Genome-wide association study reveals effect of nsSNPs on candidate genes in rice during iron deficiency.

Resource-poor areas with moisture deficit lands following aerobic and direct seeded rice (DSR) methods of cultivation face severe problems of iron deficiency. In this study, Bengal and Assam Aus rice panel was phenotyped at the seedling stage using an iron-deprived hydroponic medium for various shoot and root traits. A novel iron deficiency scoring scale was used to classify the tolerance reaction and could range anywhere between 0 and 9, indicating the most tolerant and susceptible, respectively. The GWAS results identified four putative candidate genes; OsFLA for number of leaves and shoot length, OsBIDK1 for root traits; average diameter, volume, biomass, projected area, and surface area, OsHPL3 for chlorophyll index of the third leaf and AKR2B (XBOS252) was for Fe score, (which was earlier reported in relation to Xa21). The nsSNP (nsSNPs) variations in these gene sequences were used to group the panel and identify superior haplotypes and donors. BR16 was identified as a superior donor, with higher chlorophyll index and shoot length than RA23, also higher values for root traits like root average diameter, root volume, root projected area and root surface area followed by Shete Bhado. The impact of identified nsSNPs on protein structure and stability was investigated. The conserved domains detected in the mutated proteins of the superior haplotypes are very informative, highlighting that natural selection favors abiotic stress tolerant variants in resource poor areas. Thus, justifying our choice of Aus landraces for association mapping of Fe deficiency tolerant genes in rice.

Pathogenic nsSNPs of protein kinase C-eta with hepatocellular carcinoma susceptibility.

BACKGROUND: Hepatocellular carcinoma (HCC) is a global health concern. Due to late diagnosis and limited therapeutic strategies, HCC based mortality rate is exponentially increasing globally. Genetic predisposition is a non-avoidable intrinsic factor that could alter the genome sequence, ultimately leading to HCC. Protein kinase C eta (PKCη) is involved in key physiological roles, hence alteration in PKCη could aid in cancer progression. Research indicates association between non-synonymous (ns) SNPs and HCC onset. However, effect of nsSNP variants of PKCη on HCC development has not been explored yet. Hence, this study aimed to investigate the association between pathogenic nsSNPs of PKCη with HCC. METHODS: Non-synonymous (missense) variants of PKCη were obtained from Ensembl genome browser. These variants were filtered out to obtain pathogenic nsSNPs of PKCη. Genotyping of nsSNPs was done through Tetra ARMS PCR. For that, blood samples of 348 HCC patients and 337 controls were collected. The clinical factors that influence HCC were studied. Relative risk (RR) and Odds Ratio (OR) with 95% confidence interval was calculated by Chi-square test and P-value < 0.05 was deemed significant. RESULTS: Five nsSNP variants of PKCη including rs1162102190 (T/C), rs868127012 (G/T), rs750830348 (G/T), rs768619375 (T/C), and rs752329416 (T/C) were identified. The retrieved nsSNPs were frequently identified in HCC patients. However, rs752329416 T/C was significantly prevalent in patients having HCC family history. Moreover, all the variants were found in HCC patients manifesting the stage II than the advance stages of HCC. CONCLUSION: This study can be utilized to identify potential genetic markers for early screening of HCC. Moreover, consideration of further clinical factors, and mechanistic approach would enhance the understanding that how alteration in nsSNPs could impact the HCC onset.

Computational insights into NIMA-related kinase 6: unraveling mutational effects on structure and function.

