Identification of the two-phase mechanism of arachidonic acid regulating inflammatory prostaglandin E2 biosynthesis by targeting COX-2 and mPGES-1

Abstract

Through linking inducible cyclooxygenase (COX)-2 with microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1), a Single-Chain Enzyme Complex (SCEC, COX-2-10aa-mPGES-1) was engineered to mimic a specific inflammatory PGE2 biosynthesis from omega-6 fatty acid, arachidonic acid (AA), by eliminating involvements of non-inducible COX-1 and other PGE2 synthases. Using the SCEC, we characterized coupling reactions between COX-2 and mPGES-1 at 1:1 ratio of inflammatory PGE2 production. AA demonstrated two phase activities to regulate inflammatory PGE2 production. In the first phase (<2 μM), AA was a COX-2 substrate and converted to increasing production of PGE2. In the second phase with a further increased AA level (2-10 μM), AA bound to mPGES-1 and inhibited the PGE2 production. The SCEC study was identical to the co-expression of COX-2 and mPGES-1. This was further confirmed by using mPGES-1 and PGH2 as a direct enzyme target and substrate, respectively. Furthermore, the carboxylic acid group of AA binding to R67 and R70 of mPGES-1 was identified by X-ray structure-based docking and mutagenesis. mPGES-1 mutants, R70A, R70K, R67A and R67K, lost 40-100% binding to [(14)C]-AA. To conclude, a cellular model, in which AA is involved in self-controlling initial initiating and later resolving inflammation by its two phase activities, was discussed.

Screening the active compounds of Phellodendri Amurensis cortex for treating prostate cancer by high-throughput chinmedomics

“…Arachidonic acid, Thromboxane, 16(R)-HETE, Prostaglandin A1, Prostaglandin A2, and 8-Isoprostane are involved in arachidonic acid metabolism. Arachidonic acid and its metabolites, as the main inflammatory substances, are taking part in the process of immune and inflammatory actions6061. Thromboxane could promote cancer cell proliferation through enhancing the activity of cell mitosis6263.…”

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“…Platelets also have thromboxane A synthase 1, which has been confirmed by an inhibition experiment (19). Phospholipids in the cell membrane are rich in arachidonic acid so when the cells are stimulated by external stimuli, including bradykinin, thrombin, antigen-antibody complexes or other pathological factors, a number of which have not yet been characterized, the phospholipase A2 in cell membrane will be activated, consequently hydrolyzing the phospholipids and releasing arachidonic acid, which may then synthesize PGs under a series of enzymes including COX-2 and PGs synthetase (20). In the majority of normal tissues, PG is not expressed; however, under the stimuli of factors such as cytokines, growth factors, oncogenes or tumor-promoting agents, it may be upregulated and associated with the occurrence and development of tumors (21).…”

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“…Therefore, a change in the distribution of PGH 2 to the particular isozyme is the key to control the metabolism of AA into the specific prostanoid. In recent years, using an enzymatic engineering approach to control the distribution of PGH 2 has been focused by our group to address this issue . In our previous studies, we have successfully created a single‐chain hybrid enzyme complex (SCHEC), COX‐1‐10aa‐PGIS, through the enzymatic engineering approach, which can force AA to be isomerized into PGI 2 , in order to rescue the deficiency of PGI 2 and to study the vascular protection effects of PGI 2 in cellular and animal models .…”

“…In our previous studies, we have successfully created a single‐chain hybrid enzyme complex (SCHEC), COX‐1‐10aa‐PGIS, through the enzymatic engineering approach, which can force AA to be isomerized into PGI 2 , in order to rescue the deficiency of PGI 2 and to study the vascular protection effects of PGI 2 in cellular and animal models . Another SCHEC, COX‐2‐10aa‐mPGES‐1, which can effectively pass PGH 2 to mPGES‐1, to convert AA to PGE 2 , has also been created as a model for understanding how PGE 2 is biosynthesized during inflammation . In this study, we created a novel SCHEC, linking the C‐terminus of COX‐1 to the N‐terminus of the TXAS, through a 10‐residue amino acid linker, to directly guide the metabolism of AA to TXA 2 by effectively passing the COX‐1 produced PGH 2 to TXAS.…”

“…In recent years, using an enzymatic engineering approach to control the distribution of PGH 2 has been focused by our group to address this issue. [10][11][12][13][14][15][16][17] In our previous studies, we have successfully created a single-chain hybrid enzyme complex (SCHEC), COX-1-10aa-PGIS, through the enzymatic engineering approach, which can force AA to be isomerized into PGI 2 , in order to rescue the deficiency of PGI 2 and to study the vascular protection effects of PGI 2 in cellular and animal models. [10][11][12][13] Another SCHEC, COX-2-10aa-mPGES-1, which can effectively pass PGH 2 to mPGES-1, to convert AA to PGE 2 , has also been created as a model for understanding how PGE 2 is biosynthesized during inflammation.…”