Latent TGF-β2 Structure and Activation

潜在 TGF-β2 结构和激活

• 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
• 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

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.

抽象。转化生长因子β2（TGF-β2）对心脏和血管发育至关重要 和修复。TGF-β2失调见于TGF-β2突变、系统性硬化症和川崎患者 心血管疾病，有心血管后遗症，如主动脉瘤和心脏纤维化。TGF-β1、2和3 作为前蛋白合成，其二聚化并与调节TGF-β组织的环境分子结合 定位，如跨膜蛋白糖蛋白A重复占主导地位（GARP）和潜伏 细胞外基质（ECM）中的TGF-β结合蛋白（LTBP）。前转化酶在 前结构域和生长因子（GF）结构域;然而，前结构域二聚体保持非共价缔合 与分泌后的proTGF-β-环境分子复合物中的GF。ProTGF-β-环境分子复合物是 由于前结构域包围GF并阻止与TGF-β受体结合，因此是无活性的。ProTGF-β1和3 活化是通过整合素αVβ6和αVβ8与前结构域中的RGD基序结合介导的， proTGF-β与环境分子的结合。缺乏RGD基序的proTGF-β2是如何被激活的， 一个谜目的1明确proTGF-β2的结构，了解其潜伏机制。目标2将 通过X射线晶体学和冷冻电镜确定proTGF-β2/环境分子复合物的结构，以确定如何 环境分子结合并改变TGF-β2潜伏期。我们将产生抗体作为结晶 除了使用已经开发的纳米抗体来proTGF-β2之外，还可以使用分子伴侣。互补展开 研究将检验环境分子结合稳定proTGF-β2的假设。目的3：TGF-β2的特性 activation.后续研究将鉴定天然激活TGF-β2的细胞系，并表征TGF-β2的特性。 生理相关的过程。这项资助的结果将增强我们对TGF-β2潜伏期的理解 并在细胞外环境中活化，为开发靶向proTGF-β2的治疗方法奠定基础 及其与环境分子的生理相关复合物。