Structural mechanism of integrin-mediated TGF-b activation

About This Grant

Summary/Abstract: The multifunctional cytokine TGF-β is a central mediator of the chronic inflammatory and fibrotic pathologic processes that leads to lung and airway fibrosis. Our long-term goals for this project are to acquire a deeper understanding of how TGF-β function is regulated, and to leverage that understanding to develop new strategies and treatments for fibrosing lung disease. There is an urgent need for effective therapies to treat chronic fibrosing and inflammatory diseases of the lung. Clinical trials targeting TGF-β itself or its receptors has faced challenges, most notably, toxicities. More understanding of how TGF-β functions is clearly needed to develop better and more specific methods to more effectively target the pathologic effects of TGF-β. Since TGF-β is always produced in an inactive form that must be activation to function, methods targeting its activation may more specifically target the local effects of TGF-β thus avoiding systemic toxicities. TGF-β is activated by binding to several integrins. What exactly happens biologically after these integrins bind to TGF-β remains largely speculative. Because of this lack of biologic understanding it has long been assumed that TGF-β must be released from LAP so that free TGF-β can operate as a paracrine factor and diffuse and bind its receptors on target cells. Based on our recent structural data obtained using single particle electron cryomicroscopy (cryo-EM), we have developed a new hypothesis where integrins can bind to L-TGF-β on cells presenting the L-TGF-β on their cell surface and induce autocrine signaling without release and diffusion of TGF-β. We have recently verified that such an autocrine mechanism is sufficient to maintain the essential functions of TGF-β1 in mice, and that paracrine TGF-β1 signaling is dispensable. We now have structural and cell-based data that provides a new hypothesis for TGF-β1 activation: The inherent disorder (entropy) present in specific domains of TGF-β1 and integrin αvβ8 is redistributed upon binding of L-TGF-β1 to αvβ8 which is sufficient to cause exposure of mature TGF-β1 to its receptors. Here in three aims, we address three critical questions concerning this new model of L-TGF-β activation. (1) Can entropy redistribution of the L-TGF-β/αvβ8 complex be predicted by structural methods, and if so can it be biochemically and therapeutically manipulated? (2) How does a native membrane environment affect entropy redistribution of the L-TGF-β/αvβ8 complex? (3) Can the entropy redistribution hypothesis be applied to other integrins and can it be harnessed to be therapeutically useful to treat lung fibrosis? To answer these questions, we will establish and employ state-of- the-art techniques including cryo-EM coupled to orthogonal spectroscopic, and analytical techniques to more quantitatively assess protein thermodynamics, protein engineering, recombinant antibodies to affect local protein conformational dynamics and finally testing of those antibodies in lung fibrosis models in vivo. Together, these studies will improve mechanistic understanding of TGF-β activation and therapeutic targeting strategies to inhibit it.