Project description:PurposeEphrin (Eph) receptor A2 (EPHA2) polymorphism has been associated with age-related cataract (ARC) in different populations worldwide, but the mechanisms by which this polymorphism results in the development of ARC are unclear. Here, we chose four EPHA2 single nucleotide polymorphisms (SNPs; rs35903225, rs145592908, rs137853199, and rs116506614) and studied their function in human lens epithelial cells (LECs).MethodsThe four EPHA2 mutants were overexpressed using lentiviral transduction in human LECs. Cells expressing wild-type (WT) and mutated EPHA2 were subjected to quantitative PCR (qPCR), western blot, immunoprecipitation (IP), and transwell migration assay. MG132 and chloroquine were used to inhibit the degradation of the WT and mutated EPHA2. The structural changes induced by rs137853199 were predicted and optimized using Schrödinger software. IP-mass spectrometry (IP-MS) was performed to examine the proteins that directly interact with WT and rs137853199 EPHA2. Sanger sequencing was performed to determine the frequency of rs137853199 in 184 patients with ARC (73 cortical cataracts, 56 nuclear cataracts, and 55 posterior subcapsular cataracts) and 49 normal controls.ResultsCompared with the WT and the other three mutations, the rs137853199 mutation specifically resulted in a significant decrease in the expression of EPHA2. We identified that EPHA2 rs137853199 is degraded via the ubiquitin-proteasomal pathway through a lysine-48 (K48) residue linkage. Furthermore, the knockdown of EPHA2 reduced cell migration; while the overexpression of WT EPHA2 rescued this defect, the overexpression of rs137853199 EPHA2 did not. In addition, in cells overexpressing rs137853199 EPHA2, the expression of β-catenin, a key protein that regulates cell migration, significantly decreased. We predicted that rs137853199 would induce a conformational change at a linker position in the carboxyl terminal of EPHA2. The IP-MS results showed that the main molecular functions of the proteins that specifically bind WT or rs137853199 EPHA2 are binding and catalysis, while the main protein class is the protein-modifying enzyme. Finally, we discovered that the minor allele frequency of rs137853199 was significantly higher in cortical cataract patients than it was in normal controls.ConclusionsIn summary, these findings suggest a mechanism by which a point mutation in EPHA2 disrupts protein stability, expedites protein degradation, and decreases cell mobility. Importantly, this mutant is associated with cortical cataracts.

Project description:BackgroundTuftelin 1 (TUFT1) gene is important in the development and mineralization of dental enamel. The study aimed to identify potential functionally deleterious non-synonymous SNPs (nsSNPs) in the TUFT1 gene by using different in silico tools. The deleterious missense SNPs were identified from SIFT, PolyPhen-2, PROVEAN, SNPs & GO, PANTHER, and SNAP2. The stabilization, conservation, and three-dimensional modeling of mutant proteins were analyzed by I-Mutant 3.0, Consurf, and Project HOPE, respectively. The protein-protein interaction using STRING, GeneMANIA for gene-gene interaction, and DynaMut for evaluating the impact of the mutation on protein stability, conformation, and flexibility.ResultsEight deleterious nsSNPs (E242A, R303W, K182N, K123N, R117W, H289Q, R203W, and Q107R) out of 304 were found to have high-risk damaging effects using six in silico tools. Among them, K182N and K123N alone had increased stability, whereas E242A, R303W, R117W, H289Q, Q107R, and R203W exhibited a decrease in protein stability, based on DDG values. Meanwhile, all the eight deleterious nsSNPs altered the size, charge, hydrophobicity, and spatial organization of the amino acids and predominantly had alpha helix domains. These deleterious variants were located in highly conserved regions except R203W. Protein-protein interaction predicted that TUFT1 interacted with ten proteins that are involved in enamel mineralization and odontogenesis. Gene-gene interaction network showed that TUFT1 is involved in physical interactions, gene co-localization, and pathway interactions. DynaMut ΔΔG values predicted that five nsSNPs were destabilizing the protein, ΔΔG ENCoM values showed a destabilizing effect for all mutants, and seven nsSNPs increased the molecular flexibility of TUFT1.ConclusionOur study predicted eight functional SNPs that had detrimental effects on the structure and function of the TUFT1 gene. This will aid in the development of candidate deleterious markers as a potential target for disease diagnosis and therapeutic interventions.

Dataset Information

Screening and structural evaluation of deleterious Non-Synonymous SNPs of ePHA2 gene involved in susceptibility to cataract formation.

Publications

Screening and structural evaluation of deleterious Non-Synonymous SNPs of ePHA2 gene involved in susceptibility to cataract formation.

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