The NEK6 (NIMA-related kinase 6) serine/threonine kinase is a pivotal player in a multitude of cellular processes, including the regulation of the cell cycle and the response to DNA damage. Its significance extends to disease pathogenesis, as changes in NEK6 activity have been linked to the development of cancer. Non-synonymous single nucleotide polymorphisms (nsSNPs) in NEK6 have been linked to cancer as they alter the protein's native structure and function. The association between NEK6 activity and cancer development has prompted researchers to explore the effects of genetic variations within the NEK6 gene. Therefore, we utilized advanced computational tools to analyze 155 high-confidence nsSNPs in the NEK6 gene. From this analysis, 21 nsSNPs were identified as potentially harmful, raising concerns about their impact on NEK6 activity and cancer risk. These 21 mutations were then examined for structural alterations, and eight of nsSNPs (I51M, V76A, I134N, Y152D, R171Q, V186G, L237R, and C285S) were found to destabilize the protein. Among the destabilizing mutations screened, a specific mutation, R171Q, stood out due to its conserved nature. To understand its impact on the protein and conformation, all-atom molecular dynamics simulations (MDS) for 100 ns were performed for both Wildtype NEK6 (WT-NEK6) and R171Q. The simulations revealed that the R171Q variant was unstable and led to significant conformational changes in NEK6. This study provides valuable insights into NEK6 dysfunction caused by single amino acid alterations, offering a novel understanding of the molecular mechanisms underlying NEK6-related cancer progression.

Comprehensive in-silico analysis of deleterious SNPs in APOC2 and APOA5 and their differential expression in cancer and cardiovascular diseases conditions.

Genetic variations in APOC2 and APOA5 genes involve activating lipoprotein lipase (LPL), responsible for the hydrolysis of triglycerides (TG) in blood and whose impaired functions affect the TG metabolism and are associated with metabolic diseases. In this study, we investigate the biological significance of genetic variations at the DNA sequence and structural level using various computational tools. Subsequently, 8 (APOC2) and 17 (APOA5) non-synonymous SNPs (nsSNPs) were identified as high-confidence deleterious SNPs based on the effects of the mutations on protein conservation, stability, and solvent accessibility. Furthermore, based on our docking results, the interaction of native and mutant forms of the corresponding proteins with LPL depicts differences in root mean square deviation (RMSD), and binding affinities suggest that these mutations may affect their function. Furthermore, in vivo, and in vitro studies have shown that differential expression of these genes in disease conditions due to the influence of nsSNPs abundance may be associated with promoting the development of cancer and cardiovascular diseases. Preliminary screening using computational methods can be a helpful start in understanding the effects of mutations in APOC2 and APOA5 on lipid metabolism; however, further wet-lab experiments would further strengthen the conclusions drawn from the computational study.

Molecular Dynamics Simulation of Kir6.2 Variants Reveals Potential Association with Diabetes Mellitus.

Diabetes mellitus (DM) represents a problem for the healthcare system worldwide. DM has very serious complications such as blindness, kidney failure, and cardiovascular disease. In addition to the very bad socioeconomic impacts, it influences patients and their families and communities. The global costs of DM and its complications are huge and expected to rise by the year 2030. DM is caused by genetic and environmental risk factors. Genetic testing will aid in early diagnosis and identification of susceptible individuals or populations using ATP-sensitive potassium (KATP) channels present in different tissues such as the pancreas, myocardium, myocytes, and nervous tissues. The channels respond to different concentrations of blood sugar, stimulation by hormones, or ischemic conditions. In pancreatic cells, they regulate the secretion of insulin and glucagon. Mutations in the KCNJ11 gene that encodes the Kir6.2 protein (a major constituent of KATP channels) were reported to be associated with Type 2 DM, neonatal diabetes mellitus (NDM), and maturity-onset diabetes of the young (MODY). Kir6.2 harbors binding sites for ATP and phosphatidylinositol 4,5-diphosphate (PIP2). The ATP inhibits the KATP channel, while the (PIP2) activates it. A Kir6.2 mutation at tyrosine330 (Y330) was demonstrated to reduce ATP inhibition and predisposes to NDM. In this study, we examined the effect of mutations on the Kir6.2 structure using bioinformatics tools and molecular dynamic simulations (SIFT, PolyPhen, SNAP2, PANTHER, PhD&SNP, SNP&Go, I-Mutant, MuPro, MutPred, ConSurf, HOPE, and GROMACS). Our results indicated that M199R, R201H, R206H, and Y330H mutations influence Kir6.2 structure and function and therefore may cause DM. We conclude that MD simulations are useful techniques to predict the effects of mutations on protein structure. In addition, the M199R, R201H, R206H, and Y330H variant in the Kir6.2 protein may be associated with DM. These results require further verification in protein-protein interactions, Kir6.2 function, and case-control studies.

