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Redox Homeostasis and
Signaling
Dmitri Fomenko
University of Nebraska - Lincoln
Percent of oxygen in earth atmosphere, %
Evolution and oxygen
21% Oxygen
20
Start of shelled invertebrates
15
Start of multicelled plants
10
5
Start of photosynthesis
Build up Ozone layer
Start of respiration
First living cell
0
4
3
2
1
Billion Years before Today
Bacteria
Mitochondria
0
Eukaryotes
"LUCA"
Archaea
Redox homeostasis
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 processes requires the simultaneous presence of both oxidized and reduced
forms of electron carriers.
Reductants
Oxidants
Reductants
Redox balance
Oxidants
Oxidative environment,
Oxidative stress
Oxidants
Reductants
Reductive environment,
Reductive stress
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.
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 redoxsensitive amino acids in signaling proteins
(PTP1B, PTEN).
Carroll, Nature Chemical Biology, 2011
ROS - the good and the bad sides
- Gpx1 overexpression - hyperinsulinemia and
insulin resistance
- Gpx1 knockout - low level of insulin, high
insulin sensitivity
H2O2
Haque et al, Cell, 2011
ROS sources and defense systems
Major intracellular ROS sources:
Environmental factors influencing ROS level:
- Mitochondria (respiration)
- ER (oxidative protein folding)
- NADPH oxidases
- Reduced metals (Fenton's reaction)
- Environmental oxidants (O2, H2O2)
- Ionizing radiation
Catalase
O2
ē
._ ē,2H+
O2
H2O2
ē
OH
Superoxide
dismutase
_
.
OH
ē,H+
Peroxidases – PRXs,
GPXs
Thiol oxidoreductases
H2O
General functions of thiol oxidoreductases
ROS
detoxification
Sulfur
metabolism
RNS
detoxification
Set of thiol oxidoreductases
in an organism - THIOREDOXOME
Transcription
control
Signal
transduction
Protein
degradation
Protein
modification
Oxidative
folding
120
110
100
90
80
70
60
50
40
30
20
10
0
Thiol oxidoreductases
Glycolytic enzymes
0
2000
4000
6000
Proteome size
Number of identified proteins
Number of identified proteins
Identified thioredoxomes
8000
10000
1000
100
10
Eukaryota
Bacteria
Archaea
1
500
5000
50000
Proteome size
Fomenko and Gladyshev, ARS, 2011
Thioredoxomes and environmental factors
Number of identified proteins
100
80
60
40
Hyperthermophilic
bacteria
Series1
Thermophilic mez
ps
Mesophilic bacteria
ter
Psychrophilic bacteria
20
Number of identified proteins
120
120
100
0
80
60
40
20
0
0
2000
4000
6000
8000
10000
0
Proteome size
2000
4000
6000
8000
10000
Proteome size
120
120
100
80
60
40
20
Host associated bacteria
Other bacteria
0
Number of identified proteins
Number of identified proteins
Series1
an
Aerobic bacteria
fa
Microaerobic
bacteria
ma
Facultative anaerobic bacteria
Anaerobic bacteria
100
80
60
40
Host associated bacteria
Terrestrial bacteria
Specialized bacteria
Aquatic bacteria
20
0
0
2000
4000
6000
Proteome size
8000
10000
0
2000
4000
6000
Proteome size
8000
10000
Thioredoxomes summary
Minimal Thioredoxome – 3 thiol oxidoreductases
Nanoarchaeum equitans 535 ORFs/3 thiol oxidoreductases – Peroxiredoxin,
Thioredoxin and Thioredoxin reductase
Largest Thioredoxome (194 proteins) - Arabidopsis thaliana
Fomenko and Gladyshev, ARS, 2011
Redox stress response in unicellular and multicellular organisms
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.
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
NADPH
Thioredoxin
reductase
Thioredoxin
Thioredoxin
peroxidase
Kinases
Signaling
Transcription
factors
Redox regulation
Transcription response
Other
targets
Current model
H2O2
Targets
H2O2
Molecular Mechanism for the Yap1-Gpx3 Redox System
Paulsen CE et al, Chem. & Biol., 2009
Viability of WT cells and 8∆ cells
Fraction viable
Fraction
Replicative life span
Gpx1∆
∆
Gpx2∆
∆
Gpx3∆
∆
3Gpx∆
∆
5Gpx∆
∆
7∆
∆ (all Prx∆
∆ 2Gpx∆
∆)
8∆
∆ (all Prx∆
∆ all Gpx∆
∆)
WT
1
0.8
0.6
0.4
0.2
0
0
10
20
30
Generations
Generations
40
20
30
Generations
Generations
40
WT
Tsa1∆
Tsa1∆
Tsa2∆
Tsa2∆
Tsa1∆
Tsa1∆ Tsa2∆
Tsa2∆
nPrx∆
nPrx∆
mPrx∆
mPrx∆
Ahp1∆
Ahp1∆
Fraction viable
Fraction
viable
1
0.8
0.6
0.4
0.2
0
0
10
Regulation of gene expression in response to hydrogen peroxide
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
observed in strain lacking all 8 thiol
peroxidases.
NADPH
Thioredoxin
reductase
Signaling
Redox regulation
Transcription response
Thioredoxin
Thioredoxin
peroxidase
Kinases
Transcription
factors
Other
targets
H2O2
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 strains
27
µM
M / mg of total protein
28
5
7
21
17
25
21
23
21
21
24
8
5
27
19
17 32
27
23
15 20
8
32 16
22
Hydrogen peroxide response in thioredoxin mutant strains
NADPH
Thioredoxin
reductase
Thioredoxin
peroxidase
H2O2
Kinases
Signaling
Transcription
factors
Redox regulation
Transcription response
C
Thioredoxin
D
Other
targets
E
Regulation of gene expression by H2O2 and diamide
WT activated by H2O2 (897)
787
WT repressed by H2O2 (923)
790
WT activated by diamide
(1439)
WT repressed by diamide
(1313)
WT activated by H2O2 (897)
WT repressed by H2O2 (923)
756
8∆
∆ activated by diamide
(1498)
767
8∆
∆ repressed by diamide
(1337)
W
T
Y
ap
Sk 1∆
n
Y 7∆
ap
M 1∆,
s S
4∆ n2,4 kn
∆ 7∆
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
Kinases
NADPH
Thioredoxin
reductase
Transcription
factors
Thioredoxins
Thioredoxin
peroxidases
H2O2
Other
targets
Transcription response
Redox regulation
Current model
H2O2
Targets
Signaling
New model
H2O2
Thiol peroxidases
Targets
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