The role of fibrillin in TGFB latency and integrin-mediated activation

纤维蛋白是所有哺乳动物弹性组织细胞外基质的重要组成部分，如血管、肺、关节和皮肤。纤维蛋白赋予我们的组织弹性，也是细胞信号传递所必需的，它将TGFb家族生长因子存储在基质中，以提供对正常发育和组织维护至关重要的组织存储。当纤维蛋白被破坏时，就像马凡综合征和其他由纤维蛋白基因突变引起的疾病一样，活性的TGFb会增加，从而导致广泛的组织损伤。这一悖论目前尚不清楚，但表明在没有纤维蛋白的情况下，TGFb更容易被激活。TGFb被细胞表面整合素受体激活，整合素受体也与细胞表面的纤维蛋白结合。然而，目前还不清楚纤维蛋白在TGFb激活过程中的作用，因此，我们希望了解纤维蛋白在控制TGFb的生物利用度和激活过程中的作用。为了解决这个问题，我们将形成纤维蛋白与潜伏的TGFb和整合素的复合体，用于结构和生化分析。该项目将使用蛋白质表达系统来纯化特定的蛋白质复合体，然后使用电子显微镜技术(CryoEM)来确定它们的三维结构。这些方法将得到基于细胞的TGFb信号分析的支持。将使用最先进的成像方法，包括冷冻电子显微镜，在最近的“分辨率革命”之后，现在可以从CryoEM数据中获得原子分辨率结构，使以前难以处理的蛋白质，如细胞外基质蛋白质，可以成为结构确定的目标。为了帮助复杂的形成和结构分析，该项目将使用单域抗体片段(纳米体)，这是蛋白质结构生物学中特别有用的工具，有助于构象动态蛋白质的研究。一个体外酵母表面展示平台将被用来寻找针对纤维蛋白的纳米体，以帮助结构、生化和细胞生物学研究。这个项目将使用结合分析和低温EM来确定当潜伏的TGFb与纤维蛋白结合时，整合素-TGFb结合是增强还是抑制。对这些结构的分析将有助于深入了解纤维蛋白在TGFb的储存和激活中的作用。用于提纯潜伏的TGFb、纤维蛋白和整合素胞外域aVB6的表达系统已经在实验室中可用于这些研究。这些相互作用分析将得到使用纳米荧光素酶报告分析TGFb信号的信号分析的补充，该分析也可在该项目的实验室内获得。

Fibrillin is an essential component of the extracellular matrix of all mammalian elastic tissues such as blood vessels, lung, joints and skin. Fibrillin gives our tissues elasticity and is also essential for cell signalling, storing TGFB family growth factors within the matrix to provide a tissue store which is critical for normal development and tissue maintenance. When fibrillin is disrupted, as seen in Marfan syndrome and other diseases caused by fibrillin mutations, there is an increase in active TGFB which results in widespread tissue damage. This paradox is not currently understood but indicates that in the absence of fibrillin, TGFB can be more readily activated. TGFB is activated by integrin cell surface receptors which also bind to fibrillin at the cell surface. However, it is not understood what role fibrillin plays in the process of TGFB activation, therefore, we wish to understand the role of fibrillin in controlling the bioavailability and activation of TGFB.To address this question, complexes of fibrillin with latent TGFB and integrins will be formed for structural and biochemical analysis. This project will use protein expression systems to purify specific protein complexes and then use electron microscopy techniques (CryoEM) to determine their 3-dimensional structures. These approaches will be supported by cell-based TGFB signalling assays. State-of-the-art imaging approaches will be used, including cryo-electron microscopy where following the recent "Resolution Revolution", atomic resolution structures from cryoEM data are now achievable, making previously intractable proteins such as extracellular matrix proteins accessible targets for structure determination. To aid in complex formation and structural analysis, this project will use single-domain antibody fragments ('nanobodies') which are especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins. An in vitro yeast surface display platform will be used to pan for nanobodies against fibrillin to aid the structural, biochemical and cell biology studies. This project will determine whether integrin-TGFB binding is enhanced or inhibited when latent TGFB is bound to fibrillin using binding assays and cryoEM. Analysis of these structures will provide insight into the role of fibrillin in the storage and activation of TGFB. Expression systems to purify latent TGFB, fibrillin and the extracellular domain of integrin aVB6 are already available in the lab for these studies. These interaction analyses will be complemented by signalling assays using a nano-Luciferase reporter assay for TGFB signalling which is also available within the lab for this project.