In Silico Evaluation of Coding and Non-Coding nsSNPs in the Thrombopoietin Receptor (MPL) Proto-Oncogene: Assessing Their Influence on Protein Stability, Structure, and Function.

The thrombopoietin receptor (MPL) gene is a critical regulator of hematopoiesis, and any alterations in its structure or function can result in a range of hematological disorders. Non-synonymous single nucleotide polymorphisms (nsSNPs) in MPL have the potential to disrupt normal protein function, prompting our investigation into the most deleterious MPL SNPs and the associated structural changes affecting protein-protein interactions. We employed a comprehensive suite of bioinformatics tools, including PredictSNP, InterPro, ConSurf, I-Mutant2.0, MUpro, Musitedeep, Project HOPE, STRING, RegulomeDB, Mutpred2, CScape, and CScape Somatic, to analyze 635 nsSNPs within the MPL gene. Among the analyzed nsSNPs, PredictSNP identified 28 as significantly pathogenic, revealing three critical functional domains within MPL. Ten of these nsSNPs exhibited high conservation scores, indicating potential effects on protein structure and function, while 14 were found to compromise MPL protein stability. Although the most harmful nsSNPs did not directly impact post-translational modification sites, 13 had the capacity to substantially alter the protein's physicochemical properties. Some mutations posed a risk to vital protein-protein interactions crucial for hematological functions, and three non-coding region nsSNPs displayed significant regulatory potential with potential implications for hematopoiesis. Furthermore, 13 out of 21 nsSNPs evaluated were classified as high-risk pathogenic variants by Mutpred2. Notably, amino acid alterations such as C291S, T293N, D295G, and W435C, while impactful on protein stability and function, were deemed non-oncogenic "passenger" mutations. Our study underscores the substantial impact of missense nsSNPs on MPL protein structure and function. Given MPL's central role in hematopoiesis, these mutations can significantly disrupt hematological processes, potentially leading to a variety of disorders. The identified high-risk pathogenic nsSNPs may hold promise as potential biomarkers or therapeutic targets for hematological diseases. This research lays the foundation for future investigations into the MPL gene's role in the realm of hematological health and diseases.

In silico profiling of nonsynonymous SNPs of fat mass and obesity-associated gene: possible impacts on the treatment of non-alcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver, or NAFLD, is the most common chronic liver ailment. It is characterized by excessive fat deposition in hepatocytes of individuals who consume little or no alcohol and are unaffected by specific liver damaging factors. It is also associated with extrahepatic manifestations such as chronic kidney disease, cardiovascular disease, and sleep apnea. The global burden of NAFLD is increasing at an alarming rate. However, no pharmacologically approved drugs against NAFLD are available owing to their complex pathophysiology. Genome-wide association studies have uncovered SNPs in the fat mass and obesity-associated gene (FTO) that are robustly associated with obesity and higher BMI. The prevalence of NAFLD increases in parallel with the increasing prevalence of obesity. Since FTO might play a crucial role in NAFLD development, the current study identified five potentially deleterious mutations from 383 ns-SNPs in the human FTO gene using various in silico tools. METHODS: This study aims to identify potentially deleterious nonsynonymous SNPs (ns-SNPs) employing various in silico tools. Additionally, molecular modeling approaches further studied the structural changes caused by identified SNPs. Moreover, molecular dynamics studies finally investigated the binding potentials of the phytochemicals resveratrol, rosmarinic acid, and capsaicin with different mutant forms of FTO. RESULTS: The current investigation has five potentially deleterious mutations from 383 ns-SNPs in the human FTO gene using various in silico tools. The present study identified five nsSNPs of the human gene FTO, Gly103Asp, Arg96Pro, Tyr295Cys, and Arg322Gln, with an apparent connection to the disease condition. Modulation of demethylation activity by phytomolecule scanning explains the hepatoprotective action of molecules. The current investigation also suggested that predicted mutations did not affect the binding ability of three polyphenols: rosamarinic acid, resveratrol, and capsaicin. CONCLUSION: This study showed that the predicted mutations in FTO did not affect the binding of three polyphenols. Thus, these three molecules can significantly aid drug development against FTO and NAFLD.

