Eicosanoid synthesis

Transcription

1 Seminar 12

2 Eicosanoid synthesis

3 Examples of eicosanoids prostaglandins prostacyclins thromboxanes leukotrienes epoxyeicosatrienoic acids (EET) They have roles in: inflammation fever regulation of blood pressure blood clotting immune system modulation control of reproductive processes and tissue growth regulation of sleep/wake cycle

4 Eicosanoids most are produced from arachidonic acid, a 20-carbon polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic acid) COOH Arachidonic acid

5 Eicosanoids the eicosanoids are considered local hormones they have specific effects on target cells close to their site of formation they are rapidly degraded, so they are not transported to distal sites within the body but in addition to participating in intercellular signaling, there is evidence for involvement of eicosanoids in intracellular signal cascades

6 Prostaglandins PGE 2 (prostaglandin E 2 ) is an example of a prostaglandin, produced from arachidonic acid O COOH HO OH PGE 2

7 Prostaglandins all have a cyclopentane ring O COOH HO OH PGE 2 a subscript refers to the number of double bonds in the two side-chains a letter code is based on ring modifications (e.g., hydroxyl or keto groups) thromboxanes are similar but have instead a 6-member ring

8 Prostaglandin receptors prostaglandins and related compounds are transported out of the cells that synthesize them most affect other cells by interacting with plasma membrane G-protein coupled receptors depending on the cell type, the activated G-protein may stimulate or inhibit formation of camp activate a phosphatidylinositol signal pathway leading to intracellular Ca 2+ release another prostaglandin receptor, designated PPARγ, is related to a family of nuclear receptors with transcription factor activity

9 Prostaglandin receptors prostaglandin receptors are specified by the same letter code (e.g., receptors for E-class prostaglandins are EP) thromboxane receptors are designated TP multiple receptors for a prostaglandin are specified by subscripts (e.g., EP 1, EP 2, EP 3, etc.) different receptors for a particular prostaglandin may activate different signal cascades effects of a particular prostaglandin may vary in different tissues, depending on which receptors are expressed (e.g., in different cells PGE 2 may activate either stimulatory or inhibitory or G-proteins, leading to either increase or decrease in camp formation)

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11 Prostaglandin synthesis Site of cleavage by Phospholipase A 2 O O H 2 C O C R 1 The fatty acid arachidonate is often esterified to OH on C2 of glycerophospholipids, especially phosphatidyl inositol R 2 C O C H CH 2 O Site of cleavage by Phospholipase C O P O- O H H OH OH H Phosphatidyl inositol OH H OH H H OH arachidonate is released from phospholipids by hydrolysis catalyzed by Phospholipase A 2 this enzyme hydrolyzes the ester linkage between a fatty acid and the OH at C2 of the glycerol backbone, releasing the fatty acid and a lysophospholipid as products

12 Anti-inflammatory factors corticosteroids are anti-inflammatory because they prevent inducible Phospholipase A2 expression, reducing arachidonate release there are multiple Phospholipase A2 enzymes, subject to activation via different signal cascades the inflammatory signal platelet activating factor is involved in activating some Phospholipase A2 variants attempts have been made to develop drugs that inhibit particular isoforms of Phospholipase A2, for treating inflammatory diseases (success has been limited by the diversity of Phospholipase A2 enzymes, and the fact that arachidonate may give rise to inflammatory or anti-inflammatory eicosanoids in different tissues)

13 Phospholipase C Diacylglycerol Lipase O R 1 O H 2 C O C C O CH O R 2 H 2 C O P O Phosphatidyl inositol signal cascades may lead to release of arachidonate cleavage by Phospholipase C PIP 2 phosphatidylinositol- 4,5-bisphosphate O OH H H OH H H OH H OPO 3 2 H OPO 3 2 after PI is phosphorylated to PIP 2, cleavage via Phospholipase C yields diacylglycerol (and IP 3 ) arachidonate release from diacylglycerol is then catalyzed by Diacylglycerol Lipase

