The role of ADAMTS-like proteins in fibrillin microfibril assembly

Fibrillin forms fibres that are important for providing our connective tissues, such as large blood vessels like the aorta, eye ligaments and skin, with elasticity. Symptoms of ageing associated with a loss of elasticity, for example skin wrinkles, hypertension and eye deterioration, have been linked to the degradation of fibrillin. Fibrillin binds to growth factors outside of the cell creating a tissue store needed for correct development, repair and maintenance of our tissues. Mutations in fibrillin typically cause Marfan syndrome, a common inherited disease where suffers are very tall with bone abnormalities, heart and eye defects linked to disorganised cell signalling. However, mutations in two very specific regions of fibrillin cause rarer diseases with opposite symptoms to Marfan syndrome where sufferers are very short with stiff joints and thickened stiff skin. How mutations in fibrillin can cause these two very different diseases is a conundrum but one clue is that mutations in A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS) and ADAMTS-like proteins also cause the same "short" diseases. ADAMTS/L proteins assist fibrillin assembly into tissue structures but we currently know very little of how they function. Our limited knowledge regarding the mechanism of action of ADAMTS/L proteins presents a major obstacle to understanding their function in fibrillin assembly and cell interactions. Therefore, the aims of our work are to understand how ADAMTS/L proteins enhance fibrillin assembly and using electron microscopy imaging discover the structure of ADAMTSL2 alone and in complex with fibrillin. We will show how ADAMTS/L proteins interact with proteins at the cell surface to support fibrillin assembly, and we will determine what changes ADAMTS/L proteins make to cell behaviour and protein expression. Together these findings will lead to a better understanding of how ADAMTS/L proteins influences fibrillin assembly and cell interactions. Due to their essential roles 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 how ADAMTS/L proteins aid in fibrillin assembly, whose functions influence normal bone growth and 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. 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.

Fibrillin microfibrils are vital components of connective tissues, required for elastic fibre formation which is essential for the formation and function of mammalian elastic tissues, such as large blood vessels, skin and eye ligaments. In addition to their structural role, microfibrils mediate cell adhesion via integrin and syndecan receptors, and cell signalling by sequestering TGFbeta superfamily growth factors within the matrix providing a tissue store which is critical for homeostasis and remodelling. ADAMTS/L proteins enhance fibrillin assembly and mutations in both ADAMTS/L proteins and fibrillin cause a group of "short" fibrillinopathies, with manifestations in bones, eyes, and skin. However, a lack of knowledge of the molecular mechanisms of ADAMTS/L proteins and how they influences fibrillin assembly, cell adhesion and signalling, limits our understanding of their mode of action. Therefore, the aim of this research is to understand the structure of ADAMTS/L proteins, alone and in complex with fibrillin, along with protein interaction and cell-based analyses will inform us how they influence assembly of fibrillin microfibrils and support cell adhesion.

We will use cryo-EM with image analysis to determine the 3D structure of ADAMTSL2 alone and in complex with fibrillin, and we will investigate how ADAMTS/L proteins influence assembly of fibrillin by analysing binary and tertiary molecular interactions. Finally, we will determine which ADAMTS/L proteins support focal adhesion formation and cell signalling, and whether they influence expression of elastic fibre proteins. Given the key role played by microfibrils as mediators of tissue homeostasis and the role of ADAMTS/L proteins in enhancing microfibril assembly, our findings will inform future therapeutic avenues for tissue engineering strategies to recapitulate native tissue function.

We anticipate that the results gained from this study will be of both significant intellectual and clinical benefit on the molecular mechanisms of both "short" and "tall" fibrillinopathies. The research will impact on a number of mammalian connective tissues including eyes, bone and skin and the role of the matrix in providing an instructive role in the assembly and homeostasis of these tissues. Our research findings could be of future interest to the pharmaceutical industry in developing treatments to support normal bone growth, wound healing and 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 any commercialisable research.

The results of this study will be of academic benefit to a range of research communities including connective tissues, development, growth factor and tissue regeneration 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 committed to public engagement in science. Our 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. 'Daresbury Laboratory Science Festival, July 2016, regularly host pupils for work placements e.g. Nuffield Bursary scheme, and participate in science events (e.g. Whitworth Art Gallery Science Spectacular, November 2016). We report research breakthroughs in the local, national and international press via the Faculties Media Relations Office.

Training and development of the postdoctoral researcher (PDRA) in new sought after techniques, namely cryoEM will enhance his research career. The Faculty of Biology, Medicine and Health 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.