Dual functionalities of the LTBP family in matrix assembly and growth factor regulation

Our elastic tissues such as our skin, lungs and large diameter blood vessels are made up of elastic fibres. Large proteins called fibrillin and latent TGFbeta binding protein (LTBP) are important components of elastic fibres and, as well as providing our tissues with mechanical support, they also store growth factors in our connective tissues. Transforming growth factor (TGF)beta is a powerful growth factor, 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 when disorganised can often lead to disease. The action of TGFbeta is controlled by LTBP and fibrillin, which are found outside of the cell, where they bind and store TGFbeta. This storage system is needed for correct development, repair and maintenance of our tissues.

As well as storing TGFbeta, the LTBP family of proteins are important in the tissue organisation of fibrillin and other elastic fibre proteins. However, our limited knowledge regarding the mechanism of action of the LTBP family in TGFbeta storage and tissue assembly presents a major obstacle to understanding their function. The main aim of our work therefore is to understand how the LTBP family facilitates assembly of fibrillin and other elastic fibre proteins including the role of proteoglycans in their function. We want to understand the differences between members of the LTBP family as some only store TGFbeta whereas others are only involved in matrix assembly. We want to study the structure of LTBPs in complex with fibrillin using cryo-electron microscopy, and we have found that this complex is stabilised by a covalent bond. We will also analyse the structure of the LTBP-TGFbeta complex when this is connected to fibrillin which we believe will lead to an understanding of how TGFbeta storage occurs. Together these findings will lead to a better understanding of how the LTBP proteins influences matrix assembly and growth factor storage. Due to the essential roles of LTBPs and fibrillin in normal tissue assembly, elasticity and maintenance of our tissues, being able to reconstitute or repair these tissues would provide opportunities for regenerative medicinal applications.

Understanding these molecular events for assembling and maintaining elastic tissues could have significant health and economic benefits to the UK. 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. 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 and in engineering of replacement biomaterials. Effective treatment would significantly improve the quality of life of an ageing population.

Extracellular glycoproteins, the latent TGFbeta-binding proteins (LTBP) and fibrillin, are essential components of elastic fibres, required for the formation and function of mammalian elastic tissues, such as large blood vessels, skin and lung. In addition to their mechanical role, they mediate cell signalling by sequestering TGFbeta growth factors within the matrix. The TGFbeta family 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. LTBP and fibrillin regulate the bioavailability of TGFbeta via the formation of latent complexes, thereby preventing receptor activation but an unresolved aspect of TGFbeta signalling in humans are the molecular events surrounding the regulation of growth factor bioavailability in the matrix.

Moreover, emerging functions of the LTBP family are their TGFbeta-independent roles in the assembly and stabilisation of fibrillin bundles and elastic fibres but through as of yet undefined mechanisms. Therefore, the aims of this research are to understand the structure and assembly of LTBP complexes that either mediate elastic fibre assembly or stabilise latent TGFbeta to influence growth factor bioavailability, the differences between LTBP isoforms that predispose them to function in either matrix assembly or growth factor regulation and the role fibrillin plays in these processes. We will use a multi-technique approach of structural, biochemical and biophysical techniques including cryo-electron microscopy with single particle image analysis, protein-protein interaction analyses (surface plasmon resonance and biolayer interferometry) and cell signalling assays. Given the key role played by fibrillin and LTBPs as mediators of tissue homeostasis, our findings could inform future tissue engineering strategies for storing or releasing growth factors which could lead to innovative strategies to influence cellular behaviour.

We anticipate that the results gained from this study will be of both significant intellectual and clinical benefit as the project will inform on molecular mechanisms of elastic fibre assembly and latent TGFbeta storage in the extracellular matrix. In particular, this work is of relevance to the BBSRC Strategic Research Priority "Healthy ageing across the lifecourse" and the RCUK programme on "Lifelong Health and Wellbeing" because of the vital roles elastic fibre proteins, such as LTBPs and fibrillin, play in maintaining the normal structure and function of mammalian connective tissues including the musculoskeletal system. The research will provide novel insights into molecular mechanisms relevant to tissue assembly, in particular elastic tissues such as the skin, heart, lungs and eyes and the role of the matrix in providing an instructive role in the assembly and homeostasis of these 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 growth factor signalling, proteoglycan, connective tissues 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. The University strongly supports staff in communicating their work publicly and we are also committed to public engagement in science. For example, the Wellcome Centre for Cell-Matrix Research has a dedicated public engagement programme manager to facilitate the delivery of such events. We vigorously pursue public engagement: interact with school students e.g. Whitworth Gallery Art-Science Primary School Masterclass, October 2017; regularly host pupils for work placements e.g. Nuffield Bursary scheme; interact with the local community (the annual Faculty of Biology, Medicine and Health (FBMH) Community Open Day) and participate in science events (e.g. Manchester Science Festival, October 2018). We report research breakthroughs in the local, national and international press via the Faculties Media Relations Office.

Training and development of Dr Alan Godwin, the named PDRA, in new sought after techniques such as single particle analysis of proteins and complexes using cryo-EM will enhance his research career. Indeed, this aligns with the BBSRC's strategic plan in maintaining strength and capabilities in core underpinning disciplines such as structural biology. FBMH 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.