Vanessa Cervantes

Engineering of a novel hybrid enzyme: an anti-inflammatory drug target with triple catalytic activities directly converting arachidonic acid into the inflammatory prostaglandin E2

Cyclooxygenase isoform-2 (COX-2) and microsomal prostaglandin E(2) synthase-1 (mPGES-1) are inducible enzymes that become up-regulated in inflammation and some cancers. It has been demonstrated that their coupling reaction of converting arachidonic acid (AA) into prostaglandin (PG) E(2) (PGE(2)) is responsible for inflammation and cancers. Understanding their coupling reactions at the molecular and cellular levels is a key step toward uncovering the pathological processes in inflammation. In this paper, we describe a structure-based enzyme engineering which produced a novel hybrid enzyme that mimics the coupling reactions of the inducible COX-2 and mPGES-1 in the native ER membrane. Based on the hypothesized membrane topologies and structures, the C-terminus of COX-2 was linked to the N-terminus of mPGES-1 through a transmembrane linker to form a hybrid enzyme, COX-2-10aa-mPGES-1. The engineered hybrid enzyme expressed in HEK293 cells exhibited strong triple-catalytic functions in the continuous conversion of AA into PGG(2) (catalytic-step 1), PGH(2) (catalytic-step 2) and PGE(2) (catalytic-step 3), a pro-inflammatory mediator. In addition, the hybrid enzyme was also able to directly convert dihomo-gamma-linolenic acid (DGLA) into PGG(1), PGH(1) and then PGE(1) (an anti-inflammatory mediator). The hybrid enzyme retained similar K(d) and V(max) values to that of the parent enzymes, suggesting that the configuration between COX-2 and mPGES-1 (through the transmembrane domain) could mimic the native conformation and membrane topologies of COX-2 and mPGES-1 in the cells. The results indicated that the quick coupling reaction between the native COX-2 and mPGES-1 (in converting AA into PGE(2)) occurred in a way so that both enzymes are localized near each other in a face-to-face orientation, where the COX-2 C-terminus faces the mPGES-1 N-terminus in the ER membrane. The COX-2-10aa-mPGES-1 hybrid enzyme engineering may be a novel approach in creating inflammation cell and animal models, which are particularly valuable targets for the next generation of NSAID screening.

An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector

It remains a challenge to achieve the stable and long‐term expression (in human cell lines) of a previously engineered hybrid enzyme [triple‐catalytic (Trip‐cat) enzyme‐2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003–14011], which links cyclo‐oxygenase isoform‐2 (COX‐2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s Trip‐cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)‐mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip‐cat enzyme, in which human cyclo‐oxygenase isoform‐1, which has a different C‐terminal sequence from COX‐2, was linked to PGI2 synthase and called Trip‐cat enzyme‐1. Transient expression of recombinant Trip‐cat enzyme‐1 in HEK293 cells led to 3–5‐fold higher expression capacity and better PGI2‐synthesizing activity as compared to that of the previously engineered Trip‐cat enzyme‐2. Furthermore, an HEK293 cell line that can stably express the active new Trip‐cat enzyme‐1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase‐rich plasma. This study has optimized engineering of the active Trip‐cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2‐producing therapeutic cell line for use against vascular diseases.

Novel Mechanism of the Vascular Protector Prostacyclin: Regulating MicroRNA Expression

Prostacyclin (PGI(2)) is a key vascular protector, metabolized from endogenous arachidonic acid (AA). Its actions are mediated through the PGI(2) receptor (IP) and nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ). Here, we found that PGI(2) is involved in regulating cellular microRNA (miRNA) expression through its receptors in a mouse adipose tissue-derived primary culture cell line expressing a novel hybrid enzyme gene (COX-1-10aa-PGIS), cyclooxygenase-1 (COX-1) and PGI(2) synthase (PGIS) linked with a 10-amino acid linker. The triple catalytic functions of the hybrid enzyme in these cells successfully redirected the endogenous AA metabolism toward a stable and dominant production of PGI(2). The miRNA microarray analysis of the cell line with upregulated PGI(2) revealed a significant upregulation (711, 148b, and 744) and downregulation of miRNAs of interest, which were reversed by antagonists of the IP and PPARγ receptors. Furthermore, we also found that the insulin-mediated lipid deposition was inhibited in the PGI(2)-upregulated adipocytes. The study also initiated a discussion that suggested that the endogenous PGI(2) inhibition of lipid deposition in adipocytes could involve miRNA-mediated inhibition of expression of the targeted genes. This indicated that PGI(2)-miRNA regulation could exist in broad pathophysiological processes involving PGI(2) (i.e., apoptosis, vascular inflammation, cancer, embryo implantation, and obesity).

