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Eicosanoid Synthesis Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry II.

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Presentation on theme: "Eicosanoid Synthesis Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry II."— Presentation transcript:

1 Eicosanoid Synthesis Copyright © by Joyce J. Diwan. All rights reserved. Molecular Biochemistry II

2 Prostaglandins and related compounds are collectively known as eicosanoids. Most are produced from arachidonic acid, a 20-carbon polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic acid). 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.

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

4 PGE 2 (prostaglandin E 2 ) is an example of a prostaglandin, produced from arachidonic acid.

5 PGE 2 (prostaglandin E 2 ). Prostaglandins all have a cyclopentane ring.  A letter code is based on ring modifications (e.g., hydroxyl or keto groups).  A subscript refers to the number of double bonds in the two side-chains. Thromboxanes are similar but have instead a 6-member ring.

6 Prostaglandin receptors:  Prostaglandins & 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, or may activate a phosphatidylinositol signal pathway leading to intracellular Ca ++ release.  Another prostaglandin receptor, designated PPAR , is related to a family of nuclear receptors with transcription factor activity.

7  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.

8 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 & a lysophospholipid as products. The fatty acid arachidonate is often esterified to OH on C2 of glycerophospho- lipids, especially phosphatidyl inositol.

9 Corticosteroids are anti-inflammatory because they prevent inducible Phospholipase A 2 expression, reducing arachidonate release. There are multiple Phospholipase A 2 enzymes, subject to activation via different signal cascades.  The inflammatory signal platelet activating factor is involved in activating some Phospholipase A 2 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.

10 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. Phosphatidyl inositol signal cascades may lead to release of arachidonate.

11 Prostaglandin H 2 Synthase catalyzes the committed step in the “cyclic pathway” that leads to production of prostaglandins, prostacyclins, & thromboxanes. Different cell types convert PGH 2 to different compounds. Two major pathways of eicosanoid metabolism. Cyclic pathway:

12 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.

13 PGH 2 Synthase (expressing both cyclooxygenase & peroxidase activities) is sometimes referred to as Cyclooxygenase, abbreviated COX. The interacting cyclooxygenase and peroxidase reaction pathways are complex.

14 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). The resulting tyrosine radical is proposed to extract a H atom from arachidonate, generating a radical species that reacts with O 2.

15 The signal molecule ·NO (nitric oxide) may initiate prostaglandin synthesis by reacting with superoxide anion (O 2 ·  ) to produce peroxynitrite, which oxidizes the heme iron enabling electron transfer from the active site tyrosine. Prostaglandin synthesis in response to some inflammatory stimuli is diminished by inhibitors of Nitric Oxide Synthase.

16 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. Membrane- binding domain: 4 short amphipathic  -helices that insert into one leaflet of the bilayer, facing the ER lumen.

17 Ibuprofen and related compounds block the hydrophobic channel by which arachidonate enters the cyclooxygenase active site. Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin and derivatives of ibuprofen, inhibit cyclooxygenase activity of PGH 2 Synthase.  They inhibit formation of prostaglandins involved in fever, pain, & inflammation.  They inhibit blood clotting by blocking thromboxane formation in blood platelets.

18 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.

19 Thromboxane A 2 stimulates blood platelet aggregation, essential to the role of platelets in blood clotting.  Many people take a daily aspirin for its anti-clotting 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.

20 Two isoforms of PGH 2 Synthase: COX-1 & COX-2 (Cyclooxygenase 1 & 2):  COX-1 is constitutively expressed at low levels in many cell types.  COX-2 expression is highly regulated. 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, & localization of enzymes that convert PGH 2 into particular prostaglandins/ thromboxanes, may result in COX-1 & COX-2 yielding different ultimate products.

21  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 & 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.

22 Most NSAIDs inhibit both COX I & COX II. Selective COX-2 inhibitors have been developed, e.g., Celebrex and Vioxx.  COX-2 inhibitors are anti-inflammatory & 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.

23 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. Explore the structure of PGH 2 Synthase-1 (COX-1) crystallized with bound iodosuprofen, a derivative of ibuprofen.

24 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.

25 E.g., 5-Lipoxygenase, found in leukocytes, catalyzes conversion of arachidonate to 5-HPETE (5-hydroperoxy- eicosatetraenoic acid). 5-HPETE is converted to leukotriene-A 4, which in turn may be converted to various other leukotrienes.

26 A non-heme iron is the prosthetic group of Lipoxygenase enzymes. Ligands to the Fe include 3 His N atoms & 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.

27 Lipoxygenase reaction starts with extraction of H from arachidonate, with transfer of the e  to the iron, reducing it from Fe 3+  Fe 2+. The resulting fatty acid radical reacts with O 2 to form a hydroperoxy group. Which H is extracted, & 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.

28 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.

29 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, & Phospholipase A 2 in association with the nuclear envelope has been observed during activation of leukotriene synthesis in leukocytes.

30 A  -barrel domain at the N-terminus of Lipoxygenase enzymes may have a role in membrane binding. Explore the structure of Lipoxygenase, with a substrate analog present at the active site.

31 Cytochrome P450 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.

32 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, & various signal pathways relevant to cardiovascular and renal functions. E.g., EETs inhibit apoptosis in endothelial cells. Shown is an EET produced from arachidonate by activity of a cytochrome P 450 epoxygenase. (14,15-epoxyeicosatrienoic acid)


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