Seminar 12
Seminar 12
Eicosanoid synthesis
• 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
Examples of eicosanoids
• most are produced from arachidonic acid, a 20-carbon polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic acid)
Eicosanoids
Arachidonic acid
COOH
• 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
Eicosanoids
COOH
O
HOOH PGE2
Prostaglandins
• PGE2 (prostaglandin E2) is an example of a prostaglandin, produced from arachidonic acid
Prostaglandins
• all have a cyclopentane ring
• thromboxanes are similar but have instead a 6-member 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
COOH
O
HOOH PGE2
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 Ca2+ release
• another prostaglandin receptor, designated PPARγ, is related to a family of nuclear receptors with transcription factor activity
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., EP1, EP2, EP3, 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 PGE2 may activate either stimulatory or inhibitory or G-proteins, leading to either increase or decrease in cAMP formation)
• arachidonate is released from phospholipids by hydrolysis catalyzed by Phospholipase A2
• 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
Phosphatidyl inositol
C HO
CH2O
H2C O C R1
O
CR2
O
P O
O
O-
H
H
OH
OH
HH
OH
OHH
OH H
Site of cleavage by Phospholipase A2
Site of cleavage by Phospholipase C
The fatty acid arachidonate is often esterified to OH on C2 of glycerophospho-lipids, especially phosphatidyl inositol
Prostaglandin synthesis
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)
O P
O
O
H2C
CH
H2C
OCR1
O O C
O
R2
OH
H
OPO32
H
H
OPO32
H
OH
H
O
H OH
PIP2 phosphatidylinositol- 4,5-bisphosphate
cleavage by Phospholipase C
Phospholipase C
Phosphatidyl inositol signal cascades may lead to release of arachidonate
• after PI is phosphorylated to PIP2, cleavage via Phospholipase C yields diacylglycerol (and IP3)
• arachidonate release from diacylglycerol is then catalyzed by Diacylglycerol Lipase
Diacylglycerol Lipase
leukotrienes
phospholipids arachidonate diacylglycerol
prostaglandin H2
prostacyclins thromboxanes
other prostaglandins
Lipoxyganase
PGH2 Synthase
Prostacyclin Synthase
Thromboxane Synthase
Linear pathway
Cyclic pathway Two major pathways of eicosanoid metabolism
Cyclic pathway:
Eicosamoid metabolism
• Prostaglandin H2 Synthase catalyzes the committed step in the „cyclic pathway” that leads to production of prostaglandins, prostacyclins and thromboxanes
• different cell types convert PGH2 to different compounds
• PGH2 Synthase is a heme-containing dioxygenase, bound to ER membranes (a dioxygenase incorporates O2 into a substrate)
• PGH2 Synthase exhibits 2 activities:
– cyclooxygenase
– peroxidase
COOH
COOHO
O
OH
COOHO
O
OOH
2 O2
2 e
arachidonic acid
PGG2
PGH2
Cyclooxygenase
Peroxidase
Prostaglandin synthesis
• PGH2 Synthase (expressing both cyclooxygenase and peroxidase activities) is sometimes referred to as Cyclooxygenase abbreviated COX (the interacting cyclooxygenase and peroxidase reaction pathways are complex)
Prostaglandin synthesis
COOH
COOHO
O
OH
COOHO
O
OOH
2 O2
2 e
arachidonic acid
PGG2
PGH2
Cyclooxygenase
Peroxidase
• 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 O2
COOH
COOHO
O
OH
COOHO
O
OOH
2 O2
2 e
arachidonic acid
PGG2
PGH2
Cyclooxygenase
Peroxidase
• 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)
Prostaglandin H2 Synthase
membrane binding domain
ibuprofen analog
heme
PDB 1PGE Membrane-binding domain: 4 short amphipathic -helices that insert into one leaflet of the bilayer, facing the ER lumen
Ibuprofen and related compounds block the hydrophobic channel by which arachidonate enters the cyclooxygenase active site
NSAIDs such as aspirin and derivatives of ibuprofen, inhibit cyclooxygenase activity of PGH2 Synthas
• they inhibit formation of prostaglandins involved in fever, pain and inflammation
• they inhibit blood clotting by blocking thromboxane formation in blood platelets
CH
COOHH3C
CH2
CH
CH3H3C
Ibuprofen
Non-steroidal anti-inflammatory drugs (NSAIDs)
• 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 PGH2 Synthase would restore cyclooxygenase activity
Aspirin
+
+
PGH2 Synthase (active)
COOH
O C CH3
O
Enz-Ser CH2 OH
COOH
OH
Salicylic acid
Enz-Ser CH2
Acetylated PGH2 Synthase (inactive)
O C CH3
O
Thromboxane A2 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
Thromboxanes
• COX-1 is constitutively expressed at low levels in many cell types
• COX-2 expression is highly regulated
Two isoforms of PGH2 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 PGE2 produced as a result of COX-2 activity itself leads to changes in cAMP levels
• both catalyze PGH2 formation, but differing localization within a cell and localization of enzymes that convert PGH2 into particular prostaglandins/ thromboxanes, may result in COX-1 and COX-2 yielding different ultimate products
Two isoforms of PGH2 Synthase: COX-1 and COX-2
• 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
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 (PGI2) by endothelial cells lining small blood vessels
• lack of inhibition of COX-1-mediated formation of pro-thrombotic thromboxanes in platelets
• some evidence suggests the existence of a third isoform of PGH2 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.