Structural Consequences of IRS-2 nsSNPs and Implication for Insulin Receptor Substrate-2 Protein Stability.

Single-Nucleotide Polymorphisms (SNPs) are common genetic variations implicated in human diseases. The non-synonymous SNPs (nsSNPs) affect the proteins' structures and their molecular interactions with other interacting proteins during the accomplishment of biochemical processes. This ultimately causes proteins functional perturbation and disease phenotypes. The Insulin receptor substrate-2 (IRS-2) protein promotes glucose absorption and participates in the biological regulation of glucose metabolism and energy production. Several IRS-2 SNPs are reported in association with type 2 diabetes and obesity in human populations. However, there are no comprehensive reports about the protein structural consequences of these nsSNPs. Keeping in view the pathophysiological consequences of the IRS-2 nsSNPs, we designed the current study to understand their possible structural impact on coding protein. The prioritized list of the deleterious IRS-2 nsSNPs was acquired from multiple bioinformatics resources, including VEP (SIFT, PolyPhen, and Condel), PROVEAN, SNPs&GO, PMut, and SNAP2. The protein structure stability assessment of these nsSNPs was performed by MuPro and I-Mutant-3.0 servers via structural modeling approaches. The atomic-level structural and molecular dynamics (MD) impact of these nsSNPs were examined using GROMACS 2019.2 software package. The analyses initially predicted 8 high-risk nsSNPs located in the highly conserved regions of IRS-2. The MD simulation analysis eventually prioritized the N232Y, R218C, and R104H nsSNPs that predicted to significantly compromise the structure stability and may affect the biological function of IRS-2. These nsSNPs are predicted as high-risk candidates for diabetes and obesity. The validation of protein structural impact of these shortlisted nsSNPs may provide biochemical insight into the IRS-2-mediated type-2 diabetes.

Mechanistic insights on nsSNPs on binding site of renin and cytochrome P450 proteins: A computational perceptual study for pharmacogenomics evaluation.

Past several decades, therapeutic investigations lead to the discovery of numerous antihypertensive drugs. Although it has been proved for their potency, altered efficacy is common norms in several conditions due to genetic variations. Cytochrome P450 plays a crucial role in drug metabolism and responsible for the pharmacokinetic and pharmacodynamic properties of the drug molecules. Here, we report the deleterious point mutations in the genes associated with the altered response of antihypertensive drug molecules and their metabolizers. Missense variants were filtered as potential nonsynonymous single nucleotide polymorphisms among the available data for the target genes (REN, CYP2D6, CYP3A4). The key objective of the work is to identify the deleterious single nucleotide polymorphisms (SNPs) responsible for the drug response and metabolism for the application of personalized medication. The molecular docking studies revealed that Aliskiren and other clinically approved drug molecules have a high binding affinity with both wild and mutant structures of renin, CYP2D6, and CYP3A4 proteins. The docking (Glide XP) score was observed to have in the range of -8.896 to -11.693 kcal/mol. The molecular dynamics simulation studies were employed to perceive the structural changes and conformational deviation through various analyses. Each studied SNPs was observed to have disparate scoring in the binding affinity to the specific drug molecules. As a prospective plan, we assume this study might be applied to identify the risky SNPs associated with hypertension from the patients to recommend the suitable drug for personalized hypertensive treatment. Further, extensive clinical pharmacogenomics studies are required to support the findings.

Structural and functional insights on vitamin D receptor and CYP24A1 deleterious single nucleotide polymorphisms: A computational and pharmacogenomics perpetual approach.

