Structural mechanism of integrin-mediated TGF-b activation

摘要/摘要：TGF-β 驱动导致肺和气道纤维化的纤维炎症过程。的 该项目的长期目标是深入了解 TGF-β 活性的调控，以开发 纤维化肺病的新策略和治疗方法。治疗慢性病的有效疗法很少 肺部纤维化和炎症性疾病。细胞因子 TGF-β 是纤维化的中心介质， 病理性炎症，是纤维化肺病的潜在治疗靶点。然而，实际 靶向 TGF-β 本身或其受体的效用受到啮齿类动物、灵长类动物和 人类。非常需要针对 TGF-β 的纤维炎症作用的更具体的方法。一个 更具体地针对 TGF-β 局部效应的有希望的方法是针对其“激活”，因为它总是 以潜在形式 (L-TGF-) 产生，必须被激活才能发挥作用。 L-TGF- 的另一个特点是 它可以促进更具体的靶向，因为它通过 GARP 共价结合到特定的细胞表面。 L-TGF- 与整合素αvβ8的结合对于体内TGF-β激活至关重要。对于 v8 激活机制，为 与所有其他因素一样，长期以来人们一直认为 TGF-β 必须从 LAP 中释放，以便游离的 TGF-β 可以 扩散并结合靶细胞上的受体。基于最近使用单粒子获得的结构数据 电子冷冻显微镜（cryo-EM），我们最近提出了一种新模型，其中 v8 可以与 L- 结合 TGF-β 在呈递 L-TGF-β/GARP 复合物的细胞上诱导信号传导，但不释放和扩散 TGF-。在这三个目标中，我们解决了有关 L-TGF- 新模型的三个关键问题 激活。 (1) v8/L-TGF-/GARP 三元复合物屏蔽 TGF- 的 L-TGF- 的哪些柔性结构域 来自它的受体？ (2) L-TGF-β 与 αvβ8 结合诱导的灵活性是否是介导 TGF-β 所必需的 激活？ (3) TGF-β 受体 (TGF-βR) 是否与 αvβ8/L-TGF-β/GARP 复合体中的 TGF-β 结合？至 回答这些问题后，我们将使用冷冻电镜来确定 v8/L-TGF-/GARP 复合物的结构 以确定 L-TGF- 的灵活性如何促进 TGF- 激活，最后我们将捕获 TGF-βR 与 αvβ8/LTGF-β/GARP 的多聚体复合物。这些研究将改善机制 了解 TGF-β 激活和抑制它的治疗靶向策略。

Summary/Abstract: TGF- drives the fibroinflammatory processes that leads to lung and airway fibrosis. The long-term goal of this project is to acquire a deep understanding of the regulation of TGF- activity to develop new strategies and treatments for fibrosing lung disease. There are few effective therapies to treat chronic fibrosing and inflammatory diseases of the lung. The cytokine TGF- is a central mediator of fibrosis and pathologic inflammation and is a potential therapeutic target in fibrosing lung disease. However, the practical utility of targeting TGF- itself or its receptors is limited by risk of toxicities seen in rodents, primates and humans. More specific methods to target the fibroinflammatory effects of TGF- are highly desirable. A promising method to more specifically target local effects of TGF- is to target its “activation” since it is always produced in a latent form (L-TGF-) that must be activated in order to function. Another feature of L-TGF- that could facilitate more specific targeting is that it is covalently bound to specific cell surfaces by GARP. L-TGF- binding to the integrin v8 is essential for TGF- activation in vivo. For the v8 activation mechanism, as well as all others, it has long been assumed that TGF- must be released from LAP so that free TGF- can diffuse and bind its receptors on target cells. Based on recent structural data obtained using single particle electron cryomicroscopy (cryo-EM), we have recently proposed a new model whereby v8 can bind to L- TGF- on cells presenting the L-TGF-/GARP complex and induce signaling without release and diffusion of TGF-. Here in three aims, we address three critical questions concerning this new model of L-TGF- activation. (1) Which flexible domains of L-TGF- of the v8/L-TGF-/GARP ternary complex shield TGF- from its receptors? (2) Is flexibility of L-TGF- induced by binding to v8 necessary to mediate TGF- activation? (3) Do TGF- receptors (TGF-Rs) bind to TGF- within the v8/L-TGF-/GARP complex? To answer these questions, we will use cryo-EM to determine the structure of the v8/L-TGF-/GARP complex to determine how flexibility of L-TGF- contributes to TGF- activation, and finally we will capture the multimeric complex of TGF-Rs with v8/LTGF-/GARP. These studies will improve mechanistic understanding of TGF- activation and therapeutic targeting strategies to inhibit it.