Regulation and substrate binding of the tolloid proteinase family

The tolloid family of proteins has essential roles in two fundamental processes in mammalian biology. The first is tissue assembly, for example bone growth, where tolloids cut protein precursors initiating the assembly process. Tolloids remove a region at the end of collagen molecules and this removal begins the collagen fibril assembly process which is essential for bone growth and the formation of other normal healthy tissues. Secondly, tolloids release stored 'growth factors' which are messenger molecules that can direct processes in cells, for instance telling them how much to grow or even whether they should live or die. These processes are critical for maintaining normal tissue structure and function and for essential processes in early embryonic development. We have recently determined the shape and arrangement of one member of the tolloid family that is found in humans, and shown that this tolloid works as a pair. This self-self interaction actually slows the tolloid down and may represent a novel method of internal regulation. Tolloids can also be controlled by protein enhancers that increase their capability of cutting their substrates (i.e. their activity). How tolloids interact with the many diverse molecules they cut is currently unknown, and insights into these processes are urgently needed in order to understand how they control such diverse functions in human biology. The main aim of our work therefore is to resolve the mechanism of action of tolloids which we believe will lead to an understanding of how their regulation and interactions underpin their fundamental roles in tissue assembly and embryo development. We will determine if all tolloids work in pairs and what happens when tolloids bind to their substrates, such as the collagen precursor, for example, does it remain paired or go solo. Finally, we will discover how protein enhancers work to improve the activity of tolloids. Understanding these molecular events preceding bone formation could have significant health and economic benefits to the UK. In the UK, 1 in 2 women and 1 in 5 men suffer a fracture after the age of 50 and the cost of treating all osteoporotic fractures in postmenopausal women has been predicted to increase to more than £2 billion by 2020. Our research findings could be of future interest to the pharmaceutical industry in developing novel treatments to modulate bone deposition. Effective treatment for bone loss would significantly improve the quality of life of an ageing population.

Tolloid proteinases have essential roles in tissue assembly and developmental patterning, two fundamental processes in mammalian biology. They process a diverse range of extracellular protein precursors instigating assembly or activation. The tolloid proteinases play an essential role in collagen fibrillogenesis which is necessary for processes such as bone growth. They also release growth factors from latent complexes regulating signalling which depending on the growth factor is fundamental to tissue homeostasis or developmental processes such as bone remodelling following fracture or dorsal-ventral patterning. We have shown that dimerisation of mammalian tolloid limits substrate access to the active site, a novel mechanism of internal regulation for controlling binding of substrates. However, how tolloids interact with distinct substrates is currently unknown but insights into these processes are urgently needed in order to understand how they control such diverse functions. The main aim of our work therefore is to resolve the mechanism of action of tolloid proteinases leading to an understanding of how their regulation and substrate interactions underpin their critical roles in tissue assembly and developmental patterning. Specifically, we will determine the mechanism of self-regulation by substrate exclusion and whether this is fundamental to other species; we will determine the oligomeric state of tolloids when processing substrates and whether there is a common mechanism of substrate binding between structurally diverse substrates; and determine how protein regulators act on tolloid proteinases to moderate their activity. We will use a multidisciplinary approach of structural, biochemical and biophysical techniques including electron microscopy, small angle X-ray scattering, BIAcore, dual polarisation interferometry and analytical ultracentrifugation.

We anticipate that the results gained from this study will be of both significant intellectual and clinical benefit as it 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'. This work will provide novel insights into the molecular mechanism relevant to tissue assembly, in particular bone formation. Tolloid proteinases process the soluble procollagen precursor instigating bone formation. Progress in understanding and treating diseases that result from aberrant or decreased deposition of bone is poor. Therefore our project has the potential to lead towards new management polices and treatments for fracture healing, osteopenia and osteoporosis. Currently 1 in 2 women and 1 in 5 men will suffer a fracture after the age of 50 in the UK, and the cost of treating all osteoporotic fractures is a significant financial burden to the NHS. For example, our research findings could be of future interest to the pharmaceutical industry in developing novel inhibitors or enhancers of tolloid proteinases to modulate bone deposition. Understanding the molecular events preceding bone formation could have significant health and economic benefits to the UK and effective treatment for bone loss would improve the quality of life of the older population. The results of this study will be of academic benefit to a range of research communities including connective tissues, development, metalloproteinase 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 group participates). Initiatives include schools outreach work (e.g. curriculum enrichment in the form of practical classes, workshops and career advice) and reporting research breakthroughs in the local, national and international press via the Faculties Media Relations Office. In this regard, the electron microscopy facility in FLS has regular visits from schools. Another, recent example of such activities is a series of four open days for schools in March 2010 entitled 'Wellcome to the Matrix' in which CB was involved. Due to the range and combination of techniques required for this project, the postdoctoral researcher who will be recruited is unlikely to have skills in all the techniques described. Therefore training and development of the PDRA in new techniques will enhance their 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.