The structure and extracellular regulation of the large latent TGFbeta complex

Transforming growth factor (TGF) beta is a powerful "growth factor" which is a messenger molecule that can direct processes in cells, for example telling them how much to grow, move or even whether they should live or die. These processes are crucial in maintaining normal tissue structure and function and are essential in human embryo developmental. The action of TGFbeta is controlled by large proteins found outside of the cell that bind to TGFbeta creating a tissue storage depot. This storage is needed for correct development, repair and maintenance of our tissues, such as the heart, lungs and skin.

The large proteins that bind to TGFbeta are called fibrillin and latent TGFbeta binding protein. Fibrillin forms fibres that are important for providing our tissues with elasticity such as large blood vessels like the aorta, lungs and skin. Symptoms of ageing associated with a loss of elasticity, for example skin wrinkles, hypertension and deterioration in vision, have been linked to degradation of fibrillin. Fibrillin and latent TGFbeta binding protein (LTBP) bind to TGFbeta, both these proteins are essential for the tissue storage of TGFbeta to occur correctly but there are currently no details of the fibrillin-LTBP-TGFbeta complex. Our limited knowledge regarding TGFbeta storage and the complexes they form presents a major obstacle to understanding TGFbeta function. The main aim of our work therefore is to understand the structure of the fibrillin-LTBP-TGFbeta complex which we believe will lead to an understanding of how TGFbeta storage occurs. We will determine the shape of LTBP when bound to TGFbeta, and how fibrillin and fibrillin fibres interact with this complex. Finally, we will discover how TGFbeta is released from this complex when it is needed and how these interactions underpin their important roles in tissue assembly and maintenance.

Understanding these molecular events for maintaining tissue elasticity could have significant health and economic benefits to the UK. Stiffening of the blood vessels and valves of the heart are major causes of heart disease which affects more than 6 million citizens in Europe each year. Heart disease has a huge economic impact, due to the high medical costs and work disability. In the eye, losing elasticity effects the ability to bend the lens (accommodation) which leads to the loss of up-close vision with age. This can be improved by wearing glasses but does not correct completely for this age-related deterioration in vision. Our research findings could be of future interest to the pharmaceutical industry in developing treatments to maintain the elasticity of these tissues. Effective treatment would significantly improve the quality of life of an ageing population.

The transforming growth factor (TGF) beta family of growth factors play crucial roles in embryonic development, adult tissue homeostasis, the immune response and the pathogenesis of a wide-range of diseases from fibrosis to tumour invasion. The bioavailability of this class of growth factors is regulated by the extracellular matrix, providing a tissue store which is critical for tissue homeostasis and remodelling. Two molecules are essential for the sequestration of TGFbeta in the matrix: fibrillin and latent TGFbeta binding protein (LTBP). Fibrillin microfibrils are extensible polymers that are critically important in maintaining the integrity of tissues such as blood vessels, lung and skin. Fibrillin binds to LTBP-1 which in turn is covalently attached to TGFbeta. The expression of LTBP-1 is co-regulated with TGFbeta and together they are secreted as the large latent TGFbeta complex (LLC). The lack of knowledge regarding TGFbeta regulators and the complexes they form presents a major hurdle to understanding TGFbeta bioavailability.

The aim of our work therefore is to define the structure and organisation of the large latent TGFbeta complex and its interaction with fibrillin, leading to an understanding of how TGFbeta sequestration via fibrillin is controlled in tissue assembly and homeostasis. Specifically, we will determine the nanoscale structure of the LLC and how the LLC interacts with fibrillin and fibrillin microfibrils. We will determine the molecular events surrounding release of TGFbeta from the LLC, mediated by integrins and proteases, and the role fibrillin plays in this process. We will use a multi-technique approach of structural, biochemical and biophysical techniques including electron microscopy, solution X-ray scattering, multiangle light scattering, analytical ultracentrifugation, scanning TEM mass mapping, atomic force microscopy and binding analyses (surface plasmon resonance and dual polarisation interferometry).

We anticipate that the results gained from this study will be of both significant intellectual and clinical benefit as the project will deliver high-quality biochemical research on a fundamental process in mammalian biology. In particular, this work is relevant to the BBSRC Strategic Research Priority "Ageing research: lifelong health and wellbeing" because of the vital roles fibrillin and the large latent TGFbeta complex play in maintaining the normal structure and function of tissues such as the skin, heart, lungs and eyes.

This work will provide novel insights into a molecular mechanism relevant to tissue assembly, in particular elastic tissues. This proposal is to undertake basic science underpinning the regulation of homeostatic events in tissues, but our research findings could be of future interest to the pharmaceutical industry in developing treatments to maintain the elasticity of tissues. We will utilise the Faculty Research Support Managers, part of whose remit is to facilitate interactions with industry and University of Manchester Intellectual Property (UMIP) to identify outcomes with commercial potential.

The results of this study will be of academic benefit to a range of research communities including connective tissues, development, growth factor and structural biology research communities as outlined in the academic beneficiaries section. We will disseminate the results of this research through participation at relevant conferences and through publications in peer-review journals as outlined in the previous section. We are also committed to public engagement in science. For example, the Faculty of Life Sciences (FLS) is active in promoting the communication of science to the public (in which the applicants' groups participate). Initiatives include reporting research breakthroughs in the local, national and international press via the Faculties Media Relations Office and schools outreach work (e.g. curriculum enrichment in the form of career advice, practical classes, and workshops). In this regard, the electron microscopy facility in FLS hosts regular visits from schools for pupils in years 11-13 and the EM facility runs tours during the annual Faculty of Life Sciences Community Open Day in which all lab members participate.

Training and development of Helen Troilo, the named RA, in new techniques such as cryo-EM will enhance her research career. FLS has embraced training and career development for all categories of staff and current support available to PDRAs for professional development includes monthly training bulletins, one-to-one advice and guidance and bespoke workshops. Recent workshops have included: "Planning a Fellowship", "Grant Reviewing", "Academic CV Writing" and a "Careers Day". These workshops have aimed to develop a range of skills including career planning, networking, project management, team working, critical peer review, communication and self awareness.