BIOCHEMISTRY OF SIGNALLING MOLECULES

CYTOKINES AND METABOLISM OF EICOSANOIDS

Jana Švarcová

Eicosanoids

compounds containing a 20-carbon core

Members of this group: prostaglandins prostacyclines tromboxanes leukotrienes lipoxins hydroxyeicosatetraenoic acids (HETE) hepoxilins

prostanoids

Eicosanoids biosynthesis

A path in metabolism of polyunsaturated fatty acids (PUFAs), mainly linoleic and arachidonic acid:

linoleic acid

linolenic acid

arachidonic acid

eicosapentaenoic acid

∆6 – DESATURASA

Microsomal chain elongation system (ELONGASE)

∆5 – DESATURASA

arachidonic acid is (in humans) synthesized from linoleic acid:

!!! it is not possible to synthesise de novo

Most animals cannot form double bonds behind position ∆9 linoleic and linolenic acids are essential: must be taken from food (plant oils, peanuts, soya beans, maize)

Synthesis of eicosanoids - overview

Main eicosanoid production sites

Endothelial cells Leukocytes Platelets Kidneys

Unlike e.g. histamin, eicosanoids are not synthesized in advance and stored in granules

In case of an emergent need, these are rapidly produced from a released arachidonate

Eicosanoids biosynthesis takes place in every cell type except red blood cells

Main steps of eicosanoids production

1) Activation of phospholipase A2

(PLA2)

2) Release of arachidonate into cytosol from membrane phospholipids by PLA2

3) Eicosanoids synthesis from arachidonate

COX or LO pathway + further modifications by synthases/isomerases (PGH2

conversion to other prostanoids, LTA4 conversion..) depending on cell type

PLA2 is activated by intracellular Ca2+ concentration, PLA2 phosphorylation plays a stimulating role. Ligand binding on a receptor → phospholipase C activation : PIP2 → DAG + IP3, which opens Ca2+ channels in ER. By the action of Ca2+ and phosphorylation (MAPK, CAMKII) PLA2 is translocated to the membranes of GA, ER and/or nucleus, from which arachidonate is released.

1) Activation of phospholipase A2

Ca

GK, ER, or nuclear membrane

transl

oca

tion

activ

atio

n

NOS synthesis/activation

plasmamembrane•Ligand: e.g. ATP

released dying cells

(protein-kinase C)

PLA2 expression / activity stimulate: interleukin-1 angiotensin II bradykinin thrombin epinephrine…

PLA2 expression / activity block: dexamethasone

(synthetic corticoid) annexin 1 (lipocortin) –

protein inducible by glucocorticoids

caspase-3

dexamethasone

2) Arachidonate mobilization for eicosanoid synthesis

From membrane phospholipids mostly by the action of phospholipase A2:

Release of arachidonate from phospholipids is blockedby anti-inflammatory steroids!

3 pathways: A) cyclooxygenase – produces

prostaglandins and thromboxanes B) lipoxygenase – produces leukotrienes,

lipoxins, hepoxilins and 12- and 15-HETE (hydroxyeicosatetraenoic acids)

C) cytochrome P450 enzymes (monooxygenases) – produces HETE, e.g. 20-HETE; it is a main pathway in kidney proximal tubules

Eicosanoids biosynthesis

Cyclooxygenase pathway (COX)

Prostaglandin H-synthase - exists in 2 isoforms (PGHS-1/COX-1, PGHS-2/COX-2) and has two different activities: cyclooxygenase (COX) – catalyses addition of two

molecules of O2 into a molecule of arachidonate forming PGG2

hydroperoxidase – catalyses conversion of hydroperoxy-group in PGG2 to hydroxy-group in PGH2; uses glutathione

Self-destructive enzyme

A particular cell type produces mostly one particular prostanoid type: platelets produce almost exclusively tromboxanes; vascular endothelial cells produce prostacyclins; myocardium cells produce mainly PGI2, PGE2, PGF2a

Prostaglandin H-synthase

The unstable endoperoxide is formed , PGG2.

PGH2 – precursor of thromboxanes and other prostaglandins from group 2!!!

The hydroperoxy group (C15) is quickly reduced (→OH),PGH2.

Products of COX pathway

Platelets contain tromboxane synthase that catalyses tromboxane synthesis

Endothelial cells in vessels contain prostacyclin synthase that catalyses prostacyclin production (PGI2)

6-membered ring containing one oxygen atom

(thromboxane)

(thromboxane)

(prostacyclin)

cyclopentane ring

Inhibition of COX pathway

Aspirin inhibits cyclooxygenase activity of PGHS-1 i PGHS-2 (by acetylation of enzyme serine)

Other non-steroidal anti-inflammatory drugs inhibit cyclooxygenase activity (ibuprofen – competes with arachidonate)

Anti-inflammatory corticosteroids block PGHS-2 transcription

Anti-inflammatory activity of corticosteroids

Lipooxygenase pathway

3 different lipoxygenases indroduce oxygen to position 5, 12 or 15 in arachidonate; a primary product is hydroperoxy-eicosatetraenoic acid (HPETE)

Only

5-lipoxygenase produces leuko-trienes; it requires protein FLAP

15-lipoxygenase

-GluLeukotriene D4 Leukotriene E4-Gly

peptidoleukotrienesGly–Cys–Glu

Hepoxilins(HXA3)

15-lipoxygenase12-lipoxygenase

5-lipoxygenase

15-lipoxygenase

5-lipoxygenase

Synthesis of peptidoleukotriens

It requires glutathione!!

