Intracellular Protein Degradation
and the Ubiquitin-Proteasome System
George N. DeMartino
ND12.124
214. 645.6024
george.demartino@utsouthwestern.edu
Intracellular protein degradation
 The basics
General features of intracellular protein
degradation
 Biochemical mechanisms
The ubiquitin-proteasome pathway
 Physiological and pathological examples
How the UPS does interesting and important
things
Cellular proteins are in a state of
continuous turnover
Degradation
Synthesis
Ksyn
(zero order)
ADP
ATP
ATP
ADP
Kdeg
(first order)
Amino Acids
at steady state: Ksyn = Kdeg
T1/2 = ln 2
Kdeg
GND
PD01
Cellular protein concentration
The cellular concentration of a protein can be altered by
changing relative rates of protein synthesis and degradation
Ksyn or
Steady state
Kdeg
Ksyn > Kdeg
Ksyn= Kdeg
Time
GND
PD02
Cellular protein concentration
The cellular concentration of a protein can be altered by
changing relative rates of protein synthesis and degradation
Ksyn or
Kdeg
Steady state
Ksyn > Kdeg
Ksyn= Kdeg
Steady state
Kdeg > Ksyn
Ksyn= Kdeg
Ksyn or
Time
Kdeg
GND
PD03
Really important concepts
Intracellular protein degradation is a
highly regulated process.
Rates of protein degradation
increase or decrease under many
physiological and pathological conditions.
Intracellular protein degradation is a
regulatory process.
Protein degradation regulates cellular events
by controlling levels of important proteins.
GND
PD04
Cellular proteins have different half-lives
1
k(syn1)
k(deg1)
2
k(deg2)
K(syn2)
k(syn3)
3
k(deg3)
T1/2 1 = 1 hr
T1/2 2 = 10 hrs
T1/2 3 = 100 hrs
Amino Acids
GND
PD05
Really important concept
Structural features of proteins
(degrons) determine half-lives.
GND
PD06
Really important concept
Structural features of proteins
(degrons) determine half-lives.
T1/2 1 = 1 hr
T1/2 2 = 1 hr
1
T1/2 3 = 1 hr
2
3
AGREGTYFQRA
LVYQRESTLVAY
LGFERTSIGNAL
Degron 1
Degron 2
Degron 3
GND
PD07
Structural features of proteins
(degrons) determine half-lives.
Protein 1: T ½ = 1 hr
Degron
Protein 1: T ½ = >>1 hr
GND
PD08
Structural features of proteins
(degrons) determine half-lives.
Protein 1: T ½ = 1 hr
c-jun
Degron
Protein 1: T ½ = >>1 hr
v-jun
GND
PD09
Physiological roles of
intracellular protein degradation
Quality control
Antigen processing
Regulation of
cellular function
Adaptation to physiological or
pathological states
GND
PD11
Cells selectively degrade proteins
with abnormal structures
Normal Protein 1: T ½ = 10 hrs
Abnormal Protein 1: T ½ = 1 hr
GND
PD12
Cells selectively degrade proteins
with abnormal structures
 Mutations
 Errors in translation
 Metabolic damage
(e.g. oxidation or denaturation
from high temperature, etc.)
 Proteins without partners
GND
PD13
Important clinical fact
Many neurological (and other) diseases may be caused by
aggregation rather than degradation of proteins with
abnormal structures.
 Alzheimer’s disease (β−amyloid, tau)
 Huntington’s disease (Huntingtin)
 Parkinson’s disease (α-synuclein)
Tau aggregates
(Azheimer’s disease)
 ALS “Lou Gerhig’s” disease (SOD)
 Spinal-Bulbar Muscular atrophy (androgen
receptor)
 Prion diseases “Mad Cow disease” (prion)
Lewy Bodies
(Parkinson’s Disease)
 et al, et al
GND
PD14
Physiological roles of
intracellular protein degradation
Quality control
Antigen processing
Regulation of
cellular function
Adaptation to physiological or
pathological states
GND
PD17
Really important concept
Protein degradation regulates processes
positively and negatively via the conditional
degradation of positive and negative effectors.
Negative regulation :
Process X depends
on Protein A to proceed
Degrade Protein A
to inhibit Process X
X
X
A
X
A
X
GND
PD18
Really important concept
Protein degradation regulates processes
positively and negatively via the conditional
degradation of positive and negative effectors.
Positive regulation :
Process Y is normally
inhibited by Protein B.
Y
X
B
Degrade Protein B
to promote Process Y.
Y
B
X
GND
PD19
What is the advantage
of having proteins with
short half-lives?
GND
PD22
Really important concept
Proteins with short half-lives can
change their concentrations faster
than proteins with long half-lives.