COX-3?
• 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)
leukotrienes
phospholipids arachidonate diacylglycerol
prostaglandin H2
prostacyclins thromboxanes
other prostaglandins
Lipoxyganase
PGH2 Synthase
Prostacyclin Synthase
Thromboxane Synthase
Linear pathway
Cyclic pathway
5-Lipoxygenase (found in leukocytes) catalyzes conversion of arachidonate to 5-HPETE (5-hydroperoxy-eicosatetraenoic acid)
5-HPETE is converted to leukotriene-A4, which in turn may be converted to various other leukotrienes
COOH
COOH
COOH
OOH
O
O2
H2O
Catalyzed by 5-Lipoxygenase:
5-HPETE
arachidonate
leukotriene-A4
Leucotrienes synthesis
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
O2 is thought to approach from the opposite side of the substrate than the iron, for a stereospecific reaction
15-Lipoxygenase PDB 1LOX
membrane-binding domain
substrate analog adjacent to Fe (green)
Lipoxygenase reaction starts with extraction of H from arachidonate, with transfer of the e to the iron, reducing it from Fe3+ Fe2+.
The resulting fatty acid radical reacts with O2 to form a hydroperoxy group
COOH
COOH
OOH
O2
5-HPETE
arachidonate
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
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.
• 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 A2 in association with the nuclear envelope has been observed during activation of leukotriene synthesis in leukocytes
Lipogenase
A b-barrel domain at the N-terminus of Lipoxygenase enzymes may have a role in membrane binding
15-Lipoxygenase PDB 1LOX
membrane-binding domain
substrate analog adjacent to Fe (green)
Cytochrome P450 epoxygenase pathways
• epoxyeicosatrienoic acids (EETs) and hydroxyeicosatrienoic acids are formed from arachidonate by enzymes of the cytochrome P450 family
• other members of the cytochrome P450 family participate in a variety of oxygenation reactions, including hydroxylation of sterols
COOH
O
14,15-EET Arachidonic acid
COOH
• 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)
COOH
O
14,15-EET
EET produced from arachidonate by activity of a cytochrome P450 epoxygenase (14,15-epoxyeicosatrienoic acid)
Cytokines and receptors
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 Kd receptors (10-10-10-12 M)
• cytokines regulate immune responses
• cytokines can activate many cells
• e.g. cytokines secreted by TH 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
Cytokines
Cytokine Categories
• 4 families – Interferons
– Chemokines
– Hematopoietins
– TNF
Hematopoietin family
• -helical structure prevalence
• Little or no b-sheet
• e.g. IL-2 and IL-4
• amino acid sequences vary considerably
Cells that make Cytokines and their Function
• a variety of cells are capable of making cytokines
• the biggest producers are M and TH • cytokines are involved in
– hematopoiesis
– adaptive immunity
– innate immunity
– inflammation
• 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-TH – cytokine concentrations (TH) are high locally
– only interacting APC gets activated
• Answer 3: short half life – short ½ life ensures local activity
Cytokines are Non-Specific
Functional groups of selected cytokines1
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
Hematopoietin Receptor Family (Class I)
• 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
Receptor Signalling (IFNR)
STAT and JAK interaction with selected cytokine receptors during signal transduction
• 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
Cytokine Antagonists
Viral mimics of cytokines and cytokine receptors
• CD4+ TH cells secret a variety of cytokines
• evidence for 2 subsets – TH1
– TH2
• distinction is based on cytokine secretion
• cytokine environment determines which subset will develop – IFN for TH1 (IL-12 and IL-18 from M, DCs)
– IL-4 for TH2
TH1 vs TH2
Cytokine secretion and principal functions of mouse TH1 and TH2 subsets
• T-bet expression results in TH1
• T-bet suppresses TH2
• GATA-3 results In TH2
• GATA-3 suppresses TH1
• IFN- regulates expression of T-bet (Stat 1)
• IL-4 regulates expression of GATA-3 (Stat 6)
Transcription Factors TH1 and TH2