Biomedical subjects

Ke-He Ruan

Publications and source records attributed to Ke-He Ruan.

23 records · Page 2Linked to original sources

PubMed2003

Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach.

The second extracellular loop (eLP2) of the thromboxane A(2) receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional (1)H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280). To further identify the residues involved in ligand binding, a TP receptor antagonist, SQ29,548 was used to interact with the synthetic peptide. High resolution two-dimensional (1)H NMR experiments, NOESY, and TOCSY were performed for the peptide, SQ29,548, and peptide with SQ29,548, respectively. Through completed (1)H NMR assignment and by comparing the different spectra, extra peaks were observed on the NOESY spectrum of the peptide with SQ29,548, which implied the contacts between residues of eLP2 at Val(176), Leu(185), Thr(186), and Leu(187) with SQ29,548 at position H2, H7, and H8. Site-directed mutagenesis was used to confirm the possible ligand-binding sites on native human TP receptor. Each of the four residues was mutated to the residues either in the same group, with different structure or different charged. The mutated receptors were then tested for their ligand binding activity. The receptor with V176L mutant retained binding activity to SQ29,548. All other mutations resulted in decreased or lost binding activity to SQ29,548. These mutagenesis results supported the prediction from NMR experiments in which Val(176), Leu(185), Thr(186), and Leu(187) are the possible residues involved in ligand binding. This information facilitates the understanding of the molecular mechanism of thromboxane A(2) binding to the important receptor and its signal transduction.

Amino Acid Sequence↗

PubMed2002

Solution structure and topology of the N-terminal membrane anchor domain of a microsomal cytochrome P450: prostaglandin I2 synthase.

The three-dimensional structure of a synthetic peptide corresponding to the N-terminal membrane anchor domain (residues 1-25) of prostaglandin I(2) synthase (also known as cytochrome P450 8A1), an eicosanoid-synthesizing microsomal cytochrome P450, has been determined by two-dimensional (1)H NMR spectroscopy in trifluoroethanol and dodecylphosphocholine which mimic the hydrophobic membrane environment. A combination of two-dimensional NMR experiments, including NOESY, TOCSY and double-quantum-filtered COSY, was used to obtain complete (1)H NMR assignments for the peptide. Using the NOE data obtained from the assignments and simulated annealing calculations, the N-terminal membrane domain reveals a bent-shaped structure comprised of an initial helix (residues 3-11), followed by a turn (residues 12-16) and a further atypical helix (residues 17-23). The hydrophobic side chains of the helix and turn segments (residues 1-20) are proposed to interact with the hydrocarbon interior of the phospholipid bilayer of the endoplasmic reticulum (ER) membrane. The hydrophilic side chains of residues 21-25 (Arg-Arg-Arg-Thr-Arg) point away from the hydrophobic residues 1-20 and are expected to be exposed to the aqueous environment on the cytoplasmic side of the ER membrane. The distance between residues 1 and 20 is approx. 20 A (1 A=0.1 nm), less than the thickness of a lipid bilayer. This indicates that the N-terminal membrane anchor domain of prostaglandin I(2) synthase does not penetrate the ER membrane.

Amino Acid Sequence↗

PubMed2002

Purification and characterization of a cyclooxygenase-2 and angiogenesis suppressing factor produced by human fibroblasts.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that plays an important role in several pathophysiological processes, including inflammation, angiogenesis, and tumorigenesis. We have recently observed that COX-2 induction is restrained in proliferating fibroblasts. The mechanism by which this occurs is unclear. Here, we report the detection and isolation from the conditioned medium of proliferating fibroblasts a factor that suppressed COX-2 expression. This factor, which was named cytoguardin, suppressed COX-2 protein levels induced by phorbol 12-myristate 13-acetate, interleukin-1beta, tumor necrosis factor alpha, and lipopolysaccharide (LPS) in fibroblasts and LPS-induced COX-2 protein levels and promoter activities in human endothelial cells and murine RAW 264.7 cells in a comparable concentration-dependent manner. It inhibited COX-2 expression induced by angiogenic factors and endothelial tube formation induced by angiogenic factors and colon cancer cell medium. These findings provide evidence for the control of COX-2 transcription by an endogenous cellular factor.

Angiogenesis Inhibitors↗

PubMed2002

Substrate access channel topology in membrane-bound prostacyclin synthase.

Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I(2) synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66-75 (P66-75) and 95-116 (P95-116)], and two other peptides corresponding to the surface about 10-20 A (1 A=0.1 nm) away from the opening [residues 366-382 (P366-382) and 472-482 (P472-482)] were used to prepare site-specific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the accessibility of the substrate access channel opening in transiently transfected COS-1 cells expressing recombinant human PGIS, and in spontaneously transformed human endothelial cell line ECV cells expressing endogenous human PGIS. Immunofluorescence staining was performed for cells selectively permeabilized with streptolysin O and for cells whose membranes were permeabilized with detergent. Antibodies to peptides in the immediate vicinity of the substrate channel (P66-75 and P95-116) bound to their targets only after general permeabilization with Triton X-100. In contrast, the two antibodies to peptides further from the channel opening (P366-382 and P472-482) bound to their targets even in cells with intact ER membranes. These observations support our topology model in which the PGIS substrate access channel opening is positioned close to the ER membrane.

Animals↗

PubMed2002

Prostacyclin is an autocrine regulator in the contraction of oviductal smooth muscle.

BACKGROUND: It was recently discovered that prostacyclin constituted 40-50% of prostaglandins (PG) produced by minced human oviduct. It is well established that prostacyclin relaxes vascular smooth muscle, but whether oviductal smooth muscle synthesizes prostacyclin and whether its contraction is affected by prostacyclin remain unclear. METHODS: Smooth muscle microdissected from human oviducts was used for the study. The expression of prostacyclin synthase (PGIS) and prostacyclin receptor (IP) was confirmed by Western blot analysis. Metabolites of [(3)H]PGH(2) were analysed for prostacyclin. Functional coupling of IP to adenyl cyclase was assessed by the accumulation of intracellular cAMP upon prostacyclin challenge. The presence of saturable, specific binding sites for prostacyclin was confirmed by binding assay. The identity of IP was further confirmed by RT-PCR and nucleotide sequence analysis. Finally, the effects of prostacyclin on muscle contraction were studied. RESULTS: Human oviductal smooth muscle expresses functionally active PGIS and IP. The IP expressed is the same as that cloned from human lung tissue. The ED(50) of prostacyclin to increase intracellular cAMP was 16 nmol/l. Prostacyclin dose-dependently decreased the amplitude of muscle contraction. CONCLUSIONS: Human oviductal smooth muscle produces prostacyclin, which, in turn, decreases its contractility. Prostacyclin may regulate embryo transport.

Adenylyl Cyclases↗