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

1: V

ern

ace

VA

, S

chm

idt-G

lenew

inkelT, Fig

ueiredo-P

ere

ira M

E. A

gin

g a

nd

regula

ted p

rote

in d

egra

dation: w

ho h

as the U

PP

erhand? A

gin

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ell.

2007

Oct;6(5

):599-6

06. E

pub

2007 A

ug 6

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evie

w. P

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PM

ID: 17681036.

2: R

ubin

szte

inD

C. The role

s o

f in

tracellu

lar pro

tein

-degra

dation p

ath

ways in

neuro

degenera

tion. N

atu

re. 2006 O

ct 19;4

43(7

113):780-6

. R

evie

w. P

ubM

ed

PM

ID: 17051204.

3: Layfield

R, S

haw

B. U

biq

uitin

-media

ted s

ignalling a

nd P

aget's d

isease o

f

bone. B

MC

Bio

chem

. 2007 N

ov 2

2;8

Suppl1:S

5. R

evie

w. P

ubM

ed

PM

ID:

18047742; P

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Central P

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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