Structural mechanism of integrin-mediated TGF-b activation

摘要/摘要：转化生长因子-β推动纤维炎症过程，导致肺和呼吸道纤维化。这个 本项目的长期目标是深入了解转化生长因子-β活性的调控发展 纤维化肺病的新策略和新疗法。治疗慢性阻塞性肺疾病的有效方法很少 肺纤维化和炎症性疾病。细胞因子转化生长因子-β是纤维化和 是一种病理性炎症，是治疗纤维化肺疾病的潜在靶点。然而，实际情况是 靶向转化生长因子-β本身或其受体的效用受到啮齿动物、灵长类动物和 人类。针对转化生长因子-β纤维炎症效应的更具体的方法是非常可取的。一个 有希望更具体地靶向转化生长因子-β的局部作用的方法是靶向其“激活”，因为它总是 以潜伏形式产生(L-转化生长因子-β)，必须激活才能发挥作用。L-转化生长因子-贝塔的另一个特点是 可以促进更具体的靶向是它通过GARP共价结合到特定的细胞表面。L-转化生长因子-贝塔 在体内，整合素αvBeta8与整合素αvBeta8的结合是转化生长因子-β激活所必需的。对于alphavBeta8激活机制，如 和所有其他的一样，长期以来人们一直认为转化生长因子-β必须从LAP中释放出来，这样游离的转化生长因子-β才能 将其受体扩散并结合到靶细胞上。基于使用单粒子获得的最新结构数据 电子冷冻显微镜(Cryo-EM)，我们最近提出了一种新的模型，在呈现L-转化生长因子-β/GARP复合体的细胞上，α-β8可以与L-转化生长因子-β结合并诱导信号传递，而不需要释放和扩散 转化生长因子-β。在这里，我们以三个目标来解决关于L-转化生长因子-贝塔这一新模式的三个关键问题 激活。(1)L-转化生长因子-β8/L-转化生长因子-β/GARP三元络合物的哪些柔性结构域屏蔽转化生长因子-β 来自其受体？(2)L与α-β8结合诱导的柔韧性是介导转化生长因子-β所必需的吗？ (3)转化生长因子-β受体是否与转化生长因子-β8/L-转化生长因子-β/GARP复合体中的转化生长因子-β结合？至 回答这些问题，我们将使用冷冻EM来确定αvBeta8/L-转化生长因子-β/GARP复合体的结构 为了确定L-转化生长因子-β的灵活性如何促进转化生长因子-β的激活，最后我们将捕获 转化生长因子-β与α-β8/1转化生长因子-β/GARP的多聚体复合物这些研究将改进机械学 了解转化生长因子-β的激活和抑制它的治疗靶向策略。

Summary/Abstract: TGF-Beta 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-Beta 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-Beta 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-Beta 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-Beta are highly desirable. A promising method to more specifically target local effects of TGF-Beta is to target its “activation” since it is always produced in a latent form (L-TGF-Beta) that must be activated in order to function. Another feature of L-TGF-Beta that could facilitate more specific targeting is that it is covalently bound to specific cell surfaces by GARP. L-TGF-Beta binding to the integrin alphavBeta8 is essential for TGF-Beta activation in vivo. For the alphavBeta8 activation mechanism, as well as all others, it has long been assumed that TGF-Beta must be released from LAP so that free TGF-Beta 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 alphavBeta8 can bind to L-TGF-Beta on cells presenting the L-TGF-Beta/GARP complex and induce signaling without release and diffusion of TGF-Beta. Here in three aims, we address three critical questions concerning this new model of L-TGF-Beta activation. (1) Which flexible domains of L-TGF-Beta of the alphavBeta8/L-TGF-Beta/GARP ternary complex shield TGF-Beta from its receptors? (2) Is flexibility of L-TGF-Beta induced by binding to alphavBeta8 necessary to mediate TGF-Beta activation? (3) Do TGF-Beta receptors (TGF-BetaRs) bind to TGF-Beta within the alphavBeta8/L-TGF-Beta/GARP complex? To answer these questions, we will use cryo-EM to determine the structure of the alphavBeta8/L-TGF-Beta/GARP complex to determine how flexibility of L-TGF-Beta contributes to TGF-Beta activation, and finally we will capture the multimeric complex of TGF-BetaRs with alphavBeta8/LTGF-Beta/GARP. These studies will improve mechanistic understanding of TGF-Beta activation and therapeutic targeting strategies to inhibit it.