14 Eicosamoid metabolism Two major pathways of eicosanoid metabolism Cyclic pathway: leukotrienes phospholipids arachidonate diacylglycerol prostacyclins Linear pathway Cyclic pathway Prostacyclin Synthase Lipoxyganase PGH 2 Synthase prostaglandin H 2 other prostaglandins Thromboxane Synthase thromboxanes Prostaglandin H 2 Synthase catalyzes the committed step in the cyclic pathway that leads to production of prostaglandins, prostacyclins and thromboxanes different cell types convert PGH 2 to different compounds

15 Prostaglandin synthesis PGH 2 Synthase is a heme-containing dioxygenase, bound to ER membranes (a dioxygenase incorporates O 2 into a substrate) PGH 2 Synthase exhibits 2 activities: cyclooxygenase peroxidase arachidonic acid 2 O 2 O O PGG 2 2 e O O PGH 2 Cyclooxygenase COOH COOH OOH Peroxidase COOH OH

16 Prostaglandin synthesis PGH 2 Synthase (expressing both cyclooxygenase and peroxidase activities) is sometimes referred to as Cyclooxygenase abbreviated COX (the interacting cyclooxygenase and peroxidase reaction pathways are complex) arachidonic acid 2 O 2 O O PGG 2 2 e O O PGH 2 Cyclooxygenase COOH COOH OOH Peroxidase COOH OH

17 a peroxide (such as that generated later in the reaction sequence) oxidizes the heme iron the oxidized heme accepts an electron from a nearby tyrosine residue (Tyr385) arachidonic acid 2 O 2 O O Cyclooxygenase COOH COOH the resulting tyrosine radical is proposed to extract a H atom from arachidonate, generating a radical species that reacts with O 2 PGG 2 O O PGH 2 2 e OOH Peroxidase OH COOH

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19 Membranebinding domain: 4 short amphipathic -helices that insert into one leaflet of the bilayer, facing the ER lumen PDB 1PGE heme ibuprofen analog membrane binding domain Prostaglandin H 2 Synthase arachidonate, derived from membrane lipids, approaches the heme via a hydrophobic channel extending from the membrane-binding domain (in the image above, the channel is occupied by an inhibitor, an ibuprofen analog)

20 Non-steroidal anti-inflammatory drugs (NSAIDs) NSAIDs such as aspirin and derivatives of ibuprofen, inhibit cyclooxygenase activity of PGH 2 Synthas they inhibit formation of prostaglandins involved in fever, pain and inflammation they inhibit blood clotting by blocking thromboxane formation in blood platelets Ibuprofen and related compounds block the hydrophobic channel by which arachidonate enters the cyclooxygenase active site H 3 C CH 3 CH CH 2 CH H 3 C COOH Ibuprofen

21 COOH O O C CH 3 Aspirin + Enz-Ser CH 2 OH PGH 2 Synthase (active) COOH OH + Enz-Ser CH 2 O O C CH 3 Salicylic acid Acetylated PGH 2 Synthase (inactive) aspirin acetylates a serine hydroxyl group near the active site, preventing arachidonate binding the inhibition by aspirin is irreversible however, in most body cells re-synthesis of PGH 2 Synthase would restore cyclooxygenase activity

22 Thromboxanes Thromboxane A 2 stimulates blood platelet aggregation, essential to the role of platelets in blood clotting many people take a daily aspirin for its anticlotting effect, attributed to inhibition of thromboxane formation in blood platelets this effect of aspirin is long-lived because platelets lack a nucleus and do not make new enzyme

23 Two isoforms of PGH 2 Synthase: COX-1 and COX-2 COX-1 is constitutively expressed at low levels in many cell types COX-2 expression is highly regulated

24 Two isoforms of PGH 2 Synthase: COX-1 and COX-2 transcription of the gene for COX-2 is stimulated by growth factors, cytokines, and endotoxins COX-2 expression may be enhanced by camp, and in many cells PGE 2 produced as a result of COX-2 activity itself leads to changes in camp levels both catalyze PGH 2 formation, but differing localization within a cell and localization of enzymes that convert PGH 2 into particular prostaglandins/ thromboxanes, may result in COX-1 and COX-2 yielding different ultimate products

25 COX-1 is essential for thromboxane formation in blood platelets and for maintaining integrity of the gastrointestinal epithelium COX-2 levels increase in inflammatory diseases such as arthritis Inflammation is associated with up-regulation of COX-2 and increased amounts of particular prostaglandins COX-2 expression is increased in some cancer cells Angiogenesis (blood vessel development), which is essential to tumor growth, requires COX-2 Overexpression of COX-2 leads to increased expression of VEGF (vascular endothelial growth factor) Regular use of NSAIDs has been shown to decrease the risk of developing colorectal cancer

