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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：
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/肌生长抑制素）是骨骼肌发育的有效负调控因子