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

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.