Latent TGF-β2 Structure and Activation

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.