The development of chronic kidney disease (CKD) drugs remains a challenge due to the variations in the genes. The vitamin D receptor (VDR) and Cytochrome 24A1 (CYP24A1) genetic variants might affect the drug potency, efficacy and pathway. Here we have to analyse and determine the deleterious single-nucleotide polymorphisms (nsSNPs) of VDR and CYP24A1 genes and their different population's drug responses in different populations to understand the key role in CKD. Among that the large scale of nsSNP, we used certain computational tools that predicted six missense variants are observed to be significantly damaging effect and SNP variability with large differences in various populations. Molecular docking studies were carried out by clinical and our screened compounds to VDR and CYP24A1. Docking results revealed all the compounds have a good binding affinity (Score). The screened compounds (TCM_2868 and UNPD_141613) show good binding affinity when compared to known compounds. The QM/MM study revealed that the compounds have electron transfer ability and act as a donor/acceptor to mutated proteins. The structural and conformational changes of protein complexes were analysed by molecular dynamics study. Hence, this study helps to identify suitable drugs through drug discovery in CKD treatment. The abovementioned compounds have more binding affinity, efficacy, and potency of both wild and mutant of VDR and CYP24A1.

CYP2R1 and CYP27A1 genes: An in silico approach to identify the deleterious mutations, impact on structure and their differential expression in disease conditions.

Mutations in CYP2R1 and CYP27A1 involved in the conversion of Cholecalciferol into Calcidiol were associated with the impaired 25-hydroxylase activity therefore affecting the Vitamin D metabolism. Hence, this study attempted to understand the influence of genetic variations at the sequence and structural level via computational approach. The non-synonymous mutations retrieved from dbSNP database were assessed for their pathogenicity, stability as well as conservancy using various computational tools. The above analysis predicted 11/260 and 35/489 non-synonymous mutations to be deleterious in CYP2R1 and CYP27A1 genes respectively. Native and mutant forms of the corresponding proteins were modeled. Further, interacting native and mutant proteins with cholecalciferol showed difference in hydrogen bonds, hydrophobic bonds and their binding affinities suggesting the possible influence of these mutations in their function. Also, expression of these genes in various disease conditions was investigated using GEO datasets which predicted that there is a differential expression in cancer and arthritis.

Computational Insights into the Deleterious Impacts of Missense Variants on N-Acetyl-d-glucosamine Kinase Structure and Function.

An enzyme of the mammalian amino-sugar metabolism pathway, N-acetylglucosamine kinase (NAGK), that synthesizes N-acetylglucosamine (GlcNAc)-6-phosphate, is reported to promote dynein functions during mitosis, axonal and dendritic growth, cell migration, and selective autophagy, which all are unrelated to its enzyme activity. As non-enzymatic structural functions can be altered by genetic variation, we made an effort in this study aimed at deciphering the pathological effect of nonsynonymous single-nucleotide polymorphisms (nsSNPs) in NAGK gene. An integrated computational approach, including molecular dynamics (MD) simulation and protein-protein docking simulation, was used to identify the damaging nsSNPs and their detailed structural and functional consequences. The analysis revealed the four most damaging variants (G11R, G32R, G120E, and A156D), which are highly conserved and functional, positioned in both small (G11R and G32R) and large (G120E and A156D) domains of NAGK. G11R is located in the ATP binding region, while variants present in the large domain (G120E and A156D) were found to induce substantial alterations in the structural organizations of both domains, including the ATP and substrate binding sites. Furthermore, all variants were found to reduce binding energy between NAGK and dynein subunit DYNLRB1, as revealed by protein-protein docking and MM-GBSA binding energy calculation supporting their deleteriousness on non-canonical function. We hope these findings will direct future studies to gain more insight into the role of these variants in the loss of NAGK function and their role in neurodevelopmental disorders.

Computational and Mass Spectrometry-Based Approach Identify Deleterious Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs) in JMJD6.