Ligand-specific conformation determines agonist activation and antagonist blockade in purified human thromboxane A2 receptor.

The binding of an agonist to a G protein-coupled receptor (GPCR) causes its coupling to different G proteins, which mediate signaling. However, the binding of an antagonist to the same site of the GPCR could not induce coupling. To understand the molecular mechanism involved, the structural flexibility of the purified human thromboxane A2 receptor (TP) was characterized by spectroscopic approaches, while bound to an agonist or antagonist. Circular dichroism not only revealed that the purified TP adopted more than 50% helical conformation in solution but also showed that the antagonist, SQ29,548, could induce more of a beta-sheet structure in the TP than that of the agonist, U46619. Also, fluorescence studies showed that the antagonist induced the intrinsic Trp fluorescence signal change more than the agonist. Furthermore, three of the nine tryptophan residues involved in the different ligand-based structural changes were demonstrated by NMR spectroscopy. Low pH-induced changes in the receptor conformation and molecular interaction field dramatically increased the agonist binding but did not significantly affect the antagonist binding. Different conformational changes were also observed in the TP reconstituted into phosphatidylcholine/phosphatidylserine/phosphatydylethanolamine-formed liposomes. These studies are the first to show a possible mechanism of the ligand-specific conformation-dependent agonist activation and antagonist blockage in the GPCR.

A simple, quick and high-yield preparation of the human thromboxane A2 receptor in full size for structural studies

Human thromboxane A2 receptor (TP), a G protein-coupled receptor (GPCR), is one of the most promising targets for developing the next generation of anti-thrombosis and hypertension drugs. However, obtaining a sufficient amount of the full-sized and active membrane protein has been the major obstacle for structural elucidation that reveals the molecular mechanisms of the receptor activation and drug designs. Here we report an approach for the simple, quick, and high-yield preparation of the purified and active full-sized TP in an amount suitable for structural studies. Glycosylated human TP was highly expressed in Sf-9 cells using an optimized baculovirus (BV) expression system. The active receptor was extracted and solubilized by different detergents for comparison and was finally purified to a nearly single band with a ratio of 1:0.9 +/- 0.05 (ligand:receptor molecule) in ligand binding using a Ni column with a relatively low yield. However, a high-yield purification (milligram quantity) of the TP protein, from a modulate scale of transfected Sf-9 cell culture, has been achieved by quick and simple purification steps, which include DNA digestion, dodecyl-maltoside detergent extraction, centrifugation, and FPLC purification. The purity and quantity of the purified TP, using the high-yield approach, were suitable for protein structural studies as evidenced by SDS-PAGE, Western blot analyses, ligand binding assays, and a feasibility test using high-resolution one-dimensional and two-dimensional (1)H NMR spectroscopic analyses. These studies provide a basis for the high-yield expression and purification of the GPCR for the structural and functional characterization using biophysics approaches.

A profile of the residues in the second extracellular loop that are critical for ligand recognition of human prostacyclin receptor.

The residues in the second extracellular loop (eLP2) of the prostanoid receptors, which are important for specific ligand recognition, were previously predicted in our earlier studies of the thromboxane A2 receptor (TP) using a combination of NMR spectroscopy and recombinant protein approaches. To further test this hypothesis, another prostanoid receptor, the prostacyclin receptor (IP), which has opposite biological characteristics to that of TP, was used as a model for these studies. A set of recombinant human IPs with site‐directed mutations at the nonconserved eLP2 residues were constructed using an Ala‐scanning approach, and then expressed in HEK293 and COS‐7 cells. The expression levels of the recombinant receptors were six‐fold higher in HEK293 cells than in COS‐7 cells. The residues important for ligand recognition and binding within the N‐terminal segment (G159, Q162, and C165) and the C‐terminal segment (L172, R173, M174, and P179) of IP eLP2 were identified by mutagenesis analyses. The molecular mechanisms for the specific ligand recognition of IP were further demonstrated by specific site‐directed mutagenesis using different amino acid residues with unique chemical properties for the key residues Q162, L172, R173, and M174. A comparison with the corresponding functional residues identified in TP eLP2 revealed that three (Q162, R173, and M174) of the four residues are nonconserved, and these are proposed to be involved in specific ligand recognition. We discuss the importance of G159 and P179 in ligand recognition through configuration of the loop conformation is discussed. These studies have further indicated that characterization of the residues in the eLP2 regions for all eight prostanoid receptors could be an effective approach for uncovering the molecular mechanisms of the ligand selectivities of the G‐protein‐coupled receptors.