GND
PD22
Fold decrease in protein content
Proteins with short half-lives can change
their cellular concentrations rapidly
0
Kdeg10x at 0 hrs
Protein
D
5
B
10
C
T1/2
(hrs)
A
1.0
B
5.0
C
10.0
D
50.0
A
10 20 30 40 50 60
Hours
GND
PD20
Highly regulated proteins have the
shortest constitutive half-lives
Protein
T1/2
Ornithine decarboxylase
10 mins
Tyrosine amino transferase
1.5 hrs
HMG CoA reductase
5 hrs
Catalase
20 hrs
Cytochrome C
100 hrs
Arginase
300 hrs
Hemoglobin
Highly regulated
Not highly regulated
OO
GND
PD23
Physiological roles of
intracellular protein degradation
Quality control
Antigen processing
Regulation of
cellular function
Adaptation to physiological or
pathological states
GND
PD24
Changes in relative rates of global protein synthesis
and degradation determine tissue growth or atrophy
Growth
(anabolic states)
K syn
K deg
K syn
K deg
Atrophy
(catabolic states)
GND
PD26
Protein degradation allows organisms to adapt to
certain physiological and pathological conditions
Muscle protein degradation
Protein synthesis
Catabolic states
Starvation
Diabetes, etc
Amino acids
Brain glucose
utilization
glucose
Hepatic
gluconeogenesis
GND
PD27
Biochemical mechanisms of
intracellular protein degradation
GND
PD28
Cells contain multiple systems for degrading
constituent proteins
 Ubiquitin-proteasome system
 Lysosomal system
 Intra-mitochondrial systems
 Calpains (Ca2+-dependent proteases)
 Caspases
 Membrane proteases
 et al
GND
PD29
The ubiquitin-proteasome pathway for
degradation of proteins has two phases
Protein
ATP
ADP
Substrate modification
by ubiquitin
Ub
ATP
ADP
Peptides and
amino acids
Substrate degradation
by the proteasome
GND
PD35
Ubiquitin
=
O
Carboxyl terminus
Gly-C-OH
(glycine 76)
 76 amino acids
 Mr = 8,000
 highly conserved
 widely distributed
(glycine 76)
GND
PD36
Ubiquitin is covalently attached to
free ε−amino groups of target proteins
E1, E2, E3
Ub
ATP
AMP
ε−NH2
K
Ub
C=O
ε−N-H
isopeptide
bond
K
COOH
(G76)
GND
PD37
A polyubiquitin chain is required to
target proteins for degradation
E1, E2, E3
Ub
isopeptide
bond
C=O
ATP
Ub
N-H
K
isopeptide
bond
K48
G76
GND
PD38
A polyubiquitin chain is required to
target proteins for degradation
E1, E2, E3
Ub
isopeptide
bond
C=O
Ub
G76
K48
K48-linked chain
ATP
N-H
K
K48
K48
K48
K48
K48
Degradation
signal
but
Not a degron
GND
PD39
Transfer of activated ubiquitin to
to a protein substrate
E3(n)
degron
E3 ubiquitin ligase
Ub-C-S-E2
E3
E2(n)
=O
=O
E2(n)
E3(n)
Ub-C-N-Protein
H
ε NH2 -Protein
Isopeptide bond
GND
PD43
2004 Nobel Prize for discovery of ubiquitin conjugation
Hershko
Ciechanover
Rose
Ubiquitin
=
O
-C-OH
ATP
E1
E2(n)
E1
E2(n)
E3(n)
degron
E3(n)
E2(n)
G76
GND
PD44
Really important concept
Proteins are selected for
ubiquitylation through the
recognition of specific
degrons by E3s.
Different E3s recognize
different degrons of
different substrate proteins.
THIS PROVIDES SPECIFICITY.
GND
PD45
Specificity for ubiquitination is achieved
with multiple conjugating enzymes
1 E1
Many different E2s
(dozens)
Many different E3s
(100s-1000s !!)
GND
PD49
Specificity for ubiquitination is achieved with multiple
conjugating enzymes
E3(n)
Ubiquitin
=
O
-C-OH
ATP
E1
E2(n)
E1
E2(n)
degron
E3(n)
E2(n)
G76
1 E1
Many different E2s
(dozens)
Many different E3s (100s-1000s !!)
