Embed Size (px)
Citation preview
Redox Homeostasis and Redox Homeostasis and
SignalingSignalingSignalingSignaling
Dmitri Dmitri FomenkoFomenko
University of Nebraska University of Nebraska -- LincolnLincoln
5
10
15
20
oxyg
en
in
eart
h atm
osp
here
, %
First living cell
Start of photosynthesis
Start of respiration
Start of multicelled plants
Start of shelled invertebrates
21% Oxygen
Build up Ozone layer
Evolution and oxygen
0
5
Perc
en
t o
f o
xyg
en
in
eart
h atm
osp
here
, %
Billion Years before Today
01234
First living cell
"LU
CA
"
Bacte
riaA
rch
aea
Eu
kary
ote
s
Mitochondria
Redox homeostasis is the tendency of a cell to maintain internal ROS level by
coordinated response to any situation or stimulus that would tend to disturb its normal
condition.
Redox homeostasis
Redox processes requires the simultaneous presence of both oxidized and reduced
forms of electron carriers.
Oxidants Reductants
Redox balance
Oxidants
Reductants
Oxidative environment,
Oxidative stress
Reductants
Reductive environment,
Reductive stress
Oxidants
Controlled ROS generation
Oxygen toxicity was mitigated during evolution
by the development of oxidative stress defense
systems and signaling systems for the control of
intracellular ROS. The level of ROS is controlled
not only by environmental and metabolic
processes but also by ROS generation. ROS-based
signaling that involves generation of ROS for
signal transduction has evolved as a complex and
essential process in eukaryotes.
Carroll, Nature Chemical Biology, 2011
essential process in eukaryotes.
The NADPH oxidase (NOX) family of enzymes is
important source of H2O2 for physiological redox
signaling.
A diverse array of extracellular signals can trigger
the activation of NOX enzymes.
H2O2 generated from active NOX targets redox-
sensitive amino acids in signaling proteins
(PTP1B, PTEN).
ROS - the good and the bad sides
- Gpx1 overexpression - hyperinsulinemia and
insulin resistance
- Gpx1 knockout - low level of insulin, high
insulin sensitivity
Haque et al, Cell, 2011
insulin sensitivity
H2O2
Catalase
ROS sources and defense systems
Major intracellular ROS sources:
- Mitochondria (respiration)
- ER (oxidative protein folding)
- NADPH oxidases
- Reduced metals (Fenton's reaction)
Environmental factors influencing ROS level:
- Environmental oxidants (O2, H2O2)
- Ionizing radiation
O2 O2._
OH.H2O2 H2O
ē ē,2H+ ē ē,H+
Superoxide
dismutase
Catalase
Peroxidases – PRXs,
GPXs
OH_
Thiol oxidoreductases
Sulfur
metabolism
Signal
transduction
ROS
detoxification
Set of thiol oxidoreductases
in an organism - THIOREDOXOME
RNS
detoxification
General functions of thiol oxidoreductases
Transcription
controlProtein
degradation
Protein
modification
Oxidative
folding
in an organism - THIOREDOXOME
30
40
50
60
70
80
90
100
110
120
Nu
mb
er
of
ide
nti
fie
d p
rote
ins Thiol oxidoreductases
Glycolytic enzymes
10
100
1000
Nu
mb
er
of
ide
nti
fie
d p
rote
ins
Eukaryota
Bacteria
Identified thioredoxomes
0
10
20
30
0 2000 4000 6000 8000 10000
Proteome size
Nu
mb
er
of
ide
nti
fie
d p
rote
ins
1
500 5000 50000
Nu
mb
er
of
ide
nti
fie
d p
rote
ins
Proteome size
Bacteria
Archaea
Fomenko and Gladyshev, ARS, 2011
Thioredoxomes and environmental factors
0
20
40
60
80
100
120
0 2000 4000 6000 8000 10000
Series1
mez
ps
ter
Nu
mb
er
of
ide
nti
fie
d p
rote
ins
Hyperthermophilic bacteria
Thermophilic
Mesophilic bacteria
Psychrophilic bacteria
0
20
40
60
80
100
120
0 2000 4000 6000 8000 10000
Series1
an
fa
ma
Nu
mb
er
of
ide
nti
fie
d p
rote
ins
Aerobic bacteria
Microaerobic bacteria
Facultative anaerobic bacteria
Anaerobic bacteria
0
20
40
60
80
100
120
0 2000 4000 6000 8000 10000
Host associated bacteria
Other bacteria
Proteome size
Nu
mb
er
of
ide
nti
fie
d p
rote
ins
0
20
40
60
80
100
120
0 2000 4000 6000 8000 10000
Proteome size
Nu
mb
er
of
ide
nti
fie
d p
rote
ins
Host associated bacteria
Terrestrial bacteria
Specialized bacteria
Aquatic bacteria
0 2000 4000 6000 8000 10000
Proteome size0 2000 4000 6000 8000 10000
Proteome size
Minimal Thioredoxome – 3 thiol oxidoreductases
Nanoarchaeum equitans 535 ORFs/3 thiol oxidoreductases – Peroxiredoxin,
Thioredoxin and Thioredoxin reductase
Thioredoxomes summary
Largest Thioredoxome (194 proteins) - Arabidopsis thaliana
Fomenko and Gladyshev, ARS, 2011
In bacteria and unicellular eukaryotes, the induced expression of detoxifying
enzymes in response to ROS plays a major role in protecting the cell against
oxidative damage.
