The ubiquitin-proteasomesystem
in bone biology
Dr Rob Layfield
University of Nottingham
ECTS PhD Training –July 5th2009
Images repro
duced fro
m:
http://w
ww
.bosto
nbio
chem
.com
/overv
iew
.php?pro
d=ubchain
s
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VA
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3: Layfield
R, S
haw
B. U
biq
uitin
-media
ted s
ignalling a
nd P
aget's d
isease o
f
bone. B
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Bio
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. 2007 N
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2;8
Suppl1:S
5. R
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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
What is ubiquitin?
�a sm
all protein (76 amino acids)
�found in all eukaryotes, highly conserved
�‘ubiquitiously’expressed in all tissues
�C-terminus (Glycine
76) protrudes from the globular structure
COOH
What does ubiquitindo?
�all of ubiquitin’scellular functions are mediated by its ability act as a
covalent modifier of other proteins
�ubiquitination, the modification with ubiquitin, is a common protein PTM
�side chains of selected Lysine (K) residues in other target proteins becom
e involved in covalent isopeptidebonds with ubiquitin’sC-terminal Gly76
target
protein
Ub
isopeptide
bond
G76
K
Similar to glycosylation, ‘chains’involving multiple
ubiquitins
can be assembled on proteins
�polyubiquitinchains form by the ‘ubiquitination’
of ubiquitin
�in this case, different Lys (K) residues of
ubiquitinbecom
e involved in isopeptide
linkages with Gly76 of the next ubiquitin
�all 7 of the Lys residues of ubiquitincan
be used to form
chains with different
topologies
�this gives rise to the form
ation of (in this
exam
ple) “Lys48-linked polyubiquitinchains”
K6
G76
target
proteinK
K11
K63
K48
K29UbUb
Ub
G76
K27
K33
K48
proximal
distal
The ubiquitin-proteasomesystem
(UPS)
�a major system for the selectivedegradation of intracellular proteins
E1/E2/E3 cascade
ubiquitinatestarget proteins
the principal signal for UPS
degradation is a Lys48
linked polyubiquitinchain
this is recognised by a large
multi-subunit protease
term
ed the ‘26S proteasom
e’
the substrate is degraded to
short peptides and ubiquitin
is recycled (by DUBs)
Importance
Autophagy–an alternative to the UPS
�double mem
brane-bound structures form
and engulf components of the
cytoplasm, which then fuse with lysosom
es–contents are degraded
�recent evidence suggests and interplay
between UPS and autophagy
�ubiquitinatedproteins also appear
to be degraded by autophagy
Ubiquitindoes not only signal protein degradation
ubiquitin-mediated
protein degradation
Ubiquitinregulates ‘classical’RANK-NF-κBsignalling
osteoclast
signalling pathway
leads to increased expression
of genes which encode proteins
required for osteoclastogenesis
and osteoclastactivity
regulated by both Lys48 and
Lys63-linked polyubiquitination
Ub
Lys
63
osteoclast
The ubiquitin-signal acts as a scaffold to establish new
protein-protein interactions
Iκ κκκB
-subunits of 26S proteasom
ebinds directly to
Lys48-linked ubiquitinatedproteins which are
subsequently degraded
Lys48-linked ubiquitin
(protein degradation)
TRAF6
TAB2
TAK1
Lys63-linked ubiquitin
(signal transduction)
NFκ κκκB
nucleus
‘signal’
TRAF6
kinase
activity
-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 plasm
a cells in the bone marrow
-initially confined to the bone marrow, later stages bone destruction
-involves increased osteoclastogenesisand activity
20S core contains (threonine) protease subunits which digest the substrates
26S proteasome:
Velcade(Bortezomib):
-FDA approved in treatm
ent of MM
-proteasomeinhibitor
-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 chromosom
e
Paget’s disease of bone
5q35 (PD
B3)
5q31 ( PDB4)
2q36 (PD
B5)
10p13 (PD
B6)
18q23 (PD
B7)
IBMPFD
9p13-p12
Paget’s disease of bone (PDB): background
�PD
B 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 osteoclasticactivity, lead to increased osteoblastic
activity (Pageticlesions)
�Pageticbone often denser than normal, but the abnorm
al architecture
causes the bone to be mechanically weak
�result: bone deformity & increased susceptibility to pathological fractures
anterior and posterior whole body images from a
radionuclide bone scan. m
ultiple bones (skull, sternum,
hum
erus, right hem
ipelvis, and left tibia) dem
onstrate
intense uptake, consistent with PDB
PDB: clinical presentation
�many patients are asymptom
atic
�~30% experience:bone pain,
skeletal deform
ity
deafness
neurological symptom
spathological fractures
�most serious complication is osteosarcom
a(<1% of cases)
�majority of adult osteosarcom
asin PDB patients
Inclusion body myopathyassociated with Paget's
disease of bone and frontotem
poraldem
entia (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% ofthose
affected
�pageticphenotype 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 chromosom
emutant gene
Paget’s disease of bone
5q35 (PD
B3)
SQSTM1
5q31 (PDB4)
2q36 (PD
B5)
10p13 (PD
B6)
18q23 (PD
B7)
IBMPFD
9p13-p12
VCP
-identification of the mutant genes in these disorders connects disorder ubiquitin-
mediated processes to disease progression
PDB and IBMPFD -commonalities
�SQSTM1(mutated in PD
B) encodes p62, a ubiquitin-binding protein
�VCP(mutated in IBMPFD) encodes VCP/p97, a ubiquitin-binding protein
target
protein
Ub
UBD
Ubiquitin-
binding
protein
non-covalent
covalent
ubiquitin-binding proteins bind non-covalently
to ubiquitinatedproteins
use short protein sequences called ubiquitin-
binding dom
ains (UBDs)
regulators of ubiquitin-mediated processes
PDB with SQSTM1mutations –disease mechanism
ub
iqu
itin
-mo
dif
ied
targ
et
pro
tein
ZZ
PB1
TF6-b
PEST
PEST
UB
A1
440
p62 p
rote
in
P387L P392L S399P M404V G411S G425R
...PPEADPRLIESLSQMLSMGFSDEGGWLTRLLQTKNYDIGAALDTIQYSKH
...PPDK
391X
...PPEATRG
394X-1210delT
...PPEADRG
394X-1215delC
...PPEADPRLI E396X
mis
se
nse
tru
nc
ati
ng
M404T
PD
B m
uta
nt
p62 p
rote
in
�all PD
B mutations so far tested impair ubiquitin-binding by p62 in vitro
at 37o C
Cavey
et al. [2005] JBMR 20:619-24.
