ENZYMATIC and Non-ENZYMATIC LIPID arachidonic acid (AA 20:4 ω-6) PEROXIDATION products RLP Cell Signaling Molecules

     The oxidation of PUFAs (polyunsaturated fatty acids), such as arachidonic acid, generates a broad range of oxidation products which historically have been used as markers of oxidative stress and are Cell signalling RLS (reactive lipid species) which are mediators of multiple pathophysioloical conditions and are not simply as unique biochemical attributes-products cell signalling molecules.
     The Lipid peroxidation products are electrophilic, which allows them to form stable covalent adducts with nucleophilic residues on proteins and selectively modulate protein function.
     The thiol –S-H groups on cysteine residues act as redox switches controlling cell signalling and metabolism.
Oxidative hydrogen peroxide or lipid peroxides to form the markers of oxidative damage and play a role in cell signalling as clear now.
     Early studies implied that lipid peroxidation always results in damage, but now more refined view of this process has evolved and suggests that oxidized lipids can elicit different cellular effects depending on the species present, their concentrations and their reactivity with protein targets. Thats work with two diverse mechanisms:
1. classic reversible binding and
2. irreversible covalent modification of receptors.
     Oxidized lipids are ligands: PGH2, PGE2, PGF2, PGl2 for s. PG (prostaglandin) receptors and mediate biological effects through reversible receptor–ligand interactions. This is best understood for the enzymatically produced PGs and LTs (leukotrienes TXA2, LTC4,LTB4, LTD4, LTE4, Lipoxins).
Lipid peroxidation products modulate cellular activity through irreversible covalent modification of nucleophilic amino acid residues on proteins. Signalling through the covalent modification of proteins is now accepted for a number of well-defined protein–lipid interactions, and selective modified product degradation is mediated through the proteasome.
Signalling through the covalent modification of proteins changes the relationship between the concentration of the RLS ligand molecules can accumulate over time and amplify the signal and even low levels of oxidized lipids RLS initiate strong response signalling. What designated as the covalent Signalling advantage.

How Aspirin and NSAIDs Work

PGHS isoform-1 inactivated by the potent aspirin analogue complexed 2-HYDROXYBENZOIC ACID (SALICYLIC ACID) with O-(bromoacetyl)-L-serine like 2-bromoacetoxy-benzoic acid analog of Aspirin like acylating of Ser530 L-serine residue . Aspirini
Aspirin = ACETYLSALICYLIC ACID
ACETYLSALICYLIC ACID
aspirin analogue complexed SALICYLIC ACID
SALICYLIC ACID
Salicilic Acid 1pth(1PTH), ibuprofen 1EQG, flurbiprofen 3N8Z, IndoMethacin 2OYE
1pthMarz, . . . 1PTHMarz, 1EQGMarz, . . . 3N8ZMarz , . . . . . 2OYEMarz
COX peroxidase PGHS isoform-1 as well
Aspirin and Acetaminophen (trade name Tylenol) are all NSAIDs.
The structural basis of aspirin activity through selective acetylation of serine 530
on prostaglandin H2 synthase inferred from
the crystal structure of inactivated prostaglandin H2
synthase with Salicilic Acid (purple) and
Ser530 brominated (green) or binding Arachidonic Acid as substrate - ligand for prostaglandin H2 (PGH2) production.

1DIYMarz, 3TZIMarz, COX peroxidase PGHS isoform-1 with Arachidonic Acid complexed active site.
1.Nature Structural Biology 1995 Aug;2(8):637-43. 1PTH publication 1995
2.Molecular Pharmacology February 1, 2003 vol. 63no. 2 450-455 1PTH publication 2003
3. Biochemistry.  2001 May 1;40(17):5172-80. 1EQG publication 2001
4.Biochemistry. 2010 August 24; 49(33): 7069-7079. 3N8Y publication 2010
5.September 21, 2007 The Journal of Biological Chemistry, 282, 28096-28105. 2OYE IM8 publication 2007
6.Science 15 September 2000: Vol. 289 no. 5486 pp. 1933-1937 1DIY publication 2000 7.July 13, 2012 The Journal of Biological Chemistry, 287,24619-24630. 3TZI publication 2012
Prostaglandins and NSAIDS
Prostaglandins are potent mediators of inflammation.
The first and committed step in the production
of prostaglandins from arachidonic acid is the
bis-oxygenation of arachindonate to prostaglandin PGG2.
This is followed by reduction to PGH2 in a peroxidase reaction.
Both these reactions are catalyzed by cyclooxygenase, also
known as PGH synthase.

Cyclooxygenase (COX) is shown as structures inhibited by the family of drugs known as non-steroidal anti-inflammatory drugs or NSAIDs. Aspirin, ibuprofen, flurbiprofen and acetaminophen (trade name Tylenol) are all NSAIDs. Salicilic Acid, Aspirini, ibuprofen, flurbiprofen, IndoMethacin, Tylenol

There are two isoforms of COX in animals: COX-1, which carries out normal, physiological production of prostaglandins, and COX-2, which is induced by cytokines, mitogens and endotoxins in inflammatory cells, and which is responsible for the production of prostaglandins in inflammation.