Structural and functional analysis of the C-terminus of Galphaq in complex with the human thromboxane A2 receptor provides evidence of constitutive activity.

The human thromboxane A(2) (TXA(2)) receptor (TP) is known to mediate platelet aggregation and vasoconstriction. The receptor predominantly interacts with the Gq protein, thereby activating phospholipase C and increasing the intracellular calcium level. In this study, we synthesized a 15-residue peptide corresponding to the C-terminal domain of the Gq protein alpha subunit (Galphaq-Ct peptide) and characterized its interaction with recombinant TP purified from a baculovirus expression system in the presence and absence of an agonist using fluorescence and NMR spectroscopic studies. With fluorescence binding assays, we demonstrated that the Galphaq-Ct peptide was bound to TP, in the absence of the agonist, with a K(d) value of approximately 17 muM. Interestingly, upon addition of the agonist, U46619, the Galphaq-Ct peptides binding affinity for this activated TP was reduced, thereby increasing the K(d) value to approximately 240 muM. NMR experiments demonstrated that the TP-bound Galphaq-Ct peptide shows a different affinity and conformation, in the absence and presence of the agonist, U46619. This suggested there is the possibility of ligand-free constitutive TP signaling through Galpha binding. Thus, an HEK293 cell line that stably expresses human TP and lacks the ability to produce TXA(2) was created by gene transfer and G418 selection. In comparison with the control cells, the stable cell line showed significant Galpha-mediated ligand-free calcium signaling. The study indicates a promising new outlook for the examination of prostanoid receptor-G-protein interactions in greater detail using integrated NMR spectroscopy, the purified receptor, and the stable cell line.

An active triple-catalytic hybrid enzyme engineered by linking COX-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector

It remains a challenge to achieve the stable and long‐term expression (in human cell lines) of a previously engineered hybrid enzyme [triple‐catalytic (Trip‐cat) enzyme‐2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003–14011], which links cyclo‐oxygenase isoform‐2 (COX‐2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s Trip‐cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)‐mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip‐cat enzyme, in which human cyclo‐oxygenase isoform‐1, which has a different C‐terminal sequence from COX‐2, was linked to PGI2 synthase and called Trip‐cat enzyme‐1. Transient expression of recombinant Trip‐cat enzyme‐1 in HEK293 cells led to 3–5‐fold higher expression capacity and better PGI2‐synthesizing activity as compared to that of the previously engineered Trip‐cat enzyme‐2. Furthermore, an HEK293 cell line that can stably express the active new Trip‐cat enzyme‐1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase‐rich plasma. This study has optimized engineering of the active Trip‐cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2‐producing therapeutic cell line for use against vascular diseases.

An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector: Prostacyclin-synthesizing protein with COX-1 and PGIS properties

It remains a challenge to achieve the stable and long‐term expression (in human cell lines) of a previously engineered hybrid enzyme [triple‐catalytic (Trip‐cat) enzyme‐2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003–14011], which links cyclo‐oxygenase isoform‐2 (COX‐2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s Trip‐cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)‐mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip‐cat enzyme, in which human cyclo‐oxygenase isoform‐1, which has a different C‐terminal sequence from COX‐2, was linked to PGI2 synthase and called Trip‐cat enzyme‐1. Transient expression of recombinant Trip‐cat enzyme‐1 in HEK293 cells led to 3–5‐fold higher expression capacity and better PGI2‐synthesizing activity as compared to that of the previously engineered Trip‐cat enzyme‐2. Furthermore, an HEK293 cell line that can stably express the active new Trip‐cat enzyme‐1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase‐rich plasma. This study has optimized engineering of the active Trip‐cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2‐producing therapeutic cell line for use against vascular diseases.