In multicellular organisms, the increased expression of antioxidant enzymes is not a
universal response of all cells to ROS, however, the basal levels of antioxidants is
important for maintenance of homeostatic conditions and protection of cells
against the damaging effects of oxidative stress.
Redox stress response in unicellular and multicellular organisms
Such difference may relate to the high ROS concentrations that unicellular
organisms can be exposed to within their ecological habitat, requiring appropriate
fast response and adaptations.
Human thioredoxome. 136 proteins, including 111 with catalytic Cys and 25 selenoproteins
Fomenko and Gladyshev, ARS, 2011
Saccharomyces cerevisiae thioredoxome (47 proteins)
Current model of H2O2 mediated signaling
H2O2ThioredoxinThioredoxin
peroxidase
Thioredoxin
reductaseNADPH
Transcription
factors
Kinases
Other
Signaling
Redox regulation
Transcription response
H2O2 Targets
Current model
Othertargets
Transcription response
Molecular Mechanism for the Yap1-Gpx3 Redox System
Paulsen CE et al, Chem. & Biol., 2009
Viability of WT cells and 8∆ cells
Replicative life span
0
0.2
0.4
0.6
0.8
1
0 10 20 30 40
Fra
cti
on
via
ble
Gpx1∆∆∆∆Gpx2∆∆∆∆Gpx3∆∆∆∆3Gpx∆∆∆∆5Gpx∆∆∆∆7∆∆∆∆ (all Prx∆∆∆∆ 2Gpx∆∆∆∆)8∆∆∆∆ (all Prx∆∆∆∆ all Gpx∆∆∆∆)WT
Fra
cti
on
via
ble
0 10 20 30 40Generations
0
0.2
0.4
0.6
0.8
1
0 10 20 30 40Generations
Fra
cti
on
via
ble
WT
Tsa1Tsa2
Tsa1 Tsa2
nPrxmPrx
Ahp1
WT
Tsa1∆∆∆∆
nPrx∆∆∆∆
Tsa2∆∆∆∆
Tsa1∆ ∆ ∆ ∆ Tsa2∆∆∆∆
mPrx∆∆∆∆
Ahp1∆∆∆∆Fra
cti
on
via
ble
Generations
Generations
1. Deletion of single thiol peroxidase did
not affect response to H2O2.
2. Yeast strains, which lack multiple thiol
peroxidases are unable respond to H2O2
stress.
3. Minimal H2O2 stress response was
Regulation of gene expression in response to hydrogen peroxide
3. Minimal H2O2 stress response was
observed in strain lacking all 8 thiol
peroxidases.
H2O2ThioredoxinThioredoxin
peroxidase
Thioredoxin
reductaseNADPH
Transcriptionfactors
Kinases
Othertargets
Signaling
Redox regulation
Transcription response
ROS level in WT and 8∆ cells
Catalase activity in WT and mutant strains.
SOD activity in WT and mutant strains
Exposed thiols and oxidized Cys in WT and mutant strains
Glutathione content in WT and mutant strainsM
/ m
g o
f to
tal
pro
tein
21
24
23
21
17
5
27
21
5
27
8
27
21
1732
2522
28
7
µM
/ m
g o
f to
tal
pro
tein
2427
1927
23
32
15 208
32 16
C D E
H2O2ThioredoxinThioredoxin
peroxidase
Thioredoxin
reductaseNADPH
Transcription
factors
Kinases
Othertargets
Signaling
Redox regulation
Transcription response
Hydrogen peroxide response in thioredoxin mutant strains
C D E
787 790
WT activated by H2O2 (897)
WT activated by diamide
(1439)
WT repressed by H2O2 (923)
WT repressed by diamide
(1313)
Regulation of gene expression by H2O2 and diamide
(1439)
756 767
WT activated by H2O2 (897) WT repressed by H2O2 (923)
8∆∆∆∆ activated by diamide
(1498)8∆∆∆∆ repressed by diamide
(1337)
Skn7∆∆∆∆
4∆4∆4∆4∆Yap
1∆, ∆, ∆, ∆, S
kn7∆∆∆∆
Msn
2,4∆∆∆∆
Yap
1∆∆∆∆
WT
Regulation of gene expression in response to hydrogen peroxide in the mutants lacking redox transcription factors
New model of H2O2-mediated signaling in S. cerevisiae
H2O2
Thioredoxin
reductaseNADPH
Transcription
factors
Kinases
Other
targets
ThioredoxinsThioredoxin
peroxidases
H2O2Targets H2O2
TargetsThiol peroxidases
Current model New model
Redox regulation SignalingTranscription response
Model for PRDX4-Mediated Oxidative Protein Folding in the ER Disulfide bond formation
Zito et al, Mol Cell, 2010
The role of cytosolic Prdxs 1 and 2 in peroxide-induced activation of the apoptosis signaling kinase 1 (ASK1)/p38 signaling pathway
Jarvis et al, Free Radical Biology and Medicine, 2012
