1. BIOCHEMISTRY OF SIGNALLING MOLECULES CYTOKINES AND METABOLISM OF EICOSANOIDS Jana Švarcová

2. Eicosanoids  compounds containing a 20-carbon core  Members of this group:  prostaglandins  prostacyclines  tromboxanes  leukotrienes  lipoxins  hydroxyeicosatetraenoic acids (HETE)  hepoxilins prostanoids

3. Eicosanoids biosynthesis  A path in metabolism of polyunsaturated fatty acids (PUFAs), mainly linoleic and arachidonic acid: linoleic acid linolenic acid arachidonic acid eicosapentaenoic acid

4. ∆ 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)

5. Synthesis of eicosanoids - overview

6. 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

7. Main steps of eicosanoids production 1) Activation of phospholipase A 2 (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 (PGH 2 conversion to other prostanoids, LTA 4 conversion..) depending on cell type

8.  PLA 2 is activated by  intracellular Ca 2+ concentration, PLA 2 phosphorylation plays a stimulating role. Ligand binding on a receptor → phospholipase C activation : PIP 2 → DAG + IP 3, which opens Ca 2+ channels in ER. By the action of Ca 2+ and phosphorylation (MAPK, CAMKII) PLA 2 is translocated to the membranes of GA, ER and/or nucleus, from which arachidonate is released. 1) Activation of phospholipase A 2 Ca GK, ER, or nuclear membrane translocation activation NOS synthesis/ activation plasma membrane Ligand: e.g. ATP released dying cells (protein- kinase C)

9.  PLA 2 expression / activity stimulate:  interleukin-1  angiotensin II  bradykinin  thrombin  epinephrine…  PLA 2 expression / activity block:  dexamethasone (synthetic corticoid)  annexin 1 (lipocortin) – protein inducible by glucocorticoids  caspase-3 dexamethasone

10. 2) Arachidonate mobilization for eicosanoid synthesis  From membrane phospholipids mostly by the action of phospholipase A 2 : Release of arachidonate from phospholipids is blocked by anti-inflammatory steroids!

11.  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

12. 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 O 2 into a molecule of arachidonate forming PGG 2  hydroperoxidase – catalyses conversion of hydroperoxy-group in PGG 2 to hydroxy-group in PGH 2 ; 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 PGI 2, PGE 2, PGF 2 

13. Prostaglandin H-synthase The unstable endoperoxide is formed, PGG 2. PGH 2 – precursor of thromboxanes and other prostaglandins from group 2!!! The hydroperoxy group (C15) is quickly reduced ( →OH), PGH 2.

14. Products of COX pathway  Platelets contain tromboxane synthase that catalyses tromboxane synthesis  Endothelial cells in vessels contain prostacyclin synthase that catalyses prostacyclin production (PGI 2 ) 6-membered ring containing one oxygen atom (thromboxane) ( prostacyclin ) cyclopentane ring

15. 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

16. Anti-inflammatory activity of corticosteroids

17. 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 -Glu Leukotriene D 4 Leukotriene E 4 - Gly peptidoleukotrienes Gly–Cys–Glu Hepoxilins (HXA 3 ) 15-lipoxygenase 12-lipoxygenase 5-lipoxygenase 15-lipoxygenase 5-lipoxygenase

18. Synthesis of peptidoleukotriens It requires glutathione!!

19. Synthesis of eicosanoids by enzymes CYP450  cytochrome P450 enzymes – monooxygenases: RH + O 2 + NADPH + H +  ROH + H 2 O + 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.)

20. 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)

21. 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

22. Prostanoids signalling  Through G-protein-coupled receptors :  a) G  s activate adenylate cyclase (AC) → cAMP activates protein kinase A (PKA)  b) G  i inhibit adenylate cyclase (e.g. PKA)

23.  c) G q activates phospholipase C (it requires Ca 2+ ) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP 2 ) to inositol-1,4,5- trisphosphate (IP 3 ) and diacylglycerol (DAG); DAG & Ca 2+ activate protein kinase C, IP 3 opens Ca 2+ channels in ER Prostanoids signalling +

24. Biological functions of eicosanoids Prostanoids  Mediators of inflammation – they cause:  vasodilatation  redness, heat (PGE 1, PGE 2, PGD 2, PGI 2 )  increased vessel permeability  edema (PGE 2, PGD 2, PGI 2 )  Regulate pain and fever (PGE 2 )  PGE 2, PGF 2 stimulate uterine muscles during delivery Tromboxanes  Tromboxanes are synthesized in platelets and induce vasoconstriction (TXA 2 ) and platelet aggregation

25. Biological functions of eicosanoids Leukotrienes  Leukotrienes are very strong bronchoconstrictors; LTC 4, LTD 4 and LTE 4 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 1 st phase of inflammatory response – leukotriene production (e.g. LTB 4 drive leukocyte migration to a site of damage) → elevated prostaglandin levels (PGE 2 ), which switch eicosanoid synthesis from LTs to LXs  in the 2 nd phase lipoxins are produced, which supports termination of an acute inflammation and prevents its transition to a chronic form

26. Cytokines

27. 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

28. 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

29. 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 )

30. 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.

31. Receptors for TNF  TNF is dominant cytokine in inflammatory process  Extracellular domain of the TNF receptor is rich in cystein

32. Receptors for chemokines  Typical domain with seven transmembrane parts and a characteristic motif Asp-Arg-Tyr – “DRY ”  Receptors are usually coupled with G-proteins

33. 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)

34. 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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