The structure shown at left is that of COX-1 from sheep, inactivated by bromoaspirin, the structure of which is shown. BromAspirin

The Enzyme Structure

The first 24 residues of COX-1 are a signal sequence. This domain is removed in the mature enzyme and will not be discussed here. Similarly , residues 25-32 do not yield interpretable electron density, and are not shown in the structure shown.

The remaining 551 residues of the enzyme (residues 33-583) comprise three distinct domains. The first of these, residues 33-72, form a small compact module that is similar to epidermal growth factor .

The second domain, composed of residues 73-116, forms a right-handed spiral of four yellowalpha-helical segments along one side of the protein . This domain of the protein forms a membrane-binding motif. The helical segments are amphipathic, with most of the hydrophobic residues (shown in green ) facing away from the protein, where they can interact with a lipid bilayer.

Turn off the hydrophobic residues and we will consider the third domain of the COX enzyme, the catalytic domain (in blue), a globular structure that contains both the cyclooxygenase and peroxidase active sites .

The peroxidase site includes a heme .

The iron(III) in the center of this heme is coordinated by proximal His-388
and by distal His-207 .

Let's return to our view of the whole molecule and consider the cyclooxygenase active site.
The cyclooxygenase active site lies at the end of a long, narrow, hydrophobic tunnel or channel. Three of the yellow alpha helices of the membrane-binding domain lie at the entrance to this tunnel.
The walls of the tunnel are defined by four alpha helices, formed by residues
106-123, 325-353,379-384, and 520-535. In the following animation,
these helices will flash red and orange.
Aliphatic-Hydrophobic Amino Acids colored whith surroundings for Arachidonic Acid inhibited by anti-inflamation drugs bound in the tunnel.

In this bromoaspirin-inactivated structure, Ser-530 is bromoacetylated or ACD and bound a molecule :
1. of salicylate in 1pthMarz is bound in the tunnel
2. of ibuprofen in 1EQGMarz is bound in the tunnel
3. of flurbiprofen in 3N8ZMarz is bound in the tunnel
4. of IndoMethacin in 2OYEMarz is bound in the tunnel
5. of arachidonic acid in 1DIYMarz is bound in the tunnel
6. of arachidonic acid in 3TZIMarz is bound in the tunnel

With this view, it should also be clear why aspirin and other NSAIDs block in COX enzyme molecule the synthesis of product PGH2 prostaglandin H2 as precursor for next synthesis of prostaglandin D2 PGD2. In various ways, they all act by filling and blocking the tunnel, preventing the migration of arachidonic acid to the active site at the back of the tunnel for enzyme controled radical-chain reaction of peroxidation -O-O-
There are thought to be at least four different mechanisms of action for NSAIDs.
1. Aspirin Aspirin (and also bromoaspirin) is the only one which covalently modifies - Acylate a Ser530 L-serine residue as O-(bromoacetyl)-L-serine in the tunnel, thus irreversibly inactivating both COX-1 and COX-2.
2.Ibuprofen acts instead by competing in a reversible fashion for the substrate binding site in the tunnel.
3. Flurbiprofen and IndoMethacin, members of the third class of inhibitors.Fluribiprofen and indomethacin cause a slow, time-dependent inhibition of COX-1 and COX-2, apparently via formation of a salt bridge between a carboxylate on the drug and Arg-120 (shown here in green), which lies in the tunnel .
4. The drug SC-558 acts by a fourth mechanism, specifically inhibiting COX-2. It is a weak competitive inhibitor of COX-1 but inhibits COX-2 in a slow, time-dependent process. Specific COX-2 inhibitors will likely be the drugs of the future, since they will be able to selectively block the inflammation mediated by COX-2, without the potential for stomach lesions and renal toxicity that arise from COX-1 inhibition.
Warfarin, Aspirin, BromAspirin molecule of anti-cloting human blood medicine
Warfarin , Aspirin (and also bromoaspirin) medical aplication to prevent Cardiovascular disease at pathological inflamation condition: atherosclerotic lesions, hart in stike, insults, which predominantly at inflamation are non-enzymatic, non-specific lipid peroxidation occurs in vivo.
Aspirin and warfarin transporter in blood plasma is albumin as often designated wie lipoprotein.
2BXDMarz poprotein 99% complexed 1% small molecules which are water insoluble like a fatts.
8.Journal of Molecular Biology Volume 353, Issue 1, 14 October 2005, Pages 38–52 RWF publication 2012
References
1. Biochem. J. (2012) 442 (453–464) (Printed in Great Britain)
2.Nature Structural Biology 1995 Aug;2(8):637-43. 1pth
3. Molecular Pharmacology February 1, 2003 vol. 63no. 2 450-455 1PTH
4. Biochemistry. 2001 May 1;40(17):5172-80. 1EQG
5. Biochemistry. 2010 August 24; 49(33): 7069-7079. 3N8Y
6. September 21, 2007 The Journal of Biological Chemistry, 282, 28096-28105. 2OYE IM8
7. Science 15 September 2000: Vol. 289 no. 5486 pp. 1933-1937 1DIY
8. ournal of Molecular Biology Volume 353, Issue 1, 14 October 2005, Pages 38–52 RWF
Original source MDL:Eric Marz (1997) Massachusetts University USA