The jumonji domain-containing protein 6 (JMJD6) gene catalyzes the arginine demethylation and lysine hydroxylation of histone and a growing list of its known substrate molecules, including p53 and U2AF65, suggesting a possible role in mRNA splicing and transcription in cancer progression. Mass spectrometry-based technology offers the opportunity to detect SNP variants accurately and effectively. In our study, we conducted a combined computational and filtration workflow to predict the nonsynonymous single nucleotide polymorphisms (nsSNPs) present in JMJD6, followed by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and validation. The computational approaches SIFT, PolyPhen-2, SNAP, I-Mutant 2.0, PhD-SNP, PANTHER, and SNPS&GO were integrated to screen out the predicted damaging/deleterious nsSNPs. Through the three-dimensional structure of JMJD6, H187R (rs1159480887) was selected as a candidate for validation. The validation experiments showed that the mutation of this nsSNP in JMJD6 obviously affected mRNA splicing or the transcription of downstream genes through the reduced lysyl-hydroxylase activity of its substrates, U2AF65 and p53, further indicating the accuracy of this prediction method. This research provides an effective computational workflow for researchers with an opportunity to select prominent deleterious nsSNPs and, thus, remains promising for examining the dysfunction of proteins.

Computational SNP Analysis and Molecular Simulation Revealed the Most Deleterious Missense Variants in the NBD1 Domain of Human ABCA1 Transporter.

The ATP-binding cassette transporter A1 (ABCA1) is a membrane-bound exporter protein involved in regulating serum HDL level by exporting cholesterol and phospholipids to load up in lipid-poor ApoA-I and ApoE, which allows the formation of nascent HDL. Mutations in the ABCA1 gene, when presents in both alleles, disrupt the canonical function of ABCA1, which associates with many disorders related to lipid transport. Although many studies have reported the phenotypic effects of a large number of ABCA1 variants, the pathological effect of non-synonymous polymorphisms (nsSNPs) in ABCA1 remains elusive. Therefore, aiming at exploring the structural and functional consequences of nsSNPs in ABCA1, in this study, we employed an integrated computational approach consisting of nine well-known in silico tools to identify damaging SNPs and molecular dynamics (MD) simulation to get insights into the magnitudes of the damaging effects. In silico tools revealed four nsSNPs as being most deleterious, where the two SNPs (G1050V and S1067C) are identified as the highly conserved and functional disrupting mutations located in the NBD1 domain. MD simulation suggested that both SNPs, G1050V and S1067C, changed the overall structural flexibility and dynamics of NBD1, and induced substantial alteration in the structural organization of ATP binding site. Taken together, these findings direct future studies to get more insights into the role of these variants in the loss of the ABCA1 function.

Identification of most damaging nsSNPs in human CCR6 gene: In silico analyses.

Single nucleotide polymorphisms in CCR6 (C-C chemokine receptor type 6) gene have been found to be the possible cause of many diseases like rheumatoid arthritis, psoriasis, lupus nephritis and systemic sclerosis and other autoimmune diseases. Therefore, identification of structurally and functionally important polymorphisms in CCR6 is important in order to study its potential malfunctioning and discovering therapeutic targets. Several bioinformatics tools were used to identify most damaging nsSNPs that might be vital for CCR6 structure and function. The in silico tools included PROVEAN, SIFT, SNP&GO and PolyPhen2 followed by I-Mutant MutPred and ConSurf. Phyre2 and I-TASSER were used for protein 3-D Modelling while gene-gene interaction was predicted by STRING and GeneMANIA. Our study suggested that three nsSNPs rs1376162684, rs751102128 and rs1185426631 are the most damaging in CCR6 gene while 7 missense SNPs rs1438637216, rs139697820, rs768420505, rs1282264186, rs1394647982, rs769360638 and rs1263402382 are found to revert into stop codons. Prediction of post-transcriptional modifications highlighted the significance of rs1376162684 because it effected potential phosphorylation site. Gene-gene interactions showed relation of CCR6 with other genes depicting its importance in several pathways and co-expressions. In future, studying diseases related to CCR6 should include investigation of these 10 nsSNPs. Being the first of its type, this study also proposes future perspectives that will help in precision medicines. For such purposes, CCR6 proteins from patients of autoimmune diseases should be explored. Animal models can also be of significance find out the effects of CCR6 in diseases.

Bioinformatics classification of mutations in patients with Mucopolysaccharidosis IIIA.

