Post-translational Modification Sites Research Articles

Engineering Critical Residues of SOX9 Discovers a Variant with Potent Capacity to Induce Chondrocytes.

Articular cartilage plays vital roles as a friction minimizer and shock absorber during joint movement but has a poor capacity to self-repair when damaged through trauma or disease. Cartilage tissue engineering is an innovative technique for cartilage regeneration, yet its therapeutic application requires chondrocytes in large numbers. Direct reprogramming of somatic cells to chondrocytes by expressing SOX9, KLF4, and c-MYC offers a promising option to generate chondrocytes in sufficient numbers; however, the low efficiency of the reprogramming system warrants further improvement. Here we referred to structural and functional features of SOX9 and performed alanine-scanning mutagenesis of functionally critical residues in the HMG box and at putative posttranslational modification (PTM) sites. We discovered that a SOX9 variant H131A/K398A, doubly mutated in the HMG box (H131) and at a PTM site (K398), significantly upregulated expression of chondrogenic genes and potently induced chondrocytes from mouse embryonic fibroblasts (MEFs). The H131A/K398A variant remained unsumoylated in cells and exhibited a stronger DNA-binding activity than wild-type SOX9, especially when complexed with other proteins. Our results show that the novel SOX9 variant may be useful for efficient induction of chondrocytes and illuminate the strategic feasibility of mutating a transcription factor at functionally critical residues to expedite discovery of an optimized reprogramming factor.

Intrinsic disorder in PRAME and its role in uveal melanoma

IntroductionThe PReferentially expressed Antigen in MElanoma (PRAME) protein has been shown to be an independent biomarker for increased risk of metastasis in Class 1 uveal melanomas (UM). Intrinsically disordered proteins and regions of proteins (IDPs/IDPRs) are proteins that do not have a well-defined three-dimensional structure and have been linked to neoplastic development. Our study aimed to evaluate the presence of intrinsic disorder in PRAME and the role these structureless regions have in PRAME( +) Class 1 UM.MethodsA bioinformatics study to characterize PRAME’s propensity for the intrinsic disorder. We first used the AlphaFold tool to qualitatively assess the protein structure of PRAME. Then we used the Compositional Profiler and a set of per-residue intrinsic disorder predictors to quantify the intrinsic disorder. The Database of Disordered Protein Prediction (D2P2) platform, IUPred, FuzDrop, fIDPnn, AUCpred, SPOT-Disorder2, and metapredict V2 allowed us to evaluate the potential functional disorder of PRAME. Additionally, we used the Search Tool for the Retrieval of Interacting Genes (STRING) to analyze PRAME's potential interactions with other proteins.ResultsOur structural analysis showed that PRAME contains intrinsically disordered protein regions (IDPRs), which are structureless and flexible. We found that PRAME is significantly enriched with serine (p-value < 0.05), a disorder-promoting amino acid. PRAME was found to have an average disorder score of 16.49% (i.e., moderately disordered) across six per-residue intrinsic disorder predictors. Our IUPred analysis revealed the presence of disorder-to-order transition (DOT) regions in PRAME near the C-terminus of the protein (residues 475–509). The D2P2 platform predicted a region from approximately 140 and 175 to be highly concentrated with post-translational modifications (PTMs). FuzDrop predicted the PTM hot spot of PRAME to be a droplet-promoting region and an aggregation hotspot. Finally, our analysis using the STRING tool revealed that PRAME has significantly more interactions with other proteins than expected for randomly selected proteins of the same size, with the ability to interact with 84 different partners (STRING analysis result: p-value < 1.0 × 10–16; model confidence: 0.400).ConclusionOur study revealed that PRAME has IDPRs that are possibly linked to its functionality in the context of Class 1 UM. The regions of functionality (i.e., DOT regions, PTM sites, droplet-promoting regions, and aggregation hotspots) are localized to regions of high levels of disorder. PRAME has a complex protein–protein interaction (PPI) network that may be secondary to the structureless features of the polypeptide. Our findings contribute to our understanding of UM and suggest that IDPRs and DOT regions in PRAME may be targeted in developing new therapies for this aggressive cancer.BBcyEW8tS6_KkPjyEwduzxVideo

α- and β-tubulin C-terminal tails with distinct modifications are crucial for ciliary motility and assembly.

α- