PRESENTED BY SHANOO SUROOWAN

Reactive oxygen species & signal transduction

A radical is an atom/ group of atoms having one/more unpaired electrons.

There are many types of radicals, but the most prominent in biological systems are derived from oxygen collectively known as Reactive Oxygen Species (ROS),

Oxygen in its ground state has 2 unpaired electrons........Remember? O8: 1s2 2s2 2p4

So it is easy for Oxygen to accept electrons to form free radicals (Reactive Oxygen Species in this case!)

ROS formation in cells These are formed as normal aerobic

processes that occur in the body; some as necessary intermediates of enzymatic reactions.

Most are produced in the ETC when oxygen is reduced to water in the mitochondria.

O2 reduction H2O During this conversion various reactive

oxygen species are formed!ē ē ē ē

O2 .O2 – H2O2 .OH- H2O

(oxygen) (superoxide) (hydrogen peroxide) (hydroxyl ion) (water)

Overproduction of ROS

Neutrophils are specialized in ROS formation which destroy pathogens as part of host defence.

When cells are exposed to abnormal environments such as hypoxia and hyperoxia.

From ionizing radiation in biological systems. Ionizing radiation will ionize molecular oxygen by pushing an electron in its outer orbit.

If oxygen species are overproduced it will be difficult for the cell to detoxify them and repair the damages they make! Result: Oxidative stress!

So Oxidative stress is an imbalance btw reactive oxygen species and antioxidants!

Harmful activities of ROS

Damage to a number of macromolecules such as lipids, proteins and DNA caused by ROS implicate them in many disease processes, ranging from arthritis, atherosclerosis, pulmonary fibrosis, cancer, neurodegenerative diseases, and aging

Toxic effects such as damage to cell membranes initiated by lipid peroxidation.

A common target for peroxidation is unsaturated fatty acids present in membrane phospholipids

Lipid peroxidation....

Consequences of lipid peroxidation: increased membrane rigidity decreased activity of membrane-bound enzymes (e.g. sodium

pumps) altered activity of membrane receptors. altered permiability

Antioxidants of the body

1. Intracellular antioxidants: vitamin E ascorbate(vitamin C) glutathione (glutamate-cysteine-glycine) [GSH]

2. Enzymatic antioxidants: Superoxide dismutases(SOD’s) Catalase Gluthione peroxidase

Intracellular antioxidants

Vitamin E; a lipid soluble antioxidant that traps peroxy radicals while doing so it itself becomes a radical

Vitamin C regenerates back vitamin E from its radical form

Enzymatic antioxidants Superoxide dismutases (SOD’s)

Mostly found in the mitochondria They depend on cofactors such as manganese,

copper or zinc for their antioxidant activity. They convert 2 superoxide ions into oxygen and

hydrogen peroxide

Catalase hydrolyses hydrogen peroxide into water and oxygen! Site of location; peroxisomes

Glutathione peroxidase also hydrolyze hydrogen peroxide and can convert organic peroxides to alcohol

SIGNAL TRANSDUCTION

Extracellular Signal

(growth factor/cytokines/neurotransmitter/hormone)

Binds to specific Receptor

Interaction of receptor-ligand complex

Generates a wide variety of intracellular signals:

1. Changes in ion concentration

2. Activation of trimeric GTP binding regulatory proteins

3. Activation of receptor kinases

Downstream signaling by secondary messengers

(cAMP, Ca2+, phospholipid metabolites)

Activation of transcription factors for transcription of

specific genes for diverse cellular functions

Oxidants and signal transduction

Oxidants modulate cell signaling events by modifying cell surface receptors, phosphatases and protein phosphorylation, etc.

These phenomena are important in transactivation of transcription factors

activation/inactivation of gene transcription that may regulate steps in the development of disease.

ROS and Signal Transduction ROS are important mediators in signal

transduction Receptor + hormone (upstream signaling)

Receptor-ligand complex

Intracellular ROS production (downstream signaling)

activates Other pathways

promote regions of intermediate response genes governing cell proliferation, differentiation, etc. ROS are involved in both upregulation and

downregulation pathways!

ROS and Signal Transduction

ROS are not only injurious by-products of cellular metabolism but also essential participants in cell signaling and regulation

The cellular functions/toxic properties of ROS is dependent on their concentration.

For e.g. when produced in low concentrations by nitric oxide synthase(NOS) NO· functions as a signaling molecule mediating vasodilation

While when produced in high concentrations in macrophages, it is a toxic oxidant for microbicidal killing

Cellular sources and regulation of ROS

Cellular production of ROS occurs from both enzymatic and nonenzymatic sources

any electron-transferring protein or enzymatic system can result in the formation of ROS as “by-products”

ROS are mostly produced in the mitochondria, H2O2 can diffuse out in the cytoplasm while O2.- remains trapped in.

O2 −·-generating microsomal NADH oxidoreductase may function as a potential pulmonary artery O2 sensor in pulmonary artery smooth muscle cells

Cellular sources and regulation of ROS

Nuclear membranes contain cytochrome oxidases and ETC. Electron “leaks” from these enzymatic systems gives rise to ROS that can damage cellular DNA

Peroxisomes are an important source of total cellular H2O2 production. They contain a number of H2O2-generating enzymes including glycolate oxidase, d-amino acid oxidase.

Peroxisomal catalase utilizes H2O2 produced by these oxidases to oxidize a variety of other substrates in “peroxidative” reactions e.g. detoxification of alcohol in the liver

Intracellular soluble enzymes such as xanthine oxidase, aldehyde oxidase, can generate ROS during catalytic cycling

Cellular sources and regulation of ROS

Autooxidation of small molecules such as dopamine, epinephrine can be an important source of intracellular ROS production.

Prooxidant effects of dopamine autooxidation is implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease

Plasma membrane-associated oxidases have been implicated as the sources of most growth factor- and/or cytokine-stimulated oxidant production

The phagocytic NADPH oxidase, which serves a specialized function in host defense against invading microorganisms

Cellular sources and regulation of ROS

functional components of the phagocytic NADPH are present in nonphagocytic cells.

P22phox is a component of NADPH oxidase and plays a key role in its activation

Expression of p22phox has been demonstrated in the adventitial smooth muscle cells of coronary arteries and the aorta

Increased aortic adventitial O2 −· production contributes to hypertension by blocking the vasodilatory effects of NO

ROS production in coronary arteries is related to hypertension

ROS in cell signaling

ROS signaling by receptors

The following receptors are involved in ROS signaling :- Cytokine receptors Receptor tyrosine kinases (RTKs) Receptor serine/threonine kinases G-protein coupled receptorThese receptors will generate intracellular signals for

ROS production

ROS signaling by cytokine receptors

1.TNF-α

Mediate ROS formation in mitochondria

Activates the TF nuclear factor (NF)-kB

nuclear factor (NF)-kB dependent transcription

Makes cancer cells resistant to apoptosis

Activates apoptosis signal-regulating kinase-1 (ASK 1)

How?

Oxidant dependent dimerization of ASK 1

ASK1 has been found to be involved in cancer, diabetes, cardiovascular and neurodegenerative diseases

TNF-α is an autocrine cytokine involved in ROS signaling by cytokine receptors (upstream transduction)

The ROS produced have various implications in downstream signaling such as:- the expression of cell adhesion molecules from genes production of chemokines In pathophysiological conditions such as induction of

cardiac myocyte hypertrophy

2.IFN-γ (activator of the phagocytic NADPH oxidase)

Stimulates cyclooygenase-dependent peroxide production in human hepatocyte

Resistance to bacteria

ROS signaling by RTKs

A number of growth factors that bind to RTKs generate intracellular ROS essential for mitogenic signaling :- Mitogens will be produced Produced mitogens activate MAPK ( Mitogen

activated protein kinase) that trigger mitosis

These growth factors that act on receptor tyrosine kinases (RTKs) include :- PDGF (plasma dependent growth factor) EGF (epidermal growth factor) FGF (fibroblast growth factor)

PDGF

Increases intracellular concentrations of hydrogen peroxides

Induce tyrosine phosphorylation

MAPK activation

DNA synthesis & chemotaxis

Regulates gene expression by .O2

– dependent pathways

.O2 – produced

involved in the upregulation of inducible NOS & NO dependent release of PGE2 in fibroblasts

induces fever

Receptor serine/threonine kinases These are receptors of the transforming

growth factor β (TGF-β) superfamilyTGF-β

Stimulates extracellular production of ROS

Regulates a number of physiological actions

Apoptosis, collagen synthesis, growth inhibitory effects

G protein-coupled receptors The ligands for these receptors include:-

ANG II (Angiotensin II) Serotonin (5-hydroxytryptamine) Bradykinin Thrombin

Angiotensin II

activates both NADH- and NADPH-dependent O2 −· production in vascular smooth muscle cells

A variety of physiological actions of ANG II are mediated by ROS

its vasopressor activity, smooth muscle cell hypertrophy, activation of cell survival PK Akt/PKB, induction of insulin-like growth factor-1 receptor

Mechanisms of ROS action ROS act via two mechanisms:-

1)alterations in intracellular redox state

2) oxidative modifications of proteins

Alterations in intracellular redox states The cytosol is maintained under strong

reducing conditions This is accomplished by the “redox-

buffering” capacity of intracellular thiols, primarily glutathione (GSH)and thioredoxin (TRX).

They reduce both H2O2 and lipid peroxides, reactions that are catalyzed by peroxidases

e.g. GSH peroxidase catalyzes the reaction H2O2 + 2GSH → 2H2O + GSSG

GSH and TRX are antioxidants that play important roles in cell signaling

Alterations in intracellular redox states

During oxidative stress , the concentration of the oxidized form of GSH increases[H2O2 + 2GSH → 2H2O + GSSG]

Decreased cell proliferation in vascular endothelial cells increased proliferation of fibroblasts induces the binding of some TFs to DNA.

Conclusion

• ROS are mediators of cell signaling• They cause a series of changes during cell signaling

References

http://www.jleukbio.org/content/65/3/337.full.pdf

http://www.sciencedirect.com/science/article/pii/S0014579300016690

http://jcb.rupress.org/content/194/1/7http://www.heribert-hirt.info/pdf/

prr85_ros.pdfhttp://ajplung.physiology.org/content/279/6/

L1005.full