Mucopolysaccharidosis (MPS) IIIA, also known as Sanfilippo syndrome type A, is a severe, progressive disease that affects the central nervous system (CNS). MPS IIIA is inherited in an autosomal recessive manner and is caused by a deficiency in the lysosomal enzyme sulfamidase, which is required for the degradation of heparan sulfate. The sulfamidase is produced by the N-sulphoglucosamine sulphohydrolase (SGSH) gene. In MPS IIIA patients, the excess of lysosomal storage of heparan sulfate often leads to mental retardation, hyperactive behavior, and connective tissue impairments, which occur due to various known missense mutations in the SGSH, leading to protein dysfunction. In this study, we focused on three mutations (R74C, S66W, and R245H) based on in silico pathogenic, conservation, and stability prediction tool studies. The three mutations were further subjected to molecular dynamic simulation (MDS) analysis using GROMACS simulation software to observe the structural changes they induced, and all the mutants exhibited maximum deviation patterns compared with the native protein. Conformational changes were observed in the mutants based on various geometrical parameters, such as conformational stability, fluctuation, and compactness, followed by hydrogen bonding, physicochemical properties, principal component analysis (PCA), and salt bridge analyses, which further validated the underlying cause of the protein instability. Additionally, secondary structure and surrounding amino acid analyses further confirmed the above results indicating the loss of protein function in the mutants compared with the native protein. The present results reveal the effects of three mutations on the enzymatic activity of sulfamidase, providing a molecular explanation for the cause of the disease. Thus, this study allows for a better understanding of the effect of SGSH mutations through the use of various computational approaches in terms of both structure and functions and provides a platform for the development of therapeutic drugs and potential disease treatments.

Do environmentally induced DNA variations mediate adaptation in Aspergillus flavus exposed to chromium stress in tannery sludge?

BACKGROUND: Environmental stress induced genetic polymorphisms have been suggested to arbitrate functional modifications influencing adaptations in microbes. The relationship between the genetic processes and concomitant functional adaptation can now be investigated at a genomic scale with the help of next generation sequencing (NGS) technologies. Using a NGS approach we identified genetic variations putatively underlying chromium tolerance in a strain of Aspergillus flavus isolated from a tannery sludge. Correlation of nsSNPs in the candidate genes (n = 493) were investigated for their influence on protein structure and possible function. Whole genome sequencing of chromium tolerant A. flavus strain (TERIBR1) was done (Illumina HiSeq2000). The alignment of quality trimmed data of TERIBR1 with reference NRRL3357 (accession number EQ963472) strain was performed using Bowtie2 version 2.2.8. SNP with a minimum read depth of 5 and not in vicinity (10 bp) of INDEL were filtered. Candidate genes conferring chromium resistance were selected and SNPs were identified. Protein structure modeling and interpretation for protein-ligand (CrO4- 2) docking for selected proteins harbouring non-synonymous substitutions were done using Phyre2 and PatchDock programs. RESULTS: High rate of nsSNPs (approximately 11/kb) occurred in selected candidate genes for chromium tolerance. Of the 16 candidate genes selected for studying effect of nsSNPs on protein structure and protein-ligand interaction, four proteins belonging to the Major Facilitator Superfamily (MFS) and recG protein families showed significant interaction with chromium ion only in the chromium tolerant A. flavus strain TERIBR1. CONCLUSIONS: Presence of nsSNPs and subsequent amino-acid alterations evidently influenced the 3D structures of the candidate proteins, which could have led to improved interaction with (CrO4- 2) ion. Such structural modifications might have enhanced chromium efflux efficiency of A. flavus (TERIBR1) and thereby offered the adaptation benefits in counteracting chromate stress. Our findings are of fundamental importance to the field of heavy-metal bio-remediation.

An Integrated Computational Framework to Assess the Mutational Landscape of α-L-Iduronidase IDUA Gene.