Synthesis of eicosanoids by enzymes CYP450

cytochrome P450 enzymes – monooxygenases:

RH + O2 + NADPH + H+ ROH + H2O + NADP+

Two types of compounds are produced: epoxygenases - catalyse production of

epoxyeicosatrienoic acids (EETs) which are metabolized by epoxid-hydrolases into almost inactive dihydroxyeicosatrienoic acids (DiHETEs)

hydroxylases - catalyse production of HETEs (20-HETE, 13-HETE etc.)

List of products

arachidonic acid CYP450

DiHETEs

19-, 20-, 8-, 9-, 10-, 11-, 12-, 13-, 15-, 16-, 17-, 18-HETE

cyklooxygenases

prostacyklins

prostaglandins

tromboxanes

lipoxygenases

5-, 8-, 12-, 15-HETE

lipoxins

hepoxilins

leukotrienes

EETs (epoxides)

Biological effects of eicosanoids

Eicosanoids are active at very low concentrations (like hormones)

Short half-life autocrine and paracrine signalling (unlike hormones)

Given the variety of eicosanoid molecules and the multitude of respective receptors there are many different effects of eicosanoids in the organism

Prostanoids signalling

Through G-protein-coupled receptors : a) Gs activate adenylate cyclase (AC) → cAMP

activates protein kinase A (PKA) b) Gi inhibit adenylate cyclase

(e.g. PKA)

c) Gq activates phospholipase C (it requires Ca2+) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); DAG & Ca2+ activate protein kinase C, IP3 opens Ca2+

channels in ER

Prostanoids signalling

+

Biological functions of eicosanoids

Prostanoids Mediators of inflammation – they cause:

vasodilatation redness, heat (PGE1, PGE2, PGD2, PGI2) increased vessel permeability edema (PGE2, PGD2,

PGI2)

Regulate pain and fever (PGE2)

PGE2, PGF2 stimulate uterine muscles during delivery

Tromboxanes Tromboxanes are synthesized in platelets and induce

vasoconstriction (TXA2) and platelet aggregation

Biological functions of eicosanoids

Leukotrienes Leukotrienes are very strong bronchoconstrictors; LTC4, LTD4 and LTE4

are together called slow-reacting substance of anaphylaxis (SRS-A) Increase vessel permeability Act as chemotactants and activate leukocytes Regulate vasoconstriction

Lipoxins In contrast to proinflammatory eicosanoids lipoxins retard inflammatory

reaction Hypothesis: in the 1st phase of inflammatory response – leukotriene

production (e.g. LTB4 drive leukocyte migration to a site of damage) → elevated prostaglandin levels (PGE2), which switch eicosanoid synthesis from LTs to LXs in the 2nd phase lipoxins are produced, which supports termination of an acute inflammation and prevents its transition to a chronic form

Cytokines

What are cytokines?

Group of proteins and peptides (glycopeptides)

Influence cell growth (growth factors) Signal transmission from a cell to

another cell

Important group - lymphokines (also interleukins), proteins released from activated cells of immune system which coordinate immune response of the organism

Cytokine nomenclature

Lymphokines - produced by activated T-lymphocytes, they control the response of immune system by signalization between immunocompetent cells

Interleukins (IL) - target cells for IL are leukocytes

Chemokines - specific class, mediating chemotaxis between cells; stimulate leukocyte movement and regulate their migration from blood into tissues

Monokines - produced mainly by mononuclear cells, such as macrophages

Main function of cytokines

Hematopoiesis (e.g. CSF - colony stimulating factor) Inflammatory reactions (e.g. IL1 - interleukin,

TNF - tumor necrosis factor) Chemotaxis (e.g. IL8, MIP1- macrophage

inflammatory protein 1, BLC – B-lymphocyte chemoatractant)

Imunostimulation (e.g. IL12, IFNg - interferon) Imunosupression (e.g. IL10) Angiogenesis (e.g. VEGF- vascular endothelial growth

factor) Embryogenesis (e.g. TGF-b, LT – lymphotoxin)

Signalization through cytokine receptors

•Receptor (R) activated by ligand binds kinase JAK (K).

• JAK phosphorylation and receptor phosphorylation. Phosphorylated site is intended for anchoring STAT (Signal Transducer and Activator of Transcription) (S).

•JAK catalyzes phosphorylation of tyrosine in STAT.

• Two phosphorylated molecules of STAT form an active dimer. Dimer is transferred to the nucleus, it binds then a specific part of DNA in promoter region of a target gene and activates its expression.

Receptors for TNF

TNF is dominant cytokine in inflammatory process

Extracellular domain of the TNF receptor is rich in cystein

Receptors for chemokines

Typical domain with seven transmembrane parts and a characteristic motif Asp-Arg-Tyr – “DRY”

Receptors are usually coupled with G-proteins

Interferons (IFN) and their signalization

cytokines with an antiviral activity

Their action is mediated through cell receptors

antiproliferative activity – the ability to stop growth of cells → Used for treatment of many illnesses (tumors, viral infection, autoimmune diseases)

Biological activity of IFN

IFN binds to a specific receptor on cell surface

activation of JAK/STAT signalling pathway and activation of transcrip-tion of the target gene

synthesis of a new mRNA is induced

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