Title: Paget's disease-causing mutations in sequestosome-1 affect autophagic protein degradation
Abstract: Event Abstract Back to Event Paget's disease-causing mutations in sequestosome-1 affect autophagic protein degradation E Azzam1*, A Duthie1, MH Helfrich1 and LJ Hocking1 1 University of Aberdeen, Musculoskeletal Research Programme, United Kingdom Paget's disease of bone (PDB) is characterised by focal lesions of increased bone turnover, driven by overactive osteoclasts. Mutations affecting Sequestosome-1 (SQSTM1/p62) ubiquitin-associated domain have been identified in individuals with PDB, which impair binding to ubiquitylated proteins targeted for degradation. We have previously shown elevated abundance of SQSTM1, ubiquitin and proteasomal subunits in osteoclasts from Pagetic biopsies, suggesting defects in proteasomal degradation. The aim of this study was to examine the complementary process of autophagic degradation, and to assess the effect of PDB-causing SQSTM1 mutations. {BR}Bone biopsies from four PDB patients without SQSTM1 mutations and four non-Pagetic controls were immunostained for LC3 (autophagy marker; Bond Autostainer). Osteoclast LC3 was quantified using Volocity image analysis software and differences assessed by t-test. HEK293 cell lines stably expressing exogenous wild-type and mutated SQSTM1 (P392L, E396X and G425R) were generated using the Flp-In System (Invitrogen). These cells also express endogenous SQSTM1, approximating heterozygous mutations. Cells were examined under normal growth conditions and following amino acid starvation to induce autophagy; lysates were western blotted for SQSTM1, LC3 and actin. Protein abundance was measured using LiCOR Odyssey and results expressed relative to actin, normalised to the parent cell line (endogenous SQSTM1, LC3) or wild-type SQSTM1 cell line (exogenous SQSTM1). Differences between cell lines were assessed by Kruskal-Wallis test (n=3).{BR}In bone biopsies, LC3 abundance was increased in Pagetic osteoclasts compared to controls (p= 0.04), suggesting autophagy is induced above basal levels. Between cell lines, exogenous SQSTM1 abundance differed under normal conditions (p=0.02) as well as upon starvation (p=0.02), suggesting SQSTM1 degradation is affected by PDB mutations. Endogenous SQSTM1 abundance differed between cell lines upon starvation (p=0.03) but not under normal conditions. Autophagosome-associated LC3 also differed between cell lines (p(normal)=0.03; p(starvation)=0.04). Effects varied between mutations, with missense mutations but not E396X displaying increased autophagic flux.{BR}We have shown that autophagic protein degradation is altered in osteoclasts from patients with PDB, and is affected by PDB-causing SQSTM1 mutations. Pharmacological dissection of protein degradation dynamics will provide important insights into the regulation of osteoclast function by protein degradation pathways and the influence of SQSTM1 mutations on this. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Oral Topic: Abstracts Citation: Azzam E, Duthie A, Helfrich M and Hocking L (2011). Paget's disease-causing mutations in sequestosome-1 affect autophagic protein degradation. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00003 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Mrs. E Azzam, University of Aberdeen, Musculoskeletal Research Programme, Aberdeen, United Kingdom, [email protected] Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers E Azzam A Duthie MH Helfrich LJ Hocking Google E Azzam A Duthie MH Helfrich LJ Hocking Google Scholar E Azzam A Duthie MH Helfrich LJ Hocking PubMed E Azzam A Duthie MH Helfrich LJ Hocking Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.