Role of reactive oxygen species
in physiological processes
Petr Babula
ROS = reactive oxygen species
Variation in ROS production Amount of ROS as a result of variations in ROS
production. Both, genetically (epigenetically)
and environmentally based factors are
involved in ROS production. In healthy people
(= moderate level of ROS), ROS are involved in
physiological processes including cell
signalling, host defence and biosynthetic
processes. On the other hand, reduced
amounts of ROS participate in decreased
antimicrobial defence, hypothyroidism, or
changes in blood pressure. ROS play a crucial
role in all above-mentioned physiological
processes. When ROS levels are too high,
overshoot signalling and nonspecific damage
of biomolecules (cells, tissues) occur. ROS
overproduction is involved in many
pathological processes, including chronic
inflammation and autoimmune diseases,
cancer, sensory impairment, cardiovascular
diseases, such as atherosclerosis,
hypertension, re-stenosis,
ischaemia/reperfusion injury, neurological
disorders, fibrotic disease, other ageassociated
diseases, or infectious diseases.
Adapted and modified from (Brieger et al.,
2012).
ROS signalling depends on ability of cells to
detoxify ROS
NOX (= NADPH oxidase) complex as a source of ROS and degradation cascade of ROS that contributes to ROS balance. Major enzymes involved in ROS conversions
are shown – superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and myeloperoxidase (MPO). Scheme also shows a way leading to
production of singlet oxygen and ozone. Superoxide anion radical is rapidly converted into hydrogen peroxide by superoxide dismutases with specific cellular
localization. On the other hand, hydrogen peroxide is able to oxidize cysteine residues on proteins to initiate redox biology.
Nitroxyl anion (NO), a one-electron reduction product
of nitric oxide (NO), is unlikely to arise from NO under
physiological conditions. The reaction of reactive
nitrogen species with cysteine sulphydryls can result in
either S-nitrosylation or oxidation to the sulphenic acid,
as well as disulphide-bond formation, all of which are
potentially reversible. The peroxynitrite anion (ONOO-)
and peroxynitrous acid (ONOOH) have distinct patterns
of reactivity. ONOOH spontaneously decomposes
through a series of species that resemble the reactive
radicals hydroxyl (OH) and/or nitrogen dioxide (NO2).
When the concentration of L-arginine is limiting, nitric
oxide synthase (NOS) can produce superoxide (O2-)
along with NO, which favours the formation of
peroxynitrite. Reproduced with permission from Ref. 13
© (2000) National Academy of Sciences, USA.
Physiological and pathophysiological role of
ROS Overview of physiological and pathological
(excessive) role of ROS in the cell. Under
physiological conditions, superoxide anion radical
is rapidly converted by SOD enzymes to hydrogen
peroxide. Cellular level of hydrogen peroxide is
strictly converted to water by glutathione
peroxidase (GPx), peroxiredoxins (PRx), and
catalase (CAT). Low concentration of hydrogen
peroxide is involved in the first step of cysteine
oxidation in proteins moieties from thiolate
anion (Cys-S-) to the sulfenic form (Cys-SOH). This
reaction is reversible and glutaredoxin (GRx) and
thioredoxin (TRx) play crucial role in it. Sulfenic
form of cysteine moieties in proteins causes
allosteric changes in proteins, which means
change in the protein function. On the other
hand, excess of hydrogen peroxide causes next
oxidation steps to sulfinic (SO2H) or sulfonic
(SO3H) species; these steps are irreversible and
these species cannot be converted back to initial
form of protein. Increased level of hydrogen
peroxide leads to creation of hydroxyl radicals
(HO.); the reaction is catalysed by Fe2+ ions.
Hydroxyl radicals irreversibly damage cellular
biomolecules including lipids, proteins, and
nucleic acids.
Cellular sources of ROS –
mitochondria
Sources of ROS in mitochondria and regulation of their
production – a schematic overview. Mitochondria have
several sources of ROS. Respiratory chain (RCH) produces
superoxide anion radical; production of both superoxide and
hydrogen peroxide in the intermembrane space is usually not
considered, major source of them is oxidation of
dihydroorotate to orotate with reduction of CoQ.
Intermembrane space contains Cu-Zn-SOD, which dismutates
superoxide anion radical to hydrogen peroxide. In
mitochondrial matrix, superoxide very quickly reacts with
nitric oxide to form peroxynitrite. Mitochondrial matrix
contains several systems that consume hydrogen peroxide
formed by an action of Mn-SOD: peroxiredoxins 3 and 5
(PRx3/5), catalase (CAT), and glutathione peroxidases 1 and 4
(GPx1/4), which represent together with peroxiredoxins the
most important ROS-detoxifying systems. Glutathione
reductase (GR) regenerates reduced glutathione (GSH) from
its oxidized (GSSG) form. Both superoxide anion radical and
hydrogen peroxide are transported out of mitochondria into
the cytosol. Permeability transition pore (PTP - mPTP) is
involved in the efflux of hydrogen peroxide, inner membrane
anion channel (IMAC) as the ion channel opens transiently in
response to an elevation of superoxide anion radical.
Hydrogen peroxide alters structure of many different proteins
(cytosolic or nuclear enzymes, carriers or transcription
factors), which leads to changes in activities. ROS are involved
in secondary redox signalling, which finally causes cellular
responses based on changes in MAPK signalling pathway,
epigenetic changes, changes in metabolism of substrates, or
eventually may result in cell death (apoptosis, necrosis).
Cellular sources of ROS – endoplasmic reticulum
Scheme of the ER ROS sources and their mutual
communication. Production of ROS occurs during a protein
folding. In this process, protein disulfide isomerase (PDI) and
endoplasmic reticulum oxidoreductin-1 (ERO1) play a crucial
role. Another source of ROS is NADPH oxidase 4/p22phox
(NOX4) and also NADPH-P450 reductase (NPR), which is
involved in the recycling of both endogenous and exogenous
compounds, but also amount of reduced glutathione (GSH)
itself.
An interplay between mitochondria, endoplasmic reticulum and cytoplasm
in handling ROS and calcium ions. Briefly, endoplasmic reticulum is the
crucial and major site for calcium storage in cell. Sarco-/endoplasmic
reticulum Ca2+-ATPase represents the most important transport mechanism
for influx of calcium ions. On the other hand, mitochondria represent the
second most important calcium store in the cell. However, these two
organelles are closely connected in calcium handling, mainly in response to
ROS. Increase in ROS levels in the mitochondria, where the respiratory
chain (RCH) represents the major site for creation of ROS, triggers the ER
to release calcium and sensitizes a calcium-releasing channel in the ER
membrane, sending a feedback signal. On the other hand, process of folding
proteins contributes significantly to creation ROS directly in ER. When
incorrect disulfide bonds form, they need to be reduced by GSH, resulting in
a further decrease of GSH/GSSG ratio, altering the redox state within the
ER. Alternatively, misfolded proteins can be directed to degradation through
ER-associated degradation machinery. Accumulation of misfolded proteins
in the ER initiates the unfolded protein response, which includes
involvement of protein kinase RNA-like endoplasmic reticulum kinase
(PERK), inositol-requiring enzyme (IRE), and activating transcription factor
6 (ATF6). All these proteins influence cellular responses at different levels
(transcription, translation, antioxidant defence). Calcium ions released from
ER during these processes (inositol 1,4,5-trisphosphate receptors (IP3R) and
ryanodine receptors (RyR) – accumulated in membranes with close
connection with mitochondria - in mitochondrial associated membranes
(MAMs) trigger mitochondrial ROS stimulation via stimulation of the
tricarboxylic acid cycle. Mitochondria release calcium ions via
mitochondrial sodium/calcium exchanger (mNCX), influx of calcium ions
from cytoplasm in provided by voltage-dependent anion channel (VDAC)
and calcium uniporter. Increased load of mitochondria with calcium ions
stimulate release of cytochrome c and pro-apoptic factors via mitochondrial
permeability transition pore (mPTP).
Cellular sources of ROS – peroxisomes
Other cellular sources of ROS
• Autooxidation of small molecules (dopamine, epinephrine, flavins,
and hydroquinones)
• XO, COXs, cytochrome P450 enzymes (cytochrome P450
monooxygense), LOXs, flavin-dependent demethylase, oxidases for
polyamines and amino acids, and NOSs that produce oxidants as part
of their normal enzymatic function
Nitric oxide
In the tissues, such as the heart, there are numerous pathways for the
generation of NO from nitrite, all greatly potentiated during hypoxia,
including xanthine oxidoreductase (XOR), deoxygenated myoglobin
(deoxy-Mb), enzymes of the mitochondrial chain and protons. Nitritedependent
NO formation and S-nitrosothiol formation can modulate
inflammation, inhibit mitochondrial respiration and mitochondrial
derived reactive oxygen species formation, and drive cyclic GMPdependent
signalling under anoxia. NO-dependent cytochrome c oxidase
(complex IV) inhibition can also drive reactive oxygen species (ROS)dependent
signalling. b | The formation of bioactive nitric oxide (NO)
from the inorganic anion nitrite is generally enhanced under acidic and
reducing conditions. In the acidic gastric lumen, NO is generated nonenzymatically
from nitrite in saliva after formation of nitrous acid (HNO2)
and then decomposition into NO and other reactive nitrogen oxides. This
NO helps to kill pathogenic bacteria and it also stimulates mucosal blood
flow and mucus generation, thereby enhancing gastric protection.
Detrimental effects have also been suggested, including nitritedependent
generation of nitrosamines with potentially carcinogenic
effects. c | In the blood vessels, nitrite forms vasodilatory NO after a
proposed reaction with deoxygenated haemoglobin (deoxy-Hb) and
contributes to physiological hypoxic blood flow regulation. GC, guanylate
cyclase; MPT, mitochondrial permeability transition; Oxy-Hb, oxygenated
haemoglobin; PKG, protein kinase G.
Cellular antioxidant system
• Non-enzymatic antioxidants - both lipophilic (e.g. tocopherols) and
hydrophilic (e.g. ascorbate, GSH) compounds
• a system of antioxidant enzymes, which convert superoxide anion
radical to hydrogen peroxide and water
• SOD
• CAT
• Peroxidases + TRx + glutathione systems
SOD = superoxide dismutase
SOD isoform Metal ion(s)
Molecular
mass (kDa)
Assembly of
subunits
Cellular localization
Cu-Zn-SOD
(SOD1)
Cu2+
(catalytic
activity)
88 homodimer
cytoplasm, nucleus,
mitochondrial intermembrane
space, lysosomes
Zn2+
(enzyme
stability)
Mn-SOD
(SOD2)
Mn2+
(catalytic
activity)
32 homotetramer mitochondrial matrix
Cu-Zn-SOD
(SOD3, ECSOD)
Cu2+
(catalytic
activity)
132
homotetramer,
resp.
homotetrameric
glycoprotein
plasma membrane, extracellular
fluidZn2+
(enzyme
stability)
Expression of SOD regulated by several transcription factors = nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB), activator protein 1 (AP-1), activator protein 2 (AP-2), specificity protein 1 (Sp1), or CCAATenhancer-binding
protein (C/EBP).
CAT = catalase
• enzymes that catalyse decomposition of hydrogen peroxide to water and oxygen,
therefore, it can prevent formation of hydroxyl radicals via the Fenton reaction
• CAT plays an important role in removing higher intracellular concentrations of
hydrogen peroxide
• CAT contributes to ethanol metabolism in the body after ingestion of alcohol, but
it only breaks down a small fraction of the alcohol
• CAT seems to have a specific red cell membrane location that may facilitate both
catalatic and peroxidatic roles in erythrocytes that do not contain classical
dehydrogenase systems
• Three subgroups of CAT have been classified:
• Two of them are heme-containing enzymes: monofunctional heme CAT (typical CAT) and
catalase-peroxidase (only fungi among eukaryotic organisms, activity 2 or 3 orders of
magnitude less than that of a typical CAT)
• the third one is (nonheme) manganese CAT (only several species with manganese CAT have
been identified)
PRxs = Peroxiredoxins
• = cysteine-dependent peroxide reductases
• family of enzymes that use thioredoxin (TRx) to recharge after
reducing hydrogen peroxide to water
• one of the most important mechanisms of hydrogen peroxide
detoxification !
