Numerous studies have been carried out previously to elucidate the association of the TOLLIP gene’s polymorphism with many disorders such as Leprosy or Hansen’s disease55, tuberculosis56, Visceral Leishmaniasis57, idiopathic pulmonary fibrosis58. In this study, we have examined the most damaging non-synonymous SNPs in the TOLLIP gene that may have a critical role in certain diseases.
The results have demonstrated the 196 nsSNPs, the data of other kinds of single nucleotide polymorphism extracted from the employed servers were excluded in this study. The present results have shown that the TOLLIP protein possesses the four most damaging nsSNPs. PROVEAN shows the highest score for D71N and G19D as -4.017and − 6.001 respectively, while for E72G score is -6.617, and the lower score for G32R came out to be -7.000. PolyPhen2, with a scale of 0 to 1 including G19D, G32R, D71N, and E72G scores turn out to be 0.999,1.000,0.999 and 0.995 respectively, and all had termed as probable damage by Polyphen2. We accessed Ensemble genome browser 96 to cross-check these nsSNPs via many servers, such as MetalR, REVEL, Mutation Assessors, and CADD. Mutation Assessor predicted that all four mutations are the most harmful. Likewise, the rest of the tools predicted that all of these 4 nsSNPs are toxic. CADD demonstrated the score of E72G as 34 which is the highest as compared to score of G19D, G32R, and D71N as 26,26 and 27 respectively (here the score of CADD 30 refers to the 0.1% and 20 to the 1% of the most damaging SNPs in the genome of human). The impact of characteristics like variation in the organized interface, methylation loss, and intensification in acetylation predicted by the MutPred. Out of four mutants, G19D represented 0.607 as the peak p-value, whereas three (G32R, D71N, and E72G) mutations demonstrated 0.599,0.412 and 0.571 as the lowest values. The function and structure of TOLLIP protein get affected by the presence of highlighted nsSNPs in the above results. Though, the particular stability data of TOLLIP protein obtained via I-mutant elucidated that G19D, G32R, D71N, and E72G nsSNPs reduced the stability of TOLLIP protein, See Table 3. ConSurf predicted protein conservation profile. According to Berezin59, the highly preserved residues are those which are expected to become essential in their conformational and functional aspect depending on the core and location on the surface of the protein. Likewise, Miller and Kumar60 described that amino acids involved in important biological processes are located in the most conserved region. Hence, relating to this information it is now obvious that non-synonymous SNP found on the very conserved regions can cause the maximum impairment to TOLLIP. Out of 4 nsSNPs, G19D and G32R marked on conserved and buried regions which are considered as structurally important while D71N, E72G spotted on highly conserved and exposed sections which are functionally very significant. The adverse effect of these nsSNPs on the TOLLIP protein is further confirmed. I-TASSER was employed to get the demonstration of TOLLIP protein’s structure. The FASTA sequence of the protein was utilized as the only source of input. This server is well-developed because it collects the prototypes and initiates the simulation of protein. In order to measure the RAMPAGE value of TOLLIP protein, we made the Ramachandran plot. According to (Morris) If RAMPAGE values exceed 80%, the protein configuration will be known as stronger structures. Hence, for TOLLIP the allowed wild-type RAMPAGE value came out to be 86.8 percent and for outlier residues, it turned out to be 17.4 percent. The mutant nsSNPs such as G19D, G32R, D71N, and E72G depicted the permissible percentage (82.9%, 85.2%, 82.6%, and 88.1%) and outlier values such as (13.1%, 12.6%, 17.4% and 12.9%) correspondingly. Another factor that verifies these four nsSNPs in TOLLIP as the most toxic alterations is the maximum deviation score of RMS (root mean square). Both wild and mutant structures get remarkably dissimilar when the resultant Root mean square deviation value surpasses the 2 angstroms. In the sites of TOLLIP protein PTMs, the likelihood of PTMs at these nsSNP positions was investigated. It is noteworthy that not a single site of phosphorylation and ubiquitylation was spotted in the most dangerous nsSNPs. We searched for another site on nsSNPs that identified by Modepred and NetPhos3.1. Modepred shows at twelve sites expected to be phosphorylated and NetPhos3.1 shows 31 sites which are can be phosphorylated (Table 4). ubiquitylation sites projected by BDM-PUB and UbPred. BDM-PUB both detected 7 sites.
Predictions from STRING and GeneMANIA indicate that IRK1 is the most interactive gene with the TOLLIP gene, which is associated with many diseases and also indicates its importance in many diseases.
Thus, it can be inferred that all 4 of the most destructive nsSNPs in the gene TOLLIP eventually affect and interrupt the regular work of other expressive genes based on their interaction patterns and their co-pression profiled with many diseases such as in pathways, it has relation IRAK2, IL1RAP, IRAK2, IL1RN, EHHADH, MYD88, IL1B, IRAK3, TRAF6, IL1A, IL1R1, IRAK1 and IRAK4 genes which indicates its importance.