Paget’s disease of bone (PDB) shows a strong genetic component and mutations in SQSTM1 (Sequestosome 1) are observed in about 10% of sporadic PDB patients. My PhD investigated the RIN3 gene (Ras and Rab interactor protein 3), previously implicated in the pathogenesis of PDB by GWAS. The RIN3 gene encodes a guanine exchange factor (GEF), involved in the activation of GTPases which are crucial in osteoclast activity. It also has a role in endocytosis and recycling of tyrosine kinase receptor. The role of RIN3 in bone remodelling is unclear, however some investigations revealed some associations with bone: RIN3 has been associated with high lower limb bone mineral density in children in a meta-analysis of GWAS studies, and was shown to be expressed in primary calvarial osteoblasts. The expression of RIN3 was down regulated during human primary osteoclast differentiation, and also in iliac bone biopsies from osteoporotic patients compared to healthy postmenopausal donors (Kemp et al, 2014).   In Chapter 1, I present normal bone structure, composition and remodelling before detailing PDB and its genetics. I then introduce RIN3 as a candidate gene for PDB. In Chapter 2, I describe all methods performed and materials used for the completion of this project. This includes primary cell cultures, RNA and protein work, immunostaining, immunochemistry and phenotype analysis on Rin3-/- mice. Chapter 3 presents the fine mapping of RIN3 using Sanger and next generation sequencing performed on PDB cases and controls. 18 variants were detected and one common variant (p.R279C) showed a strong association with PDB. Rare variants were also over-represented in cases, and many were shown to be on the same haplotype as p.R279C. Chapter 4 details the association study performed on a UK cohort and includes the investigation of the clinical phenotype severity in patients against the RIN3 mutations. Chapter 5 presents the expression pattern of RIN3 in bone cells and bone microenvironment. Important variations of RIN3 mRNA and protein were detected during the differentiation of bone marrow derived osteoclasts. Protein levels of RIN3 were also found in osteoclasts from human osteoclastoma, human osteosarcoma, PDB patients, giant cell tumour (GCT) and healthy controls. Within all the mouse tissues analysed, Rin3 mRNA was expressed the highest in bone after lung. Chapter 6 focuses on the work performed on mice deficient of the Rin3 gene. They showed a higher trabecular bone volume and a smaller active resorption surface occupied by osteoclasts in trabecular bone. In conclusion, the combined in vitro and in vivo analyses have uncovered that RIN3 plays a role in bone metabolism and is a strong gene candidate for PDB.

SUMOylation and ubiquitination are important post-translational modifications. While ubiquitination is well known for targeting proteins for degradation, SUMOylation often regulates the intracellular localization of substrates. In the first project of this dissertation, we developed proteomic strategies to identify novel SUMOylated proteins in mammalian cells. In the second project, we investigated the regulation of protein ubiquitination in the NF-κB signaling pathway in the context of Paget’s disease of bone (PDB). Identification of SUMOylated proteins has been a challenge because of low abundance of SUMOylation substrates. Here, we utilized a mass spectrometry (MS)-based proteomic approach to identify novel SUMOylated proteins in mammalian cells. Seventy-four unique proteins were commonly identified in the collection of four SUMO-1 plasmids, thus considered candidate SUMOylated proteins. Many of these proteins are associated with the nucleus. The results were validated by confirming SUMOylation of a novel substrate Drebrin and a well known substrate Ran-GAP1. Furthermore, the potential SUMOylation sites in Drebrin have been identified and confirmed using site-directed mutagenesis. PDB is a disorder characterized by increased bone turnover containing hyperactive osteoclasts. Mutations in Sequestosome 1 (p62) are associated with 40% of familial PDB. P62 is a scaffold protein and plays a critical role in regulating ubiquitination of TRAF family signaling molecules and mediating the activation of NF-κB by RANK and TNFα ligands. P62 also plays a critical role in shuttling substrates for autophagic degradation. The objective of this project is to determine the effects of PDB-associated p62 mutants on NF-κB signaling and autophagy. We compared the effect of wild-type (WT) p62 and PDB mutations (A381V, M404V and P392L) on the TNFα-induced NF-κB signaling using an NF-κB luciferase assay. Our results show that these p62 mutations increased the NF-κB signaling. In addition, we found that the PDB mutations did not change the interaction between p62 and the autophagy marker protein LC3. In summary, the PDB mutations in p62 are likely gain-of-function mutations that can increase NF-κB signaling and potentially contribute to disease progression. Based on the results, we proposed a model to speculate the synergetic role of p62 PDB mutant on NF-κB signaling and autophagy.