Mucopolysaccharidosis type I is a lysosomal genetic disorder caused due to the deficiency of the α-L-iduronidase enzyme (IDUA). Mutations associated with IDUA lead to mild to severe forms of diseases characterized by different clinical features. In the present study, we first performed a comprehensive analysis using various in silico prediction tools to screen and prioritize the missense mutations or nonsynonymous SNPs (nsSNPs) associated with IDUA. Subsequently, statistical analysis was empowered to examine the predictive ability and accuracy of the in silico prediction tool results supporting the disease phenotype ranging from mild to severe. Till date, no study has been carried out in IDUA in analyzing the impact of the nsSNPs at the structural level. In this context with the aid of pathogenic and stability prediction in silico tools, we identified nsSNPs R89Q, R89W, and P533R to be most deleterious and disease-causing having impact on the function of the protein. Extensive molecular dynamics analysis was performed using Gromacs to understand the deleterious nature of the mutants. Variations observed between the trajectory files of native and mutants R89Q, R89W, and P533R using Gromacs utilities enabled us to measure the adverse effects on the protein and could be the underlying reasons for the disease pathogenesis. These findings may be helpful in understanding the genotype-phenotype relationship and molecular basis of the disease to design drugs for better treatment. J. Cell. Biochem. 119: 555-565, 2018. © 2017 Wiley Periodicals, Inc.

Characterization of SNPs in the dopamine-ß-hydroxylase gene providing new insights into its structure-function relationship.

Dopamine-ß-hydroxylase (DBH, EC 1.14.17.1), an oxido-reductase that catalyses the conversion of dopamine to norepinephrine, is largely expressed in sympathetic neurons and adrenal medulla. Several regulatory and structural variants in DBH associated with various neuropsychiatric, cardiovascular diseases and a few that may determine enzyme activity have also been identified. Due to paucity of studies on functional characterization of DBH variants, its structure-function relationship is poorly understood. The purpose of the study was to characterize five non-synonymous (ns) variants that were prioritized either based on previous association studies or Sorting Tolerant From Intolerant (SIFT) algorithm. The DBH ORF with wild type (WT) and site-directed mutagenized variants were transfected into HEK293 cells to generate transient and stable lines expressing these variant enzymes. Activity was determined by UPLC-PDA and corresponding quantity by MRMHR on a TripleTOF 5600 MS respectively of spent media from stable cell lines. Homospecific activity computed for the WT and variant proteins showed a marginal decrease in A318S, W544S and R549C variants. In transient cell lines, differential secretion was observed in the case of L317P, W544S and R549C. Secretory defect in L317P was confirmed by localization in ER. R549C exhibited both decreased homospecific activity and differential secretion. Of note, all the variants were seen to be destabilizing based on in silico folding analysis and molecular dynamics (MD) simulation, lending support to our experimental observations. These novel genotype-phenotype correlations in this gene of considerable pharmacological relevance have implications for dopamine-related disorders.

Evidence for a role of glycoprotein M6a in dendritic spine formation and synaptogenesis.

Neuronal glycoprotein M6a belongs to the tetraspan proteolipid protein (PLP) family. Mutations in GPM6A gene have been related to mental disorders like schizophrenia, bipolar disorders and claustrophobia. M6a is expressed mainly in neuronal cells of the central nervous system and it has been extensively related to neuronal plasticity. M6a induces neuritogenesis and axon/filopodium outgrowth; however its mechanism of action is still unresolved. We recently reported that the integrity of the transmembrane domains (TMDs) 2 and 4 are critical for M6a filopodia induction. There is also experimental data suggesting that M6a might be involved in synaptogenesis. In this regard, we have previously determined that M6a is involved in filopodia motility, a process that is described in the first step of the filopodial model for synaptogenesis. In this work we analyzed the possible involvement of M6a in synaptogenesis and spinogenesis, and evaluated the effect of two non-synonymous SNPs present in the coding region of TMD2-GPM6A in these processes. The results showed that endogenous M6a colocalized with both, pre-synaptic (synaptophysin) and post-synaptic (NMDA-R1), markers along of neuronal soma and dendrites. M6a-overexpressing neurons displayed an increased number of synaptophysin and NMDA-R1 puncta and, also, an increased number of colocalization puncta between both markers. Conversely, the number of synaptic puncta markers in neurons expressing nsSNP variants was similar to those of control neurons. Overexpression of M6a is accompanied by an increase in spine density, particularly in mature spines, as compared with neurons expressing mGFP or GPM6A nsSNP variants. Taken together, these results suggest that M6a contributes positively to spine and, likely, synapse formation.