Transforming growth factor beta (TGF-) plays pivotal roles in development of tissues including bone and muscle, wound healing, immune regulation, and tumor-cell growth and inhibition. TGF- regulates repair in all tissues including brain, heart, and bone, and after ischemia and stroke. Mutations in the TGF-1 prodomain cause Camurati-Engelmann disease, which manifests as abnormal bone growth with muscle weakness and pain. TGF-1, 2 and 3 are synthesized as pro-proteins that dimerize and associate with presenting molecules, latent TGF- binding protein (LTBP) or glycoprotein-A repetitions predominant protein (GARP), in the endoplasmic reticulum. Furin cleaves between the prodomain and growth factor domain; however, the prodomain dimer remains noncovalently associated with the growth factor dimer in pro-TGF- after secretion. LTBP and GARP bind to the prodomains and store pro-TGF-s in the extracellular matrix and on the cell surface, respectively. Although stored in tissues, large latent complexes are biologically inactive because the prodomains encircling the TGF- dimer prevent binding to TGF- receptors. The key regulatory step in TGF- signaling is release of TGF- from latency. Integrins aV6 and aV8 bind to a specific motif that includes RGD in pro-TGF-1 and 3 and activate them, but not pro-TGF-2. Integrin-mediated activation requires pro-TGF- association with LTBP or GARP. In this grant, we study how aV integrins bind and activate pro-TGF-1 and 3, how structural differences between pro-TGF-1 and 2 correlate with different activation mechanisms, and how LTBP and GARP contribute to TGF- latency. Solving crystal structures of pro-TGF-1, integrin aV6 headpiece alone or in complex with TGF-3 peptide, and integrin aV6 head in complex with human pro-TGF- 1 macromolecule, coupled with affinity, kinetics, and thermodynamic measurements will reveal conformational changes that occur upon binding and the molecular basis for how integrin aV6 binds pro-TGF- 1 with high affinity and specificity, but not pro-TGF-2. Potential structure-based therapeutics are also emerging. To better understand TGF- structure in the context in which it is kept latent, stored, and activated in vivo, we will study po-TGF-1 and 2 and their complexes with LTBP or GARP. We will solve crystal and SAXS structures of pro-TGF-1 and 2 and probe their dynamics in solution using proteolysis and hydrogen/deuterium exchange methods. We will solve at least one complex with LTBP or GARP. These experiments will provide key structural insight into conformational changes upon binding. Furthermore, kinetics and affinity measurements on mature TGF-1 and 2 binding to free prodomains and LTBP- or GARP-associated prodomains will provide a quantitative, biochemical characterization of their respective interactions. Structural information on pro-TGF-1 and 2, and complexes with LTBP or GARP, will provide critical insight into how pro- TGF-1 and 2 differ in their latency and activation mechanisms and new therapeutic approaches.
Transforming growth factor betas (TGF-s) are secreted proteins that regulate important processes such as development of tissues including bone and muscle, wound healing, immune responses, and tumorigenesis. However, the majority of TGF-s are stored as a biologically inactive form in the extracellular matrix of cells and on cell surfaces. This grant deepens our structural and biochemical understanding of the latency and activation mechanisms of TGF-s, which will have broad implications in the development of therapeutics for treating diseases associated with elevated TGF- signaling, such as fibrosis, Marfan syndrome, Camurati-Engelmann disease, and cancer.
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