GND
PD44
Features and general structures of E3s
E2
E2 binding domain
Single protein E3s
12s (e.g. MDM2)
E3
Degron binding domain
Multisubunit E3s
100s-1000s!! (e.g. SCF, APC)
Ubiquitylated proteins are degraded
by the 26S proteasome
proteolysis
ATP
ADP
Peptides and
amino acids
Not degraded
The 26S proteasome
recognizes the
polyubiquitin chain not the protein
Monoubiquitin
GND
PD53
The 26S proteasome: an energy-dependent molecular
machine for degradation of ubiquitinated proteins
 Mr = 2,400,000
 64 subunits
 32 gene products
GND
PD54
Model for how the 26S proteasome
degrades ubiquitylated proteins
7) ubiquitin isopeptidase
(de-ubiquitylation)
1) activation by gating
2) ubiquitin chain binding
6) peptide bond hydrolysis
3) ATP hydrolysis
4) substrate unfolding
5) translocation of unfolded polypeptide chain
GND
PD63
Proteasome-inhibitor drugs are used to treat cancer
O
O
N
N
N
B
O
O
N
PS341 ( Velcade TM )
α
β
β
α
GND
V01
Ubiquitin/proteasome-dependent
protein degradation
in action
GND
PD64
Regulation of the cell cycle by protein degradation
cdc2
Cyclin B
synthesis
Mitotic events
Cyclin B
cdc2 kinase
(active)
cdc2
cdc2
Cyclin B
cdc2 kinase
(inactive)
26S
proteasome
Cyclin B degradation
GND
V03
Mitotic events
ATP
cdc2
Cyclin B
synthesis
APC
(inactive)
Cyclin B
cdc2 kinase
(active)
cdc2
E3
cdc2
Cyclin B
P
E1
cdc2 kinase
(inactive)
RING
“destruction
box” degron
26S
Cyclin B degradation
E2
Cdc20
APC
(active)
E3
Ub
securin degradation, et al
RING
P
GND
V04
Regulation of transcription by protein degradation
Hypoxia Inducible Factor (HIF) is a transcription factor
for genes required for response to hypoxia (low O2).
Under normoxic conditions, HIF concentrations are low,
and HIF is a short-lived protein, constitutively degraded
by the UPS.
Hypoxia increases HIF concentrations by decreasing
HIF degradation.
GND
V05
HIF
Prolyl
hydroxylase
pVHL
E2
O2
HO
HIF
HIF
HIF
Ub
pVHL E3 Complex
HO
E
M
L
A
HO- P
Y
degron
I
P
M
HIF
HIF
Ubiquitylated
HIF
Degradation
by the
26S Proteasome
GND
V07
Abnormal degradation of HIF promotes onogenesis
Hypoxia Inducible Factor (HIF) is a transcription factor
for genes required for response to hypoxia (low O2).
Under normoxic conditions, HIF concentrations are low,
and HIF is a short-lived protein.
Hypoxia increases HIF concentrations by decreasing
HIF degradation.
Many cancers are associated with increased transcription
of genes controlled by HIF.
Many of these cancers are associated with mutations of
the von Hippel Lindau tumor suppressor protein.
GND
V08
Mutant pVHL fails to promote
HIF ubiquitination and degradation
mutant
pVHL
E2
x
Ub
Mutant pVHL
Transcription of
hypoxic genes under
normoxic conditions;
very bad, oncogenic
HIF non-ubiquitylated HIF
HIF
x
Degradation
by the
26S Proteasome
GND
V09
Regulation of transcription by ubiquitin/proteasomedependent protein degradation
NFκB
Cytoplasm
(inactive)
Nucleus
Nucleus
NFκB-mediated
NFκB-mediated
transcription
transcription
IκB
Ubiquitin/proteasomedependent degradation
Growth, differentiation,
synthesis of proinflamatory
cytokines, et al
GND
PD67
Signal-dependent degradation of IκB
(TNF, lipopolysaccharides, etc)
receptors
Signaling cascades
NFκB
IκB kinase
IκB
IκB
Stable
PO4
PO4
Labile
GND
PD68
The degron of IκB is recognized
by an SCF-type E3 complex
IκB
P
polyubiquitin
chain
P
- - -D S G L D S - - degron
F-box
Ub
(β-TrCP)
Skp1 RING
(rbx1)
26S proteasome
E2
(cdc34)
Cul1 (cdc53)
IκB degradation
GND
PD69
Many muscle wasting conditions and diseases
result from abnormally high rates of protein degradation
 Inactivity or decreased load
immobilization (casts), denervation,
zero gravity, bed rest
 Myopathies
starvation, malnutrition
 Cancer
thyrotoxicosis, diabetes,
glucocorticoid excess, et al
Atrophy
 Nutritional state
 Sepsis
Muscular dystrophies
 Endocrinopathies
 Aging (sarcopenia)

AIDS

et al, et al
Ubiquitin/proteasome-dependent
protein degradation
GND
PD70
Proteins whose expression is altered in most muscle
wasting conditions (aka atrogenes)
26S proteasome
Ubiquitin
Certain E2s
2-4 fold increase
2-4 fold increase
2-4 fold increase
Muscle-specific E3s:
Atrogin 1 (F Box)
MURF 1 (Ring)
>10 fold increase
>10 fold increase
GND
PD71
Proteins whose expression is altered in most muscle
wasting conditions (aka atrogenes)
26S proteasome
Ubiquitin
Certain E2s
2-4 fold increase
2-4 fold increase
2-4 fold increase
Muscle-specific E3s:
Atrogin 1 (F Box)
MURF 1 (Ring)
Atrophy
Normal mouse
In catabolic state
>10 fold increase
>10 fold increase
MURF1 /atrogin1
knockout mouse
in catabolic state
No Atrophy !
GND
PD72