RESUMO
Pyruvate kinase (PKLR) is a potential candidate gene for milk production traits in cows. The main aim of this work is to investigate the potentially deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) in the PKLR gene by using several computational tools. In silico tools including SIFT, Polyphen-2, SNAP2 and Panther indicated only 18 nsSNPs out of 170 were considered deleterious. The analysis of proteins' stability change due to amino acid substitution performed by the use of the I-mutant, MUpro, CUPSTAT, SDM and Dynamut confirmed that 9 nsSNPs decreased protein stability. ConSurf analysis predicted that all 18 nsSNPs were evolutionary moderately or highly conserved. Two different domains of PKLR protein were revealed by the InterPro tool with 12 nsSNPs positioned in the Pyruvate Kinase barrel domain and 6 nsSNP present in the Pyruvate Kinase C Terminal. The PKLR 3D model was predicted by MODELLER software and validated via Ramachandran plot and Prosa which indicated a good quality model. The analysis of energy minimizations for the native and mutated structures was performed by SWISS PDB viewer with GROMOS 96 program and showed that 3 structural and 4 functional residues had total energy higher than the native model. These findings indicate that these mutant structures (rs441424814, rs449326723, rs476805413, rs472263384, rs474320860, rs475521477, rs441633284) were less stable than the native model. Molecular Dynamics simulations were performed to confirm the impact of nsSNPs on the protein structure and function. The present study provides useful information about functional SNPs that have an impact on PKLR protein in cattle.Communicated by Ramaswamy H. Sarma.
RESUMO
HGF is a protein that binds to the hepatocyte growth factor receptor to regulate cell growth, cell motility and morphogenesis in different cells and tissues. Several bioinformatics tools and in silico methods were used to identify most deleterious nsSNPs that might change the structure and function of HGF protein. The in silico tools such as SIFT, SNP&GO and PolyPhen2 were used to distinguish deleterious nsSNPs from neutral ones. Protein stability is analysed by I-Mutant, MUpro and iStable. The functional and structural effects are predicted by other tools like MutPred2, Maestro, DUET etc. Analysis of structure was performed by HOPE and Mutation3D. SWISS-MODEL. server, was used for wild type and mutant proteins 3-D Modelling. Gene-gene and protein-protein interaction were predicted by GeneMANIA and STRING, respectively. The wildtype HGF protein and these three variants were independently docked with their close interactor protein MET by the use of ClusPro. Our study suggested that out of 392 missense nsSNPs of the HGF gene, five nsSNPs (D358G, G648R, I550N, N175S and R220Q), are the most deleterious in HGF gene. Gene-gene interactions showed relation of HGF with other genes depicting its importance in several pathways and co-expressions. The protein-protein interacting network is composed of 11 nodes. Analysis of protein stability by different tools indicated that the five nsSNPS decreased the stability of the protein. Anyway these nsSNPs need a confirmation analysis by experimental investigation and GWAS studiesCommunicated by Ramaswamy H. Sarma.
