GF）二聚体组成的亲复合物 prodomains。 Prodomains可以与细胞外基质（ECM）组件相互作用，而靶向促成型成分 ECM存储。某些成员的孤立亲复合物，例如Pro-GDF8和Pro-TGF-βS1-3 潜。 pro-GDF8的激活需要TOLOID（TLD）金属蛋白酶介导的Prodomain裂解。我们的先前的研究揭示了潜在的Pro-TGF-β1的交叉武装会议和一个对比的开放式武器非LATENT PRO-BMP9的构象。我们假设与ECM组分的相互作用稳定 GDF8在交叉构型中，而TLD裂解会诱导开放式构象。我们提议揭示Pro-GDF8潜伏期和激活的基础结构机制。 AIM 1确定通过负不锈钢显微镜（EM）的GDF8亲复合物的总体结构，并检验我们的假设 TLD裂解会诱导开放式构象，而相互作用的大分子（例如糖胺聚糖稳定了交叉构象。 AIM 2问题构象的动力学 GDF8通过氢/氘交换（HDX）。 HDX将检验TLD-CEAVAGE和大分子结合改变了与裂解或结合位点局部汇率的变化，定义了这些正常由于全球构象变化（变构位点），地点，还改变了远端位置的交换。 HDX GDF8亲复合物，Prodomain和GF之间的比较将有助于识别介导的区域 Prodomain – GF相互作用。这些测量将为调节的过程提供重要的见解交叉或开臂构象。在结构和生化细节中，目标3个字符Pro-GDF8。我们将解决Pro-GDF8的晶体结构。突变和基于细胞的活动评估将测试 prodomain – GF相互作用由GDF8潜伏期的晶体结构揭示了四个神秘的作用保守的prodomain半胱氨酸。结合研究将揭示GDF8 Prodomain是否与GFS结合合作或独立，并检验了潜伏期与Prodomain -GF亲和力相关的假设。目的 4鉴定与横纹肌肉瘤细胞和肌肉组织中pro-GDF8共同关联的大分子。我们将研究如何共同关联蛋白调节总体GDF8结构，潜伏期和激活。我们在GDF8上的工作将为理解TLD-CEAVAGE和协会如何提供新的概念进步通过ECM组件调节GDF8存储，亲复合构象和潜伏期，将具有深刻的开发新疗法以治疗浪费肌肉疾病的影响。