• E.g. human erythrocytes:
• PRx2, is one of the most abundant protein and plays a preponderant role in
maintaining low endogenous levels of hydrogen peroxide produced by
haemoglobin autoxidation
GPx =
glutathione peroxidase
• family of enzymes (8 isoforms) that
catalyse conversion of lipid
hydroperoxides to corresponding
alcohols and hydrogen peroxide to
water with the use of GSH as a
typical co-substrate in the reaction
Characterization of human isoforms of GPxs – structural features, localization on chromosomes,
reducing and oxidizing substrates, and their tissue localizations. ChOOH, cholesterol
hydroperoxide; CEOOH cholesterol ester hydroperoxide; DTT, dithiotreitol; GRx, glutaredoxin;
LOOH, lipid (fatty acid) hydroperoxide; PLOOH, phospholipid hydroperoxide of different
classes; ROOH, small synthetic hydroperoxides (e.g. cumene hydroperoxide and tert-butylhydroperoxide);
TRx, thioredoxin; PDI, protein disulfide isomerase; n.d. not determined so far.
Adapted from (Brigelius-Flohe and Maiorino, 2013) and modified.
Ascorbic acid
• intracellular and extracellular aqueous-phase antioxidant capacity primarily
by scavenging oxygen free radicals
• it converts vitamin E free radicals back to vitamin E
• vitamin C possesses proactive role against some types of cancer and also
demonstrated the immunomodulatory effects enhancing host defence;
generally vitamin C is considered supportive for immune responsiveness
• vitamin C is transported in the form of dehydroxyascorbic acid (DHA) into
various cells through glucose transporters
• it is maintained in its reduced form by reaction with GSH, which can be
catalysed by protein disulfide isomerase and glutaredoxins
Tocopherols and tocotrienols – vitamin E
• Vitamin E is formed by a set of eight related tocopherols and
tocotrienols – α-, β-, γ-, δ-forms of tocopherols and tocotrienols that
vary only in the number and position of methyl substituents attached
to the chromanol ring
• it protects membranes from oxidation by reacting with lipid radicals
produced in the lipid peroxidation chain reaction
• Antioxidant activity of tocopherols and tocotrienols strongly depends
on the degree of methylation in the aromatic ring (α > β = γ > δ), size
of the heterocyclic ring, stereochemistry at position 2, and finally
length of the prenyl chain (optimum between 11 and 13 carbons)
Uric acid
• final product of purine metabolism
• accounts for roughly half antioxidant ability of plasma
• uric acid is similarly produced in the liver, adipose tissue and muscle and is
primarily excreted through the urinary tract
• high uric acid levels regulate the oxidative stress, inflammation and
enzymes associated with glucose and lipid metabolism, suggesting a
mechanism for the impairment of metabolic homeostasis
• possible neuroprotective properties based on mechanisms involving
chelating Fenton reaction transitional metals, antioxidant quenching of
superoxide and hydroxyl free radicals, and as an electron donor that
increases antioxidant enzyme activity, e.g. SOD
Carotenoids
• Carotenoids are pigments found in plants
• Primarily, β-carotene has been found to react with peroxyl, hydroxyl, and
superoxide anion radicals
• Carotenoids exhibit their antioxidant effects in low oxygen partial pressure, but
may have pro-oxidant effects at higher oxygen concentrations
• genetic polymorphisms in the beta-carotene oxygenase 1 (BCO1) = BCO1 encodes
an enzyme that is expressed in the intestine and converts provitamin A to vitamin
A-aldehyde
• carotenoids and retinoic acids are capable of regulating transcription factors - βcarotene
inhibits the oxidant-induced NF-κB activation via thiol groups of both