26 Most NSAIDs inhibit both COX-1 and COX-2 Selective COX-2 inhibitors COX-2 inhibitors are anti-inflammatory and block pain, but are less likely to cause gastric toxicity associated with chronic use of NSAIDs that block COX-1 A tendency to develop blood clots when taking some of these drugs has been attributed to: decreased production of an anti-thrombotic (clot blocking) prostaglandin (PGI 2 ) by endothelial cells lining small blood vessels lack of inhibition of COX-1-mediated formation of pro-thrombotic thromboxanes in platelets

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28 COX-3? some evidence suggests the existence of a third isoform of PGH 2 Synthase, designated COX-3, with roles in mediating pain and fever, and subject to inhibition by acetaminophen (Tylenol) acetaminophen has little effect on COX-1 or COX-2, and thus lacks anti-inflammatory activity.

29 leukotrienes Linear pathway Lipoxyganase phospholipids arachidonate diacylglycerol Cyclic pathway PGH 2 Synthase Prostacyclin Synthase prostaglandin H 2 Thromboxane Synthase prostacyclins other prostaglandins thromboxanes the 1st step of the Linear Pathway for synthesis of leukotrienes is catalyzed by Lipoxygenase (mammals have a family of Lipoxygenase enzymes that catalyze oxygenation of various polyunsaturated fatty acid at different sites. Many of the products have signal roles)

30 Leucotrienes synthesis Catalyzed by 5-Lipoxygenase: COOH 5-Lipoxygenase (found in leukocytes) catalyzes conversion of arachidonate to 5-HPETE (5-hydroperoxyeicosatetraenoic acid) O 2 OOH arachidonate COOH 5-HPETE is converted to leukotriene-a 4, which in turn may be converted to various other leukotrienes H 2 O O 5-HPETE COOH leukotriene-a 4

31 A non-heme iron is the prosthetic group of Lipoxygenase enzymes Ligands to the Fe include 3 His N atoms and the C-terminal carboxylate O The arachidonate substrate binds in a hydrophobic pocket, adjacent to the catalytic iron atom O 2 is thought to approach from the opposite side of the substrate than the iron, for a stereospecific reaction 15-Lipoxygenase membranebinding domain substrate analog adjacent to Fe (green) PDB 1LOX

32 Lipoxygenase reaction starts with extraction of H from arachidonate, with transfer of the e to the iron, reducing it from Fe 3+ Fe 2+. O 2 OOH COOH arachidonate COOH The resulting fatty acid radical reacts with O 2 to form a hydroperoxy group 5-HPETE Which H is extracted and the position of the hydroperoxy group, varies with different lipoxygenases (e.g., 5-Lipoxgenase shown here, 15-Lipoxygenase, etc.) Additional reactions then yield the various leukotrienes

33 Leukotrienes have roles in inflammation. They are produced in areas of inflammation in blood vessel walls as part of the pathology of atherosclerosis. Leukotrienes are also implicated in asthmatic constriction of the bronchioles. Some leukotrienes act via specific G-protein coupled receptors (GPCRs) in the plasma membrane. Anti-asthma medications include: inhibitors of 5-Lipoxygenase, e.g., Zyflo (zileuton) drugs that block leukotriene-receptor interactions. E.g., Singulair (montelukast) & Accolate (zafirlukast) block binding of leukotrienes to their receptors on the plasma membranes of airway smooth muscle cells.

