Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.600092
Title: The role of autophagy in the pathogenesis of Paget's disease of bone
Author: Azzam, Eman
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2013
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Abstract:
Paget's disease of bone (PDB) is characterised by focal lesions of increased bone turnover driven by overactive osteoclasts, which often contain nuclear and cytoplasmic inclusion bodies. Mutations affecting the sequestosome-1 (SQSTM1) ubiquitin-associated (UBA) domain have been identified in individuals with PDB. SQSTM1, also known as p62, is a ubiquitously expressed multidomain scaffold protein of 62 kDa that functions in multiple signalling pathways important for cell survival and osteoclast activity. The mechanisms by which SQSTM1 mutations cause PDB remain unclear. Using immunohistochemistry, I showed evidence that protein degradation pathway components, both from the UPS and the autophagy pathway, are elevated in osteoclasts in patients with PDB compared with control osteoclasts from patients without PDB. Using molecular and microscopical methods to examine Pagetic bone biopsies, osteoclast cultures and various cell lines, I have identified two isoforms of SQSTM1. In all cell types examined, four SQSTM1 transcripts were detected, differing in their 5′-untranslated region; one transcript encodes p62, while the other three encode a 55 kDa isoform of SQSTM1. The newly identified isoform also contains the UBA domain mutated in PDB. Using biochemical and microscopical methods, I found that both SQSTM1 isoforms are degraded by autophagy. The isoforms interact with each other and form aggregates upon autophagy inhibition. SQSTM1-55 is ~21× more abundant in osteoclasts than SQSTM1/p62. Biochemical and microscopical methods showed that PDB-causing mutations in SQSTM1/p62 impair its autophagic degradation. Cell lines expressing SQSTM1/p62 mutations form paracrystalline inclusion bodies that by immuno-transmission electron microscopy (TEM) were found to contain SQSTM1 and ubiquitin and were ultrastructurally identical to those found in PDB. As observed by TEM, these inclusions can be degraded by autophagy. The effects of mutations in SQSTM1-55 have yet to be characterised. Abstract Taken together, these data show that mutations in SQSTM1 isoforms impair protein degradation and can lead to inclusion body formation suggesting that PDB results from dysregulated protein degradation in osteoclasts.
Supervisor: Not available Sponsor: European Calcified Tissue Society ; Paget's Association
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.600092  DOI: Not available
Keywords: Osteitis deformans ; Autophagic vacuoles
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