Understanding TGF beta activation in health and disease

The TGF ( transforming growth factor) beta signalling pathway plays an essential role in many aspects of biology, including development of the embryo and maintenance of adult tissues. There are three different forms of TGF beta, each of which is secreted from cells in the form of an inactive or latent complex, and this complex is itself often bound to a much larger protein called latent transforming growth factor beta binding protein (LTBP). This large protein ensures that TGF beta is sequestered in the extracellular matrix, an insoluble collection of proteins and carbohydrates which surround cells, where it can be activated in response to specific signals and delivered to its receptor in a carefully regulated manner. Subsequent binding of TGFbeta to its receptor leads to many different cellular effects in tissues. This signalling pathway is disrupted in many different genetic and acquired diseases, such as cancer, autoimmune and inflammatory disorders and heritable diseases of the connective tissues including Marfan syndrome. We have recently identified one of the ways in which LTBP is localised to the extracellular matrix, by its interaction with a protein called fibrillin-1. We believe that studying this interaction in detail will provide insight into how TGF beta is released in an active form from the matrix, and how various diseases associated with excessive levels of TGF beta activation arise. Furthermore this basic science may provide new ways of designing drugs such as monoclonal antibodies or small molecule activators or inhibitors which will be invaluable in a research or clinical setting to manipulate the TGF beta signal.

This grant aims to investigate the structural and functional significance of the dynamic C-terminus of human LTBP1 and its interaction with human fibrillin-1. We wish to establish the importance of this interaction in modulating the activation of TGF beta, which is covalently bound to LTBP via its propeptide LAP at a site adjacent to this C-terminal region. We will use the well established collaboration of Handford (biochemistry) and Redfield (biophysics) to investigate i) the structural basis of the LTBP/fibrillin-1 interaction for LTBP1 and other isoforms shown to bind to fibrillin-1 (using site-directed mutagenesis, NMR, SPR and related biophysical techniques), and ii) the functional importance of this interaction for TGFbeta activation, using an established TGFbeta reporter cell line in co-culture with normal and deficient fibroblast lines secreting fibrillin-1 and LTBP variants. These data will give new mechanistic insights into the role of extracellular matrix in regulating TGF beta activation, and molecular mechanisms underlying various connective tissue diseases which lead to over-activation of TGF beta. Furthermore these studies have the potential to direct further in vivo models as well as provide a rational basis for the design of novel antagonists /agonists. Results will be presented at international conferences on TGF beta signalling and disease, and any commercial exploitation will be handled by ISIS Innovation, University of Oxford.

Since TGF beta signalling is of major importance to metazoan development and homeostasis, any new mechanistic insight into the role of the extracellular matrix ( ECM) in regulating the activity of TGFbeta should prove invaluable in understanding its role in normal biology as well as in pathological situations. Our new data presented in this application have already increased our understanding of the molecular properties of LTBPs, which sequester TGF beta in the ECM, and suggested a number of functional experiments to complement ongoing structural studies that directly address mechanisms of TGF beta activation. The bioinformatics investigations of the evolution of the LTBP/fibrillin family have also suggested a hierachy of protein/protein interactions involved in TGF beta activation which complement the experimental approach outlined in this application. We are at an early stage of dissecting the contribution of LTBP/fibrillin interactions, but our production of purified proteins for molecular studies have the potential to be used to develop monoclonal antibodies. These may prove useful as research or therapeutic tools to manipulate the LTBP/fibrillin interaction within the ECM. There is an established infrastructure and pipeline to develop such reagents through ISIS Innovation, the University of Oxford technology transfer company, in accordance with MRC policy. Handford has experience of developing such reagents and is part of a CRUK Programme to develop monoclonal antibody reagents to regulate Notch signalling. Both Handford and Redfield believe in the importance of engaging in Public Understanding of Science activities. We will willingly give support to the MRC to facilitate communication of our research discoveries, and will also engage with the public at other suitable events such as Science Open Days, School activities/ talks and visits.