IkB kinase and p65 and interleukin IL-6 and tumour necrosis factor-α (TNF-α)
production
• inhibition the production of inflammatory cytokines, such as IL-8 or prostaglandin E2
• blocking oxidative stress by interacting with the nuclear factor (erythroid-derived 2)-like 2
(Nrf2) pathway, enhancing its translocation into the nucleus, and activating phase II enzymes
and antioxidants, such as GST
Glutathione - GSH
• a cysteine-containing peptide found in most forms of aerobic life and is
highly abundant in all cell compartments and is the major soluble
antioxidant
• a part of glutathione system includes GSH, GR, GPxs, and GST
• thiol group in its cysteine moiety is a reducing agent and can be reversibly
oxidized and reduced - GSH/GSSG ratio is a major determinant of oxidative
stress
• it detoxifies H2O2 and lipid peroxides via action of GPxs
• GSH donates protons to membrane lipids and protects them from oxidant
attacks
• GSH protects cells against apoptosis by interacting with pro-apoptotic and
anti-apoptotic signalling pathways
• It also regulates and activates several transcription factors, such as AP-1,
NF-κB, and Sp-1
Glutathione – ascorbate cycle
Reactive sulphur species
Binding of sulfide anion to ferric heme results in the
formation of ferric sulfide complex, which, depending
on the polarity of the distal heme pocket, could lead to
heme iron reduction and formation of the sulfur radical.
Depending on the heme protein, either H2S or HS− can
bind ferric heme. (b) In principle, H2S can also bind
ferrous hemes, as seen with model porphyrinate
complexes.
H2S is relatively stable and is oxidized slowly
by H2O2 to give HSOH. Either HSOH or HSSH
formed from HSOH in the presence of a
second equivalent of H2S can be attacked by a
reactive cysteine on a protein to generate the
persulfide modification. The bimolecular rate
constants for HSOH and HSSH formation in
solution at pH 7.4 and 37 °C are noted. (b)
Persulfidation can result from the nucleophilic
attack of a sulfide anion on an oxidized protein
thiol (such as disulfide, mixed disulfide,
cysteine sulfenic acid or S-nitrosylated
cysteine). (c) Persulfide modifications on
proteins are reversible and, unless
sequestered, labile. They can be removed via
persulfide interchange reactions involving
glutathione (GSH), thioredoxin (Trx) or a
cysteine on the same or a different protein. In
all cases, the product is ultimately H2S, formed
upon reduction of the persulfide moiety by
either a second mole of GSH or the NADPH–
thioredoxin reductase system.
The canonical sulfide oxidation pathway found in
most tissues resides in the mitochondrion and
involves four enzymes. In the first step of the
pathway, sulfide quinone oxidoreductase (SQR)
oxidizes sulfide to persulfide, which is transferred
from the active site of SQR to a small molecule
acceptor, such as glutathione (GSH). The glutathione
persulfide (GSSH) product can be oxidized by
persulfide dioxygenase (PDO) to sulfite or can be
used in a sulfurtransferase reaction catalyzed by
rhodanese (Rhd) in the presence of sulfite, to form
thiosulfate. In the final step, sulfite is oxidized to
sulfate by sulfite oxidase (SO). (b) Heme-dependent
sulfide oxidation pathway. An alternative pathway
for sulfide oxidation involves ferric heme–dependent
conversion of H2S to a mixture of thiosulfate and
polysulfides. This newly discovered mechanism has
been established for human hemoglobin and could
be an activity of other heme proteins as well. For
clarity, the fate of the H2S sulfur atom is highlighted
in red and other reactants and reaction
stoichiometries are omitted.
