The ubiquitin-proteasome system in bone biology Dr Rob Layfield University of Nottingham ECTS PhD Training July 5 th 2009
Images reproduced from: http://www.bostonbiochem.com/overview.php?prod=ubchains 1: Vernace VA, Schmidt-Glenewinkel T, Figueiredo-Pereira ME. Aging and regulated protein degradation: who has the UPPer hand? Aging Cell. 2007 Oct;6(5):599-606. Epub 2007 Aug 6. Review. PubMed PMID: 17681036. 2: Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature. 2006 Oct 19;443(7113):780-6. Review. PubMed PMID: 17051204. 3: Layfield R, Shaw B. Ubiquitin-mediated signalling and Paget's disease of bone. BMC Biochem. 2007 Nov 22;8 Suppl 1:S5. Review. PubMed PMID: 18047742; PubMed Central PMCID: PMC2106369.
Overview an introduction to ubiquitin, the ubiquitin-proteasome system (UPS) and other ubiquitin-mediated processes how ubiquitin-mediated processes control bone cell physiology defective ubiquitin-mediated signalling and bone disorders
COOH What is ubiquitin? a small protein (76 amino acids) found in all eukaryotes, highly conserved ubiquitiously expressed in all tissues C-terminus (Glycine 76) protrudes from the globular structure
What does ubiquitin do? all of ubiquitin s cellular functions are mediated by its ability act as a covalent modifier of other proteins ubiquitination, the modification with ubiquitin, is a common protein PTM Ub G76 K isopeptide bond target protein side chains of selected Lysine (K) residues in other target proteins become involved in covalent isopeptide bonds with ubiquitin s C-terminal Gly76
Similar to glycosylation, chains involving multiple ubiquitins can be assembled on proteins polyubiquitin chains form by the ubiquitination of ubiquitin Ub K48 in this case, different Lys (K) residues of ubiquitin become involved in isopeptide linkages with Gly76 of the next ubiquitin all 7 of the Lys residues of ubiquitin can be used to form chains with different topologies K48 K63 K33 K29 G76 K11 Ub Ub K27 K6 G76 this gives rise to the formation of (in this example) Lys48-linked polyubiquitin chains K target protein distal proximal
The ubiquitin-proteasome system (UPS) a major system for the selective degradation of intracellular proteins E1/E2/E3 cascade ubiquitinates target proteins the principal signal for UPS degradation is a Lys48 linked polyubiquitin chain this is recognised by a large multi-subunit protease termed the 26S proteasome the substrate is degraded to short peptides and ubiquitin is recycled (by DUBs)
Importance
Autophagy an alternative to the UPS double membrane-bound structures form and engulf components of the cytoplasm, which then fuse with lysosomes contents are degraded recent evidence suggests and interplay between UPS and autophagy ubiquitinated proteins also appear to be degraded by autophagy
Ubiquitin does not only signal protein degradation ubiquitin-mediated protein degradation
Ubiquitin regulates classical RANK-NF-κB signalling osteoclast signalling pathway leads to increased expression of genes which encode proteins required for osteoclastogenesis and osteoclast activity osteoclast regulated by both Lys48 and Lys63-linked polyubiquitination Lys63 Ub
The ubiquitin-signal acts as a scaffold to establish new protein-protein interactions Lys48-linked ubiquitin (protein degradation) Lys63-linked ubiquitin (signal transduction) TRAF6 TAB2 TAK1 IκB signal NFκB TRAF6 kinase activity nucleus - subunits of 26S proteasome binds directly to Lys48-linked ubiquitinated proteins which are subsequently degraded - RANK activation leads to Lys63-linked ubiquitination of TRAF6 - binding to TAB2/TAK1 complex activates kinase activity of TAK1
Ubiquitin-mediated processes and bone disease Multiple myeloma: - a cancer which affects plasma cells in the bone marrow - initially confined to the bone marrow, later stages bone destruction - involves increased osteoclastogenesis and activity 20S core contains (threonine) protease subunits which digest the substrates 26S proteasome: Velcade (Bortezomib): - FDA approved in treatment of MM - proteasome inhibitor - inhibits osteoclasts (activates obs) - remarkably effective
Ubiquitin-mediated processes and bone disease - many bone diseases have a genetic contribution or are clearly genetic disorders disorded linkage to chromosome Paget s disease of bone 5q35 (PDB3) 5q31 (PDB4) 2q36 (PDB5) 10p13 (PDB6) 18q23 (PDB7) IBMPFD 9p13-p12
Paget s disease of bone (PDB): background PDB is a common condition; affects around 3% of individuals over 55 years of age in the UK and other western populations characterised by increased bone remodelling; areas of increased osteoclastic activity, lead to increased osteoblastic activity (Pagetic lesions) Pagetic bone often denser than normal, but the abnormal architecture causes the bone to be mechanically weak result: bone deformity & increased susceptibility to pathological fractures
