Estrogen circulating in blood has been proved to be a strong biomarker for breast cancer. A β-glucuronidase enzyme (GUS) from human gastrointestinal tract (GIT) microbiota including probiotics has significant involvement in enhancing the estrogen concentration in blood through deconjugation of glucuronidated estrogens. The present project has been designed to explore GIT microbiome-encoded GUS enzymes (GUSOME) repertoire in normal human and breast cancer patients. For this purpose, a total of nineteen GUS enzymes from human GIT microbes, i.e., seven from healthy and twelve from breast cancer patients have been focused on. Protein sequences of enzymes retrieved from UniProt database were subjected to ProtParam, CELLO2GO, SOPMA (secondary structure prediction method), PDBsum (Protein Database summaries), PHYRE2 (Protein Homology/AnalogY Recognition Engine), SAVES v6.0 (Structure Validation Server), MEME version 5.4.1 (Multiple Em for Motif Elicitation), Caver Web server v 1.1, Interproscan and Predicted Antigenic Peptides tool. Analysis revealed the number of amino acids, isoelectric point, extinction coefficient, instability index and aliphatic index of GUS enzymes in the range of 586–795, 4.91–8.92, 89,980–155,075, 25.88–40.93 and 71.01–88.10, respectively. Sub-cellular localization of enzyme was restricted to cytoplasm and inner-membrane in case of breast cancer patients’ bacteria as compared to periplasmic space, outer membrane and extracellular space in normal GIT bacteria. The 2-D structure analysis showed α helix, extended strand, β turn and random coil in the range of 27.42–22.66%, 22.04–25.91%, 5.39–8.30% and 41.75–47.70%, respectively. The druggability score was found to be 0.05–0.45 and 0.06–0.80 in normal and breast cancer patients GIT, respectively. The radius, length and curvature of catalytic sites were observed to be 1.1–2.8 Å, 1.4–15.9 Å and 0.65–1.4, respectively. Ten conserved protein motifs with p < 0.05 and width 25–50 were found. Antigenic propensity-associated sequences were 20–29. Present study findings hint about the use of the bacterial GUS enzymes against breast cancer tumors after modifications via site-directed mutagenesis of catalytic sites involved in the activation of estrogens and through destabilization of these enzymes.

The L-2-hydroxyglutarate dehydrogenase (L2HGDH) gene encodes an important mitochondrial enzyme. However, its altered activity results in excessive levels of L-2-hydroxyglutarate, which results in diverse psychiatric features of intellectual disability. In the current study, we executed an in-silico analysis of all reported L2HGDH missense and nonsense variants in order to investigate their biological significance. Among the superimposed 3D models, the highest similarity index for a wild-type structure was shown by the mutant Glu336Lys (87.26%), while the lowest similarity index value was shown by Arg70* (10.00%). Three large active site pockets were determined using protein active site prediction, in which the 2nd largest pocket was shown to encompass the substrate L-2-hydroxyglutarate (L2HG) binding residues, i.e., 89Gln, 195Tyr, 402Ala, 403Gly and 404Val. Moreover, interactions of wild-type and mutant L2HGDH variants with the close functional interactor D2HGDH protein resulted in alterations in the position, number and nature of networking residues. We observed that the binding of L2HG with the L2HGDH enzyme is affected by the nature of the amino acid substitution, as well as the number and nature of bonds between the substrate and protein molecule, which are able to affect its biological activity.

Polycystic ovary syndrome (PCOS) is a reproductive disorder with multiple etiologies, mainly characterized by the excess production of androgens. It is equally contributed to by genes and environment. The CYP11A1 gene is imperative for steroidogenesis, so any dysregulation or mutation in this gene can lead to PCOS pathogenesis. Therefore, nucleotide diversity in this gene can be helpful in spotting the likelihood of developing PCOS. The present study was initiated to investigate the effect of single nucleotide polymorphisms in human CYP11A1 gene on different attributes of encoded mutated proteins, i.e., sub-cellular localization, ontology, half-life, isoelectric point, instability index, aliphatic index, extinction coefficient, 3-D and 2-D structures, and transmembrane topology. For this purpose, initially coding sequence (CDS) and single nucleotide polymorphisms (SNPs) were retrieved for the desired gene from Ensembl followed by translation of CDS using EXPASY tool. The protein sequence obtained was subjected to different tools including CELLO2GO, ProtParam, PHYRE2, I-Mutant, SIFT, and PolyPhen. It was found that out of seventy-eight SNPs analyzed in this project, seventeen mutations, i.e., rs750026801 in exon 1, rs776056840, rs779154292 and rs1217014229 in exon 2, rs549043326 in exon 3, rs755186597 in exon 4, rs1224774813, rs757299093 and rs1555425667 in exon 5, rs1454328072 in exon 7, rs762412759 and rs755975808 in exon 8, and rs754610565, rs779413653, rs765916701, rs1368450780, and rs747901197 in exon 9 considerably altered the structure, sub-cellular localization, and physicochemical characteristics of mutated proteins. Among the fifty-nine missense SNPs documented in present study, fifty-five and fifty-three were found to be deleterious according to SIFT and PolyPhen tools, respectively. Forty-nine missense mutations were analyzed to have a decreasing effect on the stability of mutant proteins. Hence, these genetic variants can serve as potential biomarkers in human females for determining the probability of being predisposed to PCOS.