Cavey
et al. [2006] CTI 78:271-7.
PDB with SQSTM1mutations –disease mechanism
UBA dom
ain
structure
free p62 UBA
bound p62 UBA
Binding
mechanism
UBA UBA* UBA*:Ub
slow exchange
fast exchange
mutations affecting
binding interface e.g. M404V
mutations causing instability
of UBA* dom
ain e.g. S399P
mutations stabilising UBA
dom
ain e.g. G425R
PDB with SQSTM1mutations and IBMPFD
–disease mechanisms
�both may involve
hyper-activation of osteoclast
RANK-NF-κBsignalling
Ub
Lys
63
osteoclast
p62 regulates the Lys63-
linked ubiquitination
ofTRAF6 (and NEMO), which
potentiatessignalling via
activation of TAB2/TAK1
VCP regulates the proteasomal
degradation of IκB(Lys48-
ubiquitinated), which allows
NF-κBto enter nucleus and
potentiate
signalling
mutations appear to be gain-
-of-function wrt
signalling –this
stimulates osteoclastactivity
2oosteoblast
effects
sporadic PDB
viruses, environmental??
familial PD
Bother genes
BONE MATRIX
ma
ture
ost
eoc
las
t
oste
ob
last
bone resorbtion
RA
NK
L
RA
NK
TRA
F6
SQST
M1
NFκ κκκB
+
nucle
us
ost
eoc
las
tp
rec
urs
or
RAN
KL
RAN
K
nuc
leus
TR
AF6
UBA
TF6-b
PB
1
SQ
ST
M1
aPK
C
Ub
Ub
Ub
Ub
TA
B1
TA
B2
TA
K1
NEM
OIKKβ IK
Kα
K6
3
P+
+
+
IκB N
FκB
PP
Ub
Ub
Ub
Ub
K4
8
+
26S
p
rote
aso
me
G13
13A
(G
425R
)G
1313
A (
G42
5R)
G13
13A
(G
425R
)
SQSTM1
gene
mutations
mutant
p62
protein
dysregulated
osteoclast
RANK-NF-κB
signalling
increased
osteoclastogenesis
PDB
PDB with SQSTM1mutations –disease mechanisms
Mutations in other com
ponents of the RANK-NF-κB
pathway cause PDB-like syndromes
disorded
linkage to chromosom
emutant gene
Paget’s disease of bone
5q35 (PD
B3)
SQSTM1
5q31 (PDB4)
2q36 (PD
B5)
10p13 (PD
B6)
18q23 (PD
B7)
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 com
ponents of the RANK-NF-κB
pathway cause PDB-like syndromes
osteoclast
PDB
IBMPFD
juvenile hyperphosphatasia
caused by OPG deletions
JH
FEO and ESH caused by
signal peptide insertion
mutations in RANK
FEO/ESH
-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 velcadein MM
-appears to impinge on the RANK-NF-κBpathway
-reduced proteasom
aldegradation of IκB
-stabilisation of p62 levels
Recent developments
-p62 binds to LC3-PE, a component of the autophagosom
almem
brane
-this also allows ubiquitinatedproteins to be delivered for degradation by autophagy
-autophagyis known to be defective in many hum
an diseases
p62
UBA
Ubnated
protein
LC3-PE
newly-form
ing
autophagosome
Sum
mary
-ubiquitinis a protein modifier that can
signal selective degradation
of other proteins viathe proteasome(the UPS)
-modification with ubiquitindoes not alwayssignal protein degradation
-ubiquitinregulates the RANK-NF-κBsignalling pathway in osteoclasts
-some hum
an bone disordersaffect ubiquitin-binding proteins and
involve dysfunction
of RANK-NF-kBsignalling
-proteasomeinhibitors are effective in multiple myelomaand also
target the RANK-NF-kBpathway