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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：
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
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/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

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（GDF 8/myostatin）是骨骼肌发育的负调控因子以及治疗与遗传性、急性或慢性疾病GDF 8属于33个成员的转化生长因子β（TGF-β）家族，其调节胚胎模式、组织和器官发育以及体内平衡。成员在内部或在一个实施方案中，所述细胞在细胞外转化为由生长因子（GF）二聚体组成的前复合物，所述生长因子二聚体与两个前结构域。前结构域可以与细胞外基质（ECM）组分相互作用并靶向前复合物，用于存储的ECM。一些成员的分离的前复合物，例如，pro-GDF 8和pro-TGF-β 1-3是潜伏的pro-GDF 8的活化需要Tolloid（Tld）金属蛋白酶介导的前结构域的切割。我们以前的研究揭示了潜在的pro-TGF-β1的交叉臂构象和对比的开放臂构象，非潜伏性前BMP 9的构象。我们假设，与ECM成分的相互作用稳定了前- GDF 8处于交叉臂构象，而Tld裂解诱导开放臂构象。我们提出揭示pro-GDF 8潜伏期和激活的结构机制。目标1确定 GDF 8前体复合物的整体结构通过负染色电子显微镜（EM）和测试我们的假设 Tld裂解诱导开放臂构象，而相互作用的大分子，糖胺聚糖稳定交叉臂构象。目的2探讨前体的构象动力学 GDF 8通过氢/氘交换（HDX）。HDX将检验Tld-切割和Tld-切割的假设。大分子的结合改变了切割或结合位点的交换速率，位点，并且还由于全局构象变化（变构位点）而改变远端位点中的交换。HDX GDF 8前复合物、前结构域和GF之间的比较将有助于鉴定介导前结构域-GF相互作用。这些测量将提供重要的洞察过程，调节交叉或开放臂构象。目的3在结构和生物化学细节上表征pro-GDF 8。我们将解析pro-GDF 8的晶体结构。突变和基于细胞的活性测定将测试前体结构域-GF相互作用揭示的晶体结构对于GDF 8潜伏期和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GDF 8前结构域是否与GF结合合作或独立地，并测试潜伏期与前结构域-GF亲和力相关的假设。目的 4鉴定了横纹肌肉瘤细胞和肌肉组织中与pro-GDF 8共缔合的大分子。我们将研究共缔合蛋白如何调节整体pro-GDF 8结构，潜伏期和激活。我们对GDF 8的研究将为理解Tld-切割和结合的机制提供新的概念性进展。 ECM成分调节GDF 8储存，前复合物构象和潜伏期，并将具有深远的意义。开发新的疗法来治疗肌肉萎缩的条件的影响。