Latent TGF-β2 Structure and Activation

潜在 TGF-β2 结构和激活

基本信息

批准号：
10586060
负责人：
TIMOTHY A SPRINGER
金额：
$ 70.65万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10586060
关键词：
2019-nCoV Actins Affinity Antibodies Aortic Aneurysm Binding Biochemical Biological Biological Process Blood Vessels Cardiovascular Physiology Cardiovascular system Cell Line Cell surface Cells Chimeric Proteins Complex Cryoelectron Microscopy Crystallization Crystallography Cytoplasmic Tail Cytoskeleton Defect Detection Development Dimerization Disease Extracellular Matrix Family Follow-Up Studies Foundations Grant Growth Factor Heart Human Integral Membrane Protein Integrin Binding Integrins Knowledge LRRC32 gene Length Loeys-Dietz Syndrome Mediating Membrane Modeling Molecular Chaperones Molecular Conformation Mucocutaneous Lymph Node Syndrome Mus Mutagenesis Mutate Mutation N-terminal Patients Peptide Hydrolases Peptides Physiological Pilot Projects Preparation Process Protein Secretion RGD (sequence)Rana Reagent Research Role Scleroderma Serine Protease Shapes Site Structure Systemic Scleroderma Testing Therapeutic Time Tissues Transforming Growth Factor Beta 2 Transforming Growth Factor beta Transforming Growth Factor beta Receptors Vertebrates Virus X-Ray Crystallography cardiac repair cofactor coronary fibrosis dimer disulfide bond extracellular latent TGF-beta binding protein member monomer nanobodies novel novel therapeutics prevent receptor repaired therapeutic target

项目摘要

Abstract. Transforming growth factor β2 (TGF-β2) is critically important for heart and vascular development and repair. TGF-β2 dysregulation is seen in patient TGF-β2 mutations, systemic sclerosis, and Kawasaki disease, which have cardiovascular sequelae such as aortic aneurysms and cardiac fibrosis. TGF-β1, 2 and 3 are synthesized as proproteins that dimerize and associate with milieu molecules that regulate TGF-β tissue localization, such as the transmembrane protein glycoprotein A repetitions predominant (GARP) and latent TGF-β binding proteins (LTBPs) in the extracellular matrix (ECM). Proconvertases cleave between the prodomain and growth factor (GF) domain; however, the prodomain dimer remains non-covalently associated with the GF in a proTGF-β–milieu molecule complex after secretion. ProTGF-β–milieu molecule complexes are inactive because the prodomains encircle the GF and prevent binding to TGF-β receptors. ProTGF-β1 and 3 activation is mediated by binding of integrins αVβ6 and αVβ8 to an RGD-motif in the prodomain and requires proTGF-β association with a milieu molecule. How proTGF-β2, which lacks an RGD-motif, is activated remains a mystery. Aim 1 will define the structure of proTGF-β2 to understand its mechanism of latency. Aim 2 will determine proTGF-β2/milieu molecule complex structures by X-ray crystallography and cryo-EM to define how milieu molecules bind and alter TGF-β2 latency. We will generate antibodies to use as crystallization chaperones in addition to using already developed nanobodies to proTGF-β2. Complementary unfolding studies will test the hypothesis that milieu molecule binding stabilizes proTGF-β2. Aim 3 characterizes TGF-β2 activation. Follow-up studies will identify cell-lines that natively activate TGF-β2 and characterize the physiologically relevant process. The results of this grant will enhance our understanding of TGF-β2 latency and activation in extracellular milieus and lay the foundation for developing therapeutics that target proTGF-β2 and its physiologically relevant complexes with milieu molecules.

摘要：转化生长因子 β2 (TGF-β2) 对于心脏和血管发育至关重要。 TGF-β2 失调见于 TGF-β2 突变、系统性硬化症和川崎病患者。疾病，具有心血管后遗症，如主动脉瘤和心脏纤维化。被合成为二聚体蛋白原，并与调节 TGF-β 组织的环境分子结合定位，例如跨膜蛋白糖蛋白 A 重复优势 (GARP) 和潜在重复细胞外基质 (ECM) 中的 TGF-β 结合蛋白 (LTBP) 在原转化酶之间裂解。前结构域和生长因子 (GF) 结构域；然而，前结构域二聚体仍然是非共价关联的分泌后，ProTGF-β-环境分子复合物中的GF与ProTGF-β-环境分子复合物相结合。失活是因为前结构域包围 GF 并阻止与 TGF-β 受体结合。激活是通过整合素 αVβ6 和 αVβ8 与前结构域中的 RGD 基序结合介导的，并且需要 proTGF-β 与环境分子的关联仍然是如何激活缺乏 RGD 基序的 proTGF-β2。目标 1 将定义 proTGF-β2 的结构，以了解其潜伏机制。通过 X 射线晶体学和冷冻电镜确定 proTGF-β2/环境分子复合物结构，以定义如何环境分子结合并改变 TGF-β2 潜伏期我们将产生用作结晶的抗体。除了使用已经开发的纳米抗体来补充 proTGF-β2 外，还使用分子伴侣。研究将检验环境分子结合稳定 proTGF-β2 的假设，Aim 3 是 TGF-β2 的特征。后续研究将鉴定天然激活 TGF-β2 的细胞系并表征其特征。此项资助的结果将增强我们对 TGF-β2 潜伏期的理解。并在细胞外环境中激活，为开发靶向 proTGF-β2 的疗法奠定基础及其与环境分子的生理相关复合物。