Bone remodelling is continuous throughout the lifetime of vertebrates, whereby two cell types, the bone-forming osteoblasts and bone-resorbing osteoclasts, are continually repairing damaged or worn-out areas of the skeleton. Tight regulation of the process is essential to ensure maintenance of the strength and shape of bones. Any abnormalities in remodelling can result in skeletal deformities and are the causes a number of diseases of the bone, including osteopetrosis, osteoporosis, rheumatoid arthritis and Paget’s disease.
Three proteins whose interactions play critical roles in remodelling are receptor activator of nuclear factor-kappa B (RANK), its cognate ligand RANKL and osteoprotegerin (OPG). RANK is a transmembrane receptor expressed on osteoclasts and osteoclast precursors and RANKL is expressed by osteoblasts and stromal cells. The binding of RANKL to RANK activates signalling pathways leading to osteoclast formation and activity; OPG regulates this interaction by acting as a decoy receptor competing with RANK binding to RANKL.
A number of disease-related mutations in these proteins have been identified that cause phenotypes of varying degrees of severity. This project aims to characterise the effects of such mutations on the three-dimensional structures and protein:protein interactions between these proteins. While atomic-level structural data are available for human RANKL and OPG from X-ray crystallography, interpretation of data for mutations in the extracellular region of the receptor (eRANK) is limited to models based upon the structure murine eRANK, no human structure being available.
The research will employ a biophysical and structural approach to obtain atomic level structural data of wild-type and mutant human eRANK to characterise their properties, and will investigate specific intra-, and intermolecular interactions at the domain level in order to fully understand the role of eRANK in signal transduction in health and in disease. Structure-guided, site-directed mutagenesis will be employed to confirm the functional importance of specific residues and interactions revealed by the structural work. In parallel, osteoclast assays will be performed to assess the functional consequences of genetic variants on osteoclast differentiation and function.
The student will receive interdisciplinary training in the two Schools at Edinburgh. Early in the project, training will be in molecular biology and recombinant protein technology, in order to over-express and purify recombinant proteins from various expression systems. The student will also be trained and gain invaluable experience in a range of biophysical techniques, including NMR and/or crystallography, mass spectrometry, dynamic and static light scattering, circular dichroism and isothermal titration calorimetry.
Further training will develop skills with mammalian cell culture and assays, where osteoclast assays will be performed to assess the functional importance of mutants, both clinical variants and structure-guided mutants, to support the biophysical studies. The project will utilise state-of-the-art equipment, working within the Institute of Quantitative Biology, Biochemistry and Biotechnology; and the Institution and School Centre for Genomic and Experimental Medicine, both at the University of Edinburgh.
The student will have access to all the PhD training programmes in both Schools, including journal clubs, method clubs, scientific writing skills and presentation skills.
RANK receptor oligomerisation in the regulation of NFkB signalling (2014). Das S, Sepahi I, Duthie A, Clark S. and Crockett JC. J. Mol. Endocrin. 53, 81-91.
RANKL Employs Distinct Binding Modes to Engage RANK and the Osteoprotegerin Decoy Receptor(2012) Nelson C, Warren JT, Wang, MW-H, Teitelbaum SL and Fremont DH. Structure, 20, 1971-1982