In-Silico Investigation of Effects of Single-Nucleotide Polymorphisms in PCOS-Associated CYP11A1 Gene on Mutated Proteins

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

In-silico characterization of GABAT protein found in gut-brain axis associated bacteria of healthy individuals and multiple sclerosis patients

Background: Multiple sclerosis (MS) is a neurodegenerative disease characterized by inflammation and demyelination of neurons. There is evidence to suggest that level of a neurotransmitter gamma-aminobutyric acid (GABA), due to the degradation by γ-aminobutyric acid transaminase (GABAT), is reduced in certain areas of the brain in MS patients. MS is always accompanied by gut bacteria dysbiosis. In healthy individuals, Faecalibacterium sp. while in MS patients A. calcoaceticus, Clostridium sp. and S. typhimurium are found abundantly. Although all these microbes produce GABAT but only in MS patients this enzyme significantly degrades GABA. Objective: Present study is an attempt to characterize the GABAT protein sequences of these bacteria. Methodology: Sequences of GABAT protein were retrieved from Uniprot database. Sequences were analyzed by Protparam, Gneg-mPLoc, SOSUI, PFP-FunDSeqE, Pepwheel program, PROTEUS and Alphafold and SAVES servers, MEME suite and HDOCK server. Results: In healthy individuals gastrointestinal tract (GIT) bacteria, GABAT protein was present in inner-membrane with α helix content (61 and 62%) and β sheet content (5%), 4-helical cytokines functional domains. It has greater number of B-cell epitopes and more complex 3D configuration as compared to MS patients GIT bacterial enzymes. Conclusion: Present study might enable us to modify the GABAT encoding gene and enzyme through site-directed mutagenesis in pathogenic bacteria thus reducing their potential of causing MS

In-Silico Characterization of Estrogen Reactivating β-Glucuronidase Enzyme in GIT Associated Microbiota of Normal Human and Breast Cancer Patients

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

Exploring the association of ESR1 and ESR2 gene SNPs with polycystic ovary syndrome in human females: a comprehensive association study

Abstract Background Polycystic Ovary Syndrome (PCOS) affects a significant proportion of human females worldwide and is characterized by hormonal, metabolic, and reproductive dysfunctions, including infertility, irregular menstrual cycles, acanthosis nigricans, and hirsutism. Mutations in the estrogen receptor genes ESR1 and ESR2, involved in normal follicular development and ovulation, can contribute to development of the PCOS. The present study focuses on investigating the potential correlation between single nucleotide polymorphisms (SNPs) of ESR1 and ESR2 genes and the incidence of this syndrome. Methods For this study, SNPs in ESR1 and ESR2 genes were retrieved from the ENSEMBL database and analyzed for their effect on mutated proteins using different bioinformatics tools including SIFT, PolyPhen, CADD, REVEL, MetaLR, I-Mutant, CELLO2GO, ProtParam, SOPMA, SWISS-MODEL and HDDOCK. Results All the SNPs documented in the present study were deleterious. All the SNPs except rs1583384537, rs1450198518, and rs78255744 decreased protein stability. Two variants rs1463893698 and rs766843910 in the ESR2 gene altered the localization of mutated proteins i.e. in addition to the nucleus, proteins were also found in mitochondria and extracellular, respectively. SNPs rs104893956 in ESR1 and rs140630557, rs140630557, rs1596423459, rs766843910, rs1596405923, rs762454979 and rs1384121511 in ESR2 gene significantly changed the secondary structure of proteins (2D). SNPs that markedly changed 3D configuration included rs1554259481, rs188957694 and rs755667747 in ESR1 gene and rs1463893698, rs140630557, rs1596423459, rs766843910, rs1596405923, rs762454979 and rs1384121511 in ESR2 gene. Variants rs1467954450 (ESR1) and rs140630557 (ESR2) were identified to reduce the binding tendency of ESRα and β receptors with estradiol as reflected by the docking scores i.e. -164.97 and -173.23, respectively. Conclusion Due to the significant impact on the encoded proteins, these variants might be proposed as biomarkers to predict the likelihood of developing PCOS in the future and for diagnostic purposes

Body mass index and altered lipid profile as major risk markers for breast cancer progression: a cross-sectional study of postmenopausal women in Pakistan

Abstract Background In Pakistan, the death rate for post-menopausal women with breast cancer is significant due to late detection and delayed referral to proper facilities. There are a few reports on Pakistan’s epidemiology and breast cancer risk factors. There are modifiable and non-modifiable risk factors associated with the development of breast carcinoma; of which body mass index (BMI), central obesity, and lipid profile are considered as major risk markers. Methods This was a cross-sectional analytical study. A total of 384 women constituted the present study sample. Purposive sampling was used to collect 192 confirmed new breast cancer cases throughout the study. By using basic random sampling, an equal number of controls were chosen. Studied parameters included age, fasting blood sugar, cholesterol, triglyceride, serum high-density lipoprotein, cholesterol, serum low-density lipoprotein cholesterol, weight, height, BMI, waist circumference, and waist-to-hip ratio. The inclusion criteria of this study were post-menopausal women (45–65 years) in Pakistan. The confirmation of breast carcinoma was done through histopathology. Breast cancer occurrence was taken as a dependent variable, whereas BMI, central obesity, and lipid profile were taken as independent variables. Results Studied risk factors (cholesterol, BMI, and central obesity) significantly correlated with breast cancer. Cholesterol has a significantly high positive correlation (0.646) with breast cancer. BMI has a positive significant correlation (0.491) with breast cancer, and central obesity has a low but positive significant correlation (0.266) with breast cancer. Moreover, the binary logistic regression model also showed a significant association between biochemical factors and breast cancer occurrence. Regression analysis depicted a linear relationship between a dependent variable (breast cancer occurrence) and independent variables (central obesity, cholesterol, BMI). Conclusion Postmenopausal overweight (central obesity), increased BMI and high cholesterol levels are major risk factors for breast cancer. Moreover, high total cholesterol proved to be the most significant risk marker for the occurrence of breast cancer in post-menopausal women of Pakistan

A pan-cancer analysis of GINS complex subunit 4 to identify its potential role as a biomarker in multiple human cancers

This study was initiated to explore the expression variation, clinical significance, and biological importance of the GINS complex subunit 4 (GINS4) in different human cancers as a shared biomarker via pan-cancer analysis through different platforms including UALCAN, Kaplan Meier (KM) plotter, TNMplot, GENT2, GEPIA, DriverDBv3, Human Protein Atlas (HPA), MEXPRESS, cBioportal, STRING, DAVID, MuTarge, Enrichr, TIMER, and CTD. Our findings have verified the up-regulation of GINS4 in 24 major subtypes of human cancers, and its overexpression was found to be substantially associated with poor overall survival (OS), relapse-free survival (RFs), and metastasis in ESCA, KIRC, LIHC, LUAD, and UCEC. This suggested that GINS4 plays a significant role in the development and progression of these five cancers. Furthermore, we noticed that GINS4 is also overexpressed in ESCA, KIRC, LIHC, LUAD, and UCEC patients with different clinicopathological characteristics. Enrichment analysis revealed the involvement of GINS4 associated genes in a variety of diverse GO and KEGG terms. We also explored few significant correlations between GINS4 expression and promoter methylation, genetic alterations, CNVs, other mutant genes, tumor purity, and immune cells infiltration. In conclusion, our results elucidated that GINS4 can serve as a shared diagnostic, prognostic biomarker, and a potential therapeutic target in ESCA, KIRC, LIHC, LUAD, and UCEC patients with different clinicopathological characteristics