34 Lipogenase 5-Lipoxygenase requires the membrane protein FLAP (5-lipoxygenase-activating protein) FLAP binds arachidonate, facilitating its interaction with the enzyme Translocation of 5-Lipoxygenase from the cytoplasm to the nucleus, and formation of a complex including 5-Lipoxygenase, FLAP and Phospholipase A 2 in association with the nuclear envelope has been observed during activation of leukotriene synthesis in leukocytes

35 A b-barrel domain at the N-terminus of Lipoxygenase enzymes may have a role in membrane binding membranebinding domain substrate analog adjacent to Fe (green) 15-Lipoxygenase PDB 1LOX

36 COOH COOH Arachidonic acid 14,15-EET O Cytochrome P 450 epoxygenase pathways epoxyeicosatrienoic acids (EETs) and hydroxyeicosatrienoic acids are formed from arachidonate by enzymes of the cytochrome P 450 family other members of the cytochrome P 450 family participate in a variety of oxygenation reactions, including hydroxylation of sterols

37 COOH EET produced from arachidonate by activity of a cytochrome P 450 epoxygenase 14,15-EET O (14,15-epoxyeicosatrienoic acid) EETs are modified by additional enzyme-catalyzed reactions to produce many distinct compounds they may be incorporated into phospholipids and released by action of phospholipases EETs have roles in regulating cellular proliferation, inflammation, peptide hormone secretion and various signal pathways relevant to cardiovascular and renal functions (e.g., EETs inhibit apoptosis in endothelial cells)

38 Cytokines and receptors

39 What is a Cytokine? low molecular weight proteins (30 KDa) bind receptors, alter gene expression can bind the secreting cell (autocrine) can bind another cell close by (paracrine) few cases bind another cell far away (endocrine) very low K d receptors ( M) cytokines regulate immune responses

40 Cytokines cytokines can activate many cells e.g. cytokines secreted by T H can affect B-cells, CTLs, M, NK a cytokine can be pleiotropic (different effect on different cells), synergism, redundancy, antagonism interleukins, monokines, lymphokines, chemokines, term CYTOKINE includes all of them

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42 4 families Interferons Chemokines Hematopoietins TNF Cytokine Categories

43 Hematopoietin family -helical structure prevalence Little or no b-sheet e.g. IL-2 and IL-4 amino acid sequences vary considerably

44 Cells that make Cytokines and their Function a variety of cells are capable of making cytokines the biggest producers are M and T H cytokines are involved in hematopoiesis adaptive immunity innate immunity inflammation

45 Cytokines are Non-Specific how does immune specificity fit with Non-Specific Cytokines? Answer 1: through receptors receptors expressed on antigen activated cells Answer 2: close proximity to cytokine secreting cells e.g. APC-T H cytokine concentrations (T H ) are high locally only interacting APC gets activated Answer 3: short half life short ½ life ensures local activity

46 Functional groups of selected cytokines1

47 Cytokine Receptors 5 Major Families Immunoglobulin Superfamily Hematopoietin Receptor Family (Class I) Interferon Receptor Family (Class II) TNF Receptor Family Chemokine Receptor Family Class I and II (majority of receptors) multimeric upon receptor engagement, tyrosine phosphorylation

48 Hematopoietin Receptor Family (Class I)

49 Receptor Signalling (IFN R) ligand binds -subunit binding causes dimerization of receptor JAKs get activated phosphorylation of tyrosine residues on receptor phosphorylation of JAKs themselves STATS dock receptor phosphorylation of STATs by JAKs dimerized STATs translocate to nucleus gene expression

50 STAT and JAK interaction with selected cytokine receptors during signal transduction

51 Cytokine Antagonists antagonists exist in 2 forms receptor antagonists (bind receptor = no activation) bind cytokine (prevent cytokine from binding receptor) in many cases antagonist is a soluble receptor derived from proteolytic cleavage of extracellular domain of particular receptor e.g. IL-2, IL-4, IFN, IFN viruses produce cytokine mimics or cytokine binding proteins e.g. poxviruses produce IL-1 binding protein and TNF binding protein these agents offer viruses an advantage

52 Viral mimics of cytokines and cytokine receptors

53 T H 1 vs T H 2 CD4 + T H cells secret a variety of cytokines evidence for 2 subsets T H 1 T H 2 distinction is based on cytokine secretion cytokine environment determines which subset will develop IFN for T H 1 (IL-12 and IL-18 from M, DCs) IL-4 for T H 2

54 Cytokine secretion and principal functions of mouse T H 1 and T H 2 subsets

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57 Transcription Factors T H 1 and T H 2 T-bet expression results in T H 1 T-bet suppresses T H 2 GATA-3 results In T H 2 GATA-3 suppresses T H 1 IFN- regulates expression of T-bet (Stat 1) IL-4 regulates expression of GATA-3 (Stat 6)