H2S in cellular signalling
ROS in cellular signaling
• MAPK signalling pathway
• regulates many physiological processes:
• bone development and homeostasis of bone tissue
• regeneration of connective tissue
• epidermal homeostasis
• haematopoiesis
• circadian rhythms (in this case, MAPK pathways can function as inputs allowing the
endogenous clock to entrain to 24 h environmental cycles)
• It also modulates effect of some hormones, e.g. glucocorticoids, on target
tissues and participates in regulation of homeostasis of glucose
• PI3K signalling pathway
• PI3K/Akt/mTOR pathway regulates cell
cycle in response to growth factors,
hormone, and cytokine stimulations and
primarily is involved in the regulation of
cellular quiescence, proliferation, cancer,
and longevity
• PI3K pathway regulates several
physiological processes:
• immune functions, mainly anti-viral and
anti-tumour responses mediated by natural
killer cells
• activity of ion channels, which influences
neuronal excitability and synaptic plasticity
• regeneration of neural tissue
• insulin-mediated cardioprotection
• regulation of ovarian function including
quiescence, activation, and survival of
primordial follicles, granulosa cell
proliferation and differentiation, and
meiotic maturation of oocytes
• Nrf2 and Ref1-mediated redox
cellular signaling
• Nrf2 and Ref1-mediated redox
cellular signalling is important in
antioxidant signalling in order to
prevent oxidative stress
• induction of many cytoprotective
enzymes in response to oxidation
stress is regulated primarily at the
transcriptional level
• this transcriptional response is
mediated by a cis-acting element
termed antioxidant response
element (ARE) initially found in the
promoters of genes encoding the
detoxification enzymes, such as
GST and NADPH quinone
oxidoreductase-1
Signalling
pathway
Signal
molecules
Molecules modified by ROS and
subsequent processes
Cell responses
MAPK
- ERK1/2
- JNK/p38
- ERK5
Cytokines
Growth
factors
Hormones
Genotoxic
stress
Oxidative
stress
Abiotic stress
stimuli
ASK1 (homo-oligomerization):
1. 2 Cys residues and creation
disufhide bond (Cys32-Cys35)
2. oligomerization and
autophosphorylation
3. TRAF binding
ASK1 (hetero-oligomerization)
ASK1/ASK2
Non-apoptic:
- Cell
differentiation
- Immune
signalling
PKG*
1. Oxidation of Cys42 by H2O2 in
PKG1 (redox sensor)
2. Creation of homodimer via
disulfide bonds
Regulation of MAPK
cascade?
PKC*
1. Creation of intramolecular
disulfide bonds
PKA*
1. Activation via redox mechanism
JKN-inactivating phosphatases
Inactivation of phosphatases involved in
p38 pathway
1. Reversible oxidation of catalytic
Cys to sulfenic acid
2. Possible role of thioredoxin or
glutathione in reducing sulfenic
acid residues and reversing the
oxidative inactivation
Transducing and sustaining
growth factor signals
- Apoptosis
- Autophagy
- Inflammation?
- Cell cycle control
- Cell
differentiation
PI3K EGF
PDGF
NGF
Insulin
PTEN phosphatase
1. reversible redox regulation
(inactivation) by ROS generated
by growth factor stimulation
Regulation of:
- Cell cycle
- Cell quiescence
- Cell proliferation
The cellular signalling pathways in which
ROS play key role. The table summarizes
individual signalling pathways, signal
molecules involved in these pathways,
molecules that are modified by ROS and
processes that are subsequently initiated,
and cell responses. ARE - antioxidant
response element, ATM - ataxia–
telangiectasia mutated, EGF - epidermal
growth factor, IRP-1/2 - iron regulatory
protein-1 and -2, IRE - iron-responsive
element, MAPK - mitogen-activated
protein kinase cascade. NGF - nerve
growth factor, Nrf2 – nuclear factor-like 2,
PI3K - phosphatidylinositol-4,5bisphosphate
3-kinase, PDGF - plateletderived
growth factor, PKA – protein
kinase A, PKC – protein kinase C, PKG –
protein kinase G, PTEN - phosphatase and
tensin homolog, Ref1 – redox factor 1,
TfR1 - transferrin receptor 1, VEGF vascular
endothelial growth factor. G. * activation
of MAPK signalling. According
to citations in the chapter.
