Analysis of plasma myeloperoxidase levels and functional gene –463G>A and –129G>A polymorphisms with early onset of coronary artery disease in South Indian population

Introduction. The present investigation is pursued to study the possible association of –463G>A and –129G>A polymorphism in MPO gene and assessment of plasma MPO levels with the risk of developing coronary artery disease.Material and methods. A total of 200 angiographically documented CAD patients and 200 age, gender ethnicity matched healthy controls were recruited for the study. Plasma MPO levels were assessed using enzyme-linked immunosorbent assay (ELISA) kit and genotypes were determined by PCR-RFLP technique.Results. The MPO levels were found to be significantly increased in CAD patients when compared with controls (p < 0.04) but there were no significant effect of –463G>A gene polymorphism on MPO levels. A significant association of –463G>A polymorphism was observed with coronary artery disease. The frequency of recessive genotype “AA” at –463 promoter site was considerably lesser in patients (4%) relative to controls (11%) (odds ratio [OR] = 0.3371, 95% confidence interval [CI] 0.1463–0.7766, p = 0.012). However we did not find significant association of –129G>A polymorphism with CAD. Additionally, haplotype analysis revealed that single nucleotide polymorphisms (SNP) 1 of AA genotype and SNP 2 of GG genotype showed significant protective effect with disease (OR = 0.64; 95% CI [0.42–0.96], p = 0.032).Conclusion. The results revealed that –463G>A polymorphism in the MPO gene lowers the CAD related condition in patients by down regulating serum MPO concentration, which is known to aggravate the atherosclerotic events observed in CAD

Molecular dynamic simulations reveal suboptimal binding of salbutamol in T164I variant of β2 adrenergic receptor.

The natural variant C491T (rs1800088) in ADRB2 gene substitutes Threonine to Isoleucine at 164th position in β2AR and results in receptor sequestration and altered binding of agonists. Present investigation pursues to identify the effect of T164I variation on function and structure of β2AR through systematic computational approaches. The study, in addition, addresses altered binding of salbutamol in T164I variant through molecular dynamic simulations. Methods involving changes in free energy, solvent accessibility surface area, root mean square deviations and analysis of binding cavity revealed structural perturbations in receptor to incur upon T164I substitution. For comprehensive understanding of receptor upon substitution, OPLS force field aided molecular dynamic simulations were performed for 10 ns. Simulations revealed massive structural departure for T164I β2AR variant from the native state along with considerably higher root mean square fluctuations of residues near the cavity. Affinity prediction by molecular docking showed two folds reduced affinity of salbutamol in T164I variant. To validate the credibility docking results, simulations for ligand-receptor complex were performed which demonstrated unstable salbutamol-T164I β2AR complex formation. Further, analysis of interactions in course of simulations revealed reduced ligand-receptor interactions of salbutamol in T164I variant. Taken together, studies herein provide structural rationales for suboptimal binding of salbutamol in T164I variant through integrated molecular modeling approaches

The overlapping cartoon depicts binding cavities of wild (green solid) and T164I (red mesh) β2AR.

<p>Volume of the cavity in wild β2AR is 404.48 ų, upon substitution the cavity expands to 520.70 ų. Poses of salbutamol (Sea green in wild, and pink in T164I variant) are shown in the binding cavity.</p

Calculation of binding site properties of wild and T164I variant β2AR.

<p>Calculation of binding site properties of wild and T164I variant β2AR.</p

Energy descriptors determining the binding efficiency of salbutamol in wild and T164I variant.

<p>Energy descriptors determining the binding efficiency of salbutamol in wild and T164I variant.</p

Contact fractions of ligand-receptor interactions in course of simulation.

<p>Simulation interaction diagram showing contact fractions of residues interacting with salbutamol in (A) wild and (B) T164I variant of β2AR.</p

Structural perturbation incurred in β2AR upon T164I variation analyzed by simulations.

<p>(A) RMSD calculated for 10 ns of simulation trajectory for wild and T64I β2AR. β2AR is rendered with high conformational flexibility upon T164I substitution as observed from gradual rise in RMSD after 2.7 ns for T64I β2AR. (B) RMSF calculated for residues in wild and T164I variant. Purple bars indicate active site residues; arrow pointing the peak represents the site of variation. (C) Compactness of wild and T164I β2AR assessed by calculating radius of gyration. Trajectory lines for wild and T164I β2AR are represented in red and black color respectively.</p

Timeline representation of ligand—Receptor interactions.

<p>Residues interacting (all the interactions including H-bonds, Hydrophobic, Ionic, Water bridges) with salbutamol in (A) wild and (B) T164I variant of β2AR.</p

Number of H bonds contacts established by salbutamol with wild (red) and T164I β2AR (black) in each frame of simulation.

<p>Reduced H bond contacts for T164I variant can be observed.</p

Trajectory analysis of salbutamol in complex with wild and T164I β2AR.

<p>(A) RMSD of salbutamol with respect to the reference conformation in wild and T164I β2AR. (B) ‘Fit on protein' line shows atomic fluctuations (RMSF) with respect to the receptor. Corresponding atoms of salbutamol is shown as 2D structure in the top panel. (C) Solvent accessible surface area of salbutamol in course of simulation. Trajectory lines for wild and T164I β2AR are represented in red and black color respectively.</p