Calcified Tissue International

, Volume 78, Issue 5, pp 271–277 | Cite as

Loss of Ubiquitin Binding Is a Unifying Mechanism by Which Mutations of SQSTM1 Cause Paget’s Disease of Bone

  • J. R. Cavey
  • S. H. Ralston
  • P. W. Sheppard
  • B. Ciani
  • T. R. A. Gallagher
  • J. E. Long
  • M. S. Searle
  • R. LayfieldEmail author
Clinical Investigations


Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget’s disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.


Ubiquitin Ubiquitin-associated domain SQSTM1 p62 Paget’s disease of bone 



This work was supported in part by grants from the Wellcome Trust (to R.L. and J.C.), the Biotechnology and Biological Sciences Research Council (BBSRC) (to M.S.S., JEL R.L., and B.C.), and the Arthritis Research Campaign (ARC) (to S.H.R.).


  1. 1.
    Cody JD, Singer FR, Roodman GD, et al. (1997) Genetic linkage of Paget disease of the bone to chromosome 18q. Am J Hum Genet 61:1117–1122CrossRefPubMedGoogle Scholar
  2. 2.
    Good DA, Busfield F, Fletcher BH, et al. (2001) Linkage of Paget disease of bone to a novel region on human chromosome 18q23. Am J Hum Genet 70:517–525CrossRefPubMedGoogle Scholar
  3. 3.
    Haslam SI, Van Hul W, Morales-Piga A, et al. (1998) Paget’s disease of bone: evidence for a susceptibility locus on chromosome 18q and for genetic heterogeneity. J Bone Miner Res 13:911–917CrossRefPubMedGoogle Scholar
  4. 4.
    Hocking LJ, Herbert CA, Nicholls RK, et al. (2001) Genomewide search in familial Paget disease of bone shows evidence of genetic heterogeneity with candidate loci on chromosomes 2q36, 10p13, and 5q35. Am J Hum Genet 69:1055–1061CrossRefPubMedGoogle Scholar
  5. 5.
    Hughes AE, Shearman AM, Weber JL, et al. (1994) Genetic linkage of familial expansile osteolysis to chromosome 18q. Hum Mol Genet 3:359–361PubMedGoogle Scholar
  6. 6.
    Laurin N, Brown JP, Lemainque A, et al. (2001) Paget disease of bone: mapping of two loci at 5q35-qter and 5q31. Am J Hum Genet 69:528–543CrossRefPubMedGoogle Scholar
  7. 7.
    Eekhoff EW, Karperien M, Houtsma D, et al. (2004) Familial Paget’s disease in the Netherlands: occurrence, identification of new mutations in the sequestosome 1 gene, and their clinical associations. Arthritis Rheum 50:1650–1654CrossRefPubMedGoogle Scholar
  8. 8.
    Falchetti A, Di Stefano M, Marini F, et al. (2004) Two novel mutations at exon 8 of Sequestosome 1 gene (SQSTM1) in an Italian serie of patients affected by Paget’s disease of bone (PDB). J Bone Miner Res 19:1013–1017CrossRefPubMedGoogle Scholar
  9. 9.
    Hocking LJ, Lucas GJA, Daroszewska A, et al. (2002) Domain specific mutations in Sequestosome 1 (SQSTM1) cause familial and sporadic Paget’s disease. Hum Mol Genet 11:2735–2739CrossRefPubMedGoogle Scholar
  10. 10.
    Hocking LJ, Lucas GJA, Daroszewska A, et al. (2004) Novel UBA domain mutations of SQSTM1 in Paget’s disease of bone: genotype phenotype correlation, functional analysis, and structural consequences. J Bone Miner Res 19:1122–1127CrossRefPubMedGoogle Scholar
  11. 11.
    Johnson-Pais TL, Wisdom JH, Weldon KS, et al. (2003) Three novel mutations in SQSTM1 identified in familial Paget’s disease of bone. J Bone Miner Res 18:1748–1753CrossRefPubMedGoogle Scholar
  12. 12.
    Laurin N, Brown JP, Morissette J, Raymond V (2002) Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget disease of bone. Am J Hum Genet 70:1582–1588CrossRefPubMedGoogle Scholar
  13. 13.
    Duran A, Serrano M, Leitges M, et al. (2004) The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis. Dev Cell 6:303–309CrossRefPubMedGoogle Scholar
  14. 14.
    Raasi S, Orlov I, Fleming KG, Pickart CM (2004) Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A. J Mol Biol 341:1367–1379CrossRefPubMedGoogle Scholar
  15. 15.
    Wilkinson CR, Seeger M, Hartmann-Petersen R, et al. (2001) Proteins containing the UBA domain are able to bind to multi-ubiquitin chains. Nat Cell Biol 3:939–943CrossRefPubMedGoogle Scholar
  16. 16.
    Ciani B, Layfield R, Cavey JR, Sheppard PW, Searle MS (2003) Structure of the UBA domain of p62 (SQSTM1) and implications for mutations which cause Paget’s disease of bone. J Biol Chem 278:37409–37412CrossRefPubMedGoogle Scholar
  17. 17.
    Shin J (1998) p62 and the sequestosome, a novel mechanism for protein metabolism. Arch Pharm Res 21:629–633PubMedCrossRefGoogle Scholar
  18. 18.
    Vadlamudi RK, Joung I, Strominger JL, Shin J (1996) p62, a phosphotyrosine-independent ligand of the SH2 domain of p56lck, belongs to a new class of ubiquitin-binding proteins. J Biol Chem 271:20235–20237CrossRefPubMedGoogle Scholar
  19. 19.
    Kanayama A, Seth RB, Sun L, et al. (2004) TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell 15:535–548CrossRefPubMedGoogle Scholar
  20. 20.
    Layfield R, Hocking LJ (2004) SQSTM1 and Paget’s disease of bone. Calcif Tissue Int 75:347–357CrossRefPubMedGoogle Scholar
  21. 21.
    Wooten MW, Geetha T, Seibenhener ML, et al. (2005) The p62 scaffold regulates NGF-induced NF-kappa B activation by influencing TRAF6 polyubiquitination. J Biol Chem 280:35625–35629CrossRefPubMedGoogle Scholar
  22. 22.
    Cavey JR, Ralston SH, Hocking LJ, et al. (2005) Loss of ubiquitin-binding associated with Paget’s disease of bone p62 (SQSTM1) mutations. J Bone Miner Res 20:619–624CrossRefPubMedGoogle Scholar
  23. 23.
    Thrower JS, Hoffman L, Rechsteiner M, Pickart CM (2000) Recognition of the polyubiquitin proteolytic signal. EMBO J 19:94–102CrossRefPubMedGoogle Scholar
  24. 24.
    Mueller TD, Feigon J (2002) Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions. J Mol Biol 319:1243–1255CrossRefPubMedGoogle Scholar
  25. 25.
    Teitelbaum SL, Ross FP (2003) Genetic regulation of osteoclast development and function. Nat Rev Genet 4:638–649CrossRefPubMedGoogle Scholar
  26. 26.
    Babu JR, Geetha T, Wooten MW (2005) Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradation. J Neurochem 94:192–203CrossRefPubMedGoogle Scholar
  27. 27.
    Seibenhener ML, Babu JR, Geetha T, Wong HC, Krishna NR, Wooten MW (2004) Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol Cell Biol 24:8055–8068CrossRefPubMedGoogle Scholar
  28. 28.
    Raasi S, Varadan R, Fushman D, Pickart CM (2005) Diverse polyubiquitin interaction properties of ubiquitin-associated domains. Nat Struct Mol Biol 12:708–714CrossRefPubMedGoogle Scholar
  29. 29.
    Raasi S, Pickart CM (2003) Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains. J Biol Chem 278:8951–8959CrossRefPubMedGoogle Scholar
  30. 30.
    Walters KJ, Lech PJ, Goh AM, Wang Q, Howley PM (2003) DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a. Proc Natl Acad Sci USA 100:12694–12699CrossRefPubMedGoogle Scholar
  31. 31.
    Merkley N, Shaw GS (2004) Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain assembly. J Biol Chem 279:47139–47147CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • J. R. Cavey
    • 1
  • S. H. Ralston
    • 2
  • P. W. Sheppard
    • 3
  • B. Ciani
    • 4
  • T. R. A. Gallagher
    • 4
  • J. E. Long
    • 4
  • M. S. Searle
    • 4
  • R. Layfield
    • 1
    Email author
  1. 1.School of Biomedical SciencesUniversity of Nottingham Medical SchoolNottinghamUnited Kingdom
  2. 2.Rheumatic Diseases UnitUniversity of Edinburgh, Western General HospitalEdinburghUnited Kingdom
  3. 3.BIOMOL International, Palatine HouseMatford CourtUnited Kingdom
  4. 4.Centre for Biomolecular Sciences, School of ChemistryUniversity ParkNottinghamUnited Kingdom

Personalised recommendations