PI3K EGF
PDGF
NGF
Insulin
VEGF
PTEN phosphatase
1. reversible redox regulation
(inactivation) by ROS generated
by growth factor stimulation
2. PTEN inactivation by H2O2
3. disulfide bond formation between
Cys124 and Cys71 in catalytic
domain
4. Possible role of peroxiredoxin 3 in
reversible reaction
5. Note: PTEN knockdown
enhances transcription of AREregulated
antioxidant genes
Regulation of:
- Cell cycle
- Cell quiescence
- Cell proliferation
- Cancer
Nrf2 and Ref-1mediated
redox
cellular signalling
Genotoxic
agents
Oxidants
Various
stimuli
Ref-1 redox activity on several
transcription factors:
- AP-1
- p53
- NFB
- HIF-1a
1. increased DNA binding and
transcriptional activation of target
genes
Ref-1-mediated transcriptional activation
of Nrf2-target genes under oxidative stress
1. reversible oxidation of cysteine to
sulfenic or sulfinic acids
2. Regulatory role of thioredoxin
3. Translocation of cytoplasmic Ref-
1 to the nucleus under oxidative
stress
Protection against DNA
damage and oxidative
stress
p53-p66Shc
signalling
Genotoxic
stress
- UVC
Oxidative
stress
- H2O2
Phosphorylation of p66Shc
1. p66Shc = redox protein
2. Ser-54 and Thr-386 in a p38
dependent manner
3. phosphorylation of p66Shc at Ser-
36 by PKC-β
4. interaction of the prolyl isomerase
Pin1 with p66Shc
5. isomerization of a p66shc
phospho-Ser36-Pro37 bond
6. translocation of p66Shc into
mitochondria
Alternatively:
1. redox-dependent reversible
tetramerization
2. Cu-dependent ROS generation
3. initiation of apoptosis
4. Regulatory role of glutathione or
thioredoxin
Regulation of
mitochondrial ROS
metabolism
Oxidative stress responses
and control of cell redox
status
Regulation of rate of DNA
oxidative damage
Regulation of steady-state
levels of intracellular ROS
Regulation of p53-induced
ROS up-regulation and
cytochrome c release
selective regulation of p53dependent
apoptosis
IRE-IRP
regulatory
network
Oxidative
stress
NO
Hypoxia
Interaction of IRP-1/2 with IRE
1. Regulation of expression of
different genes (also oxidases)
2. 4Fe–4S iron–sulfur cluster
3. Destabilisation of 4Fe–4S iron–
sulfur cluster with H2O2 via
posttranslational modifications of
IRP1
4. Phosphorylation of IRP1 by PKC
at Ser-138
5. Regulation of iron storage and
export proteins – Fenton reaction
6. Role of ARE transcriptional
activation of the ferritin gene
Regulation of ferritin and
TfR1 mRNA expression –
iron homeostasis
ATM-mediated
DNA damage
responses
Genotoxic
stress
Oxidation
stress (H2O2)
Activation of ATM by H2O2
1. formation of active ATM dimers
via intermolecular disulfide bond
2. Cys2991 oxidation
Suppression of protein
synthesis and induction of
autophagy under oxidative
stress
ROS in physiological processes
• Cardiovascular physiology
• differentiation and contractility of vascular smooth muscle cells
• hydrogen peroxide, as signalling moiety, induces increase in intracellular calcium level
and contraction in pulmonary artery smooth muscle cells
• responses induced by different vasoconstrictor stimuli, including hypoxia (pulmonary
arteries constrict in response to hypoxia)
• involvement of ROS in the development of pulmonary hypertension
• control of vascular endothelial cell proliferation and migration
• platelet activation and haemostasis
• ROS, in turn, activate membrane transporters, as sodium/hydrogen exchanger
(NHE-1) and sodium/bicarbonate cotransporter (NBC) via stimulation of the
ROS-sensitive MARK cascade and finally stimulates of such effectors leads to
an increase in cardiac contractility
• ROS and respiration
• sensory plasticity of the carotid body (glomus caroticum)
• ROS and skeletal muscles
• circulation during exercise in humans
• requirements of adapting to osmotic challenges, hyperthermia challenges, and loss
of circulating fluid volume
• stimulation of glucose transport in isolated skeletal muscle preparations during
intense repeated contractions
• ROS and nervous system
• ROS are generated by microglia and astrocytes
• modulate synaptic and non-synaptic communication between neurons and glia
• synaptic long-term potentiation, a form of activity-dependent synaptic plasticity and
memory consolidation
• ROS and kidneys
• production of superoxide, hydrogen peroxide, and nitric oxide in the renal medullary
thick ascending limb of Henle regulates medullary blood flow, sodium homeostasis,
and long- term control of blood pressure
hypoxic pulmonary vasoconstriction Mitochondrial redox oxygen sensing. The sensor-effector
mechanism of hypoxic pulmonary vasoconstriction (HPV).
