In Silico Analysis of Functionally Significant Non Synonymous Single Nucleotide Polymorphisms (nsSNPs) of NOS3 Gene in Cardiovascular Diseases

Abstract

NOS3 gene encodes the enzyme endothelial nitric oxide synthase (eNOS) that is involved in cardiovascular homeostasis. Defects in eNOS have been associated with many cardiovascular diseases. Missense non-synonymous single nucleotide polymorphisms (nsSNPs) of NOS3 have the potential to damage eNOS and cause disease. This study aimed to identify the most damaging missense nsSNPs i.e., functionally significant of NOS3 and study their effect on the structure and function of eNOS through in silico approaches. Deleterious nsSNPs were predicted using PredictSNP. Out of these nsSNPs, the ones that replace amino acids determined as conserved by ConSurf were considered to be the most damaging. Upon analyzing the impact of these 48 nsSNPs on eNOS stability through I-mutant, 44 were found to be destabilizing. HOPE found the 48 nsSNPs to replace wild-type amino acids with amino acids having different properties, affecting protein structure and function. 41 nsSNPs reside in domains and can prevent their functioning. Two nsSNPs can affect domain functioning through loss of phosphorylation sites predicted there. 48 mutated eNOS structures corresponding to the nsSNPs were generated using Phyre2 and structurally compared with native eNOS structure through TM-align. A proline to serine substitution at position 1002 (rs1563233862) caused the greatest structural deviation from normal eNOS, followed by a glycine to leucine substitution at position 1115 (rs746892263). 3D structures of native eNOS and these two mutants were modeled by I-TASSER. The mutant and native structures were superimposed, showing a clear structural difference between them. This can be attributed to the loss of important structural properties of proline and glycine. These findings can be validated by experimental studies. Such studies can also further explore the effect of these nsSNPs on eNOS and their involvement in eNOS related diseases. This will help in better understanding of the role of eNOS in disease and development of therapeutic strategies.