PDB: clinical presentation many patients are asymptomatic ~30% experience: bone pain, skeletal deformity deafness neurological symptoms pathological fractures most serious complication is osteosarcoma (<1% of cases) majority of adult osteosarcomas in PDB patients anterior and posterior whole body images from a radionuclide bone scan. multiple bones (skull, sternum, humerus, right hemipelvis, and left tibia) demonstrate intense uptake, consistent with PDB
Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) a multisystem disorder which can involve muscle myopathy, early-onset PDB and neurodegeneration patients with the full spectrum of the disease make up ~ 12% of those affected pagetic phenotype very similar to classical PDB
Ubiquitin-mediated processes and bone disease - many bone diseases have a genetic contribution or are clearly genetic disorders disorded linkage to chromosome mutant gene Paget s disease of bone 5q35 (PDB3) SQSTM1 5q31 (PDB4) 2q36 (PDB5) 10p13 (PDB6) 18q23 (PDB7) IBMPFD 9p13-p12 VCP - identification of the mutant genes in these disorders connects disorder ubiquitinmediated processes to disease progression
PDB and IBMPFD - commonalities SQSTM1 (mutated in PDB) encodes p62, a ubiquitin-binding protein VCP (mutated in IBMPFD) encodes VCP/p97, a ubiquitin-binding protein non-covalent Ub UBD Ubiquitinbinding protein covalent ubiquitin-binding proteins bind non-covalently to ubiquitinated proteins target protein use short protein sequences called ubiquitinbinding domains (UBDs) regulators of ubiquitin-mediated processes
PDB with SQSTM1 mutations disease mechanism M404T P387L P392L S399P M404V G411S G425R...PPEADPRLIESLSQMLSMGFSDEGGWLTRLLQTKNYDIGAALDTIQYSKH...PPDK 391X...PPEATRG 394X-1210delT...PPEADRG 394X-1215delC...PPEADPRLI E396X missense truncating 1 440 UBA PDB mutant p62 protein PB1 ZZ TF6-b PEST PEST ubiquitin-modified target protein all PDB mutations so far tested impair ubiquitin-binding by p62 in vitro at 37 o C Cavey et al. [2005] JBMR 20:619-24. Cavey et al. [2006] CTI 78:271-7.
PDB with SQSTM1 mutations disease mechanism free p62 UBA UBA domain structure Binding mechanism bound p62 UBA mutations stabilising UBA domain e.g. G425R mutations causing instability of UBA* domain e.g. S399P mutations affecting binding interface e.g. M404V slow exchange fast exchange UBA UBA* UBA*:Ub
PDB with SQSTM1 mutations and IBMPFD disease mechanisms both may involve hyper-activation of osteoclast RANK-NF-κB signalling p62 regulates the Lys63- linked ubiquitination of TRAF6 (and NEMO), which potentiates signalling via activation of TAB2/TAK1 Lys63 VCP regulates the proteasomal degradation of IκB (Lys48- ubiquitinated), which allows NF-κB to enter nucleus and potentiate signalling Ub mutations appear to be gain- -of-function wrt signalling this stimulates osteoclast activity osteoclast
PDB with SQSTM1 mutations disease mechanisms G1313A (G425R) SQSTM1 gene mutations mutant p62 protein PB1 TRAF6 RANKL RANK mature osteoclast UBA BONE MATRIX osteoblast TRAF6 SQSTM1 NFκB osteoclast precursor RANKL RANK nucleus nucleus 2 o osteoblast effects sporadic PDB viruses, environmental?? familial PDB other genes bone resorbtion + TF6-b apkc + SQSTM1 NEMO P IKKβ IKKα K63 Ub Ub Ub Ub + + + TAB1 TAB2 TAK1 K48 P P Ub Ub Ub IκB Ub NFκB 26S proteasome dysregulated osteoclast RANK-NF-κB signalling increased osteoclastogenesis PDB
Mutations in other components of the RANK-NF-κB pathway cause PDB-like syndromes disorded linkage to chromosome mutant gene Paget s disease of bone 5q35 (PDB3) SQSTM1 5q31 (PDB4) 2q36 (PDB5) 10p13 (PDB6) 18q23 (PDB7) IBMPFD 9p13-p12 VCP familial expansile 18q21-22 RANK osteolysis (FEO) expansile skeletal 18q21-22 RANK hyperphosphatasia (ESH) juvenile hyperphosphatasia 8q24 OPG
Mutations in other components of the RANK-NF-κB pathway cause PDB-like syndromes FEO/ESH JH osteoclast FEO and ESH caused by signal peptide insertion mutations in RANK PDB juvenile hyperphosphatasia caused by OPG deletions IBMPFD - again, mutations appear to be gain-of-function wrt signalling - same pathway affected at different levels - other PDB genes linked to this pathway?
Mechanism of action of velcade in MM - appears to impinge on the RANK-NF-κB pathway - reduced proteasomal degradation of IκB - stabilisation of p62 levels
Recent developments - p62 binds to LC3-PE, a component of the autophagosomal membrane - this also allows ubiquitinated proteins to be delivered for degradation by autophagy - autophagy is known to be defective in many human diseases Ubnated protein p62 UBA LC3-PE newly-forming autophagosome
Summary - ubiquitin is a protein modifier that can signal selective degradation of other proteins via the proteasome (the UPS) - modification with ubiquitin does not always signal protein degradation - ubiquitin regulates the RANK-NF-κB signalling pathway in osteoclasts - some human bone disorders affect ubiquitin-binding proteins and involve dysfunction of RANK-NF-kB signalling - proteasome inhibitors are effective in multiple myeloma and also target the RANK-NF-kB pathway