(A) Under normoxic conditions, generation of reactive
oxygen species (ROS) occurs at mitochondrial electron
transport chain (ETC) complexes I and III, producing
superoxide (O2–), which is converted to hydrogen peroxide
(H2O2) by superoxide dismutase 2 (SOD2). Hydrogen
peroxide, along with the oxidized redox couples (eg,
nicotinamide adenine dinucleotide [NAD+], nicotinamide
adenine dinucleotide phosphate [NADP+], and flavin
adenine dinucleotide [FAD2+]) maintain Kv1.5 sulfhydryl
group oxidation and channel open state, resulting in tonic
egress of K+. This efflux of K+ sustains the resting
membrane potential (ΔΨ) of the cell at –60 mV and
inhibits voltage-gated calcium channel [CaL]-mediated
Ca2+ influx into the cell. (B) During hypoxia, the limited
presence of oxygen (1) prevents generation of hydrogen
peroxide, (2) decreases the ratio of oxidized/reduced
redox couples, and (3) reduces sulfhydryl groups on Kv1.5
channels, causing them to close. The subsequent buildup
of K+ increases the resting membrane potential of the cell
to –20 mV. This stimulates the opening of CaL, influx of
Ca2+, and subsequent activation of the contractile
apparatus (ie, vasoconstriction). ADP = adenosine
diphosphate; FADH2 = flavin adenine dinucleotide; NADH =
nicotinamide adenine dinucleotide.
ROS and endocrine system
ROS and wound healing Reactive oxygen species (ROS) and its role in
wound healing. The schematic diagram depicts
the multiple roles of ROS during acute wound
healing (note that this refers to homeostatic,
not excessive, levels of ROS). (i) ROS are
important in initial wound protection by
reducing blood flow and local cell signalling for
thrombus formation; (ii) local ROS release
attracts blood vessel-bound local neutrophils
to the wound site for bacterial protection; (iii)
phagocytosis releases ROS to stunt bacterial
growth and provide further signals supporting
the wound response; (iv) other immunocytes,
including monocytes, migrate towards the
wound site to help attack invading pathogens;
(v) wound edge and general release of ROS
stimulates endothelial cell division and
migration for blood vessel reformation,
fibroblast division and migration for new ECM
formation (including collagen synthesis) and
promote keratinocyte proliferation and
migration.
ROS and wound healing
ROS and cell death
Cell features Apoptosis Autophagy Necrosis Oncosis
Cell size reduced -shrinkage
reduced/massive
vacuolization of
cytoplasm
(accumulation
of autophagic
vacuoles)
increased - swelling increased - swelling
Plasma membrane
intact, changes in
membrane
symmetry, changes
in orientation of
lipids
intact disrupted
intact in the early
phase; increased
throughput
depending on the
phase of oncosis
Nucleus
condensation of
chromatin, changes
in shape of nucleus,
fragmentation of
DNA (the end of the
process)
no chromatin
condensation;
karyolysis and
caspase independent
DNA fragmentation,
lysis of nucleolus
(the beginning of the
process)
nucleus dilatation
and clumping of
chromatin, reticular
nucleolus
Specific features
apoptotic bodies;
pseudopod
retraction; spherical
shape of cells
presence of
autophagic
vacuoles
increasingly
translucent
cytoplasm; swelling
of ER and loss of
ribosomes; swollen
mitochondria with
amorphous densities;
lysosome rupture;
plasma membrane
rupture; myelin
figures
swelling of
organelles;
membrane blebs
Energy balance
retained ATP
production
retained ATP
production
ATP depletion ATP depletion
Adjacent
inflammation
rare no frequent frequent
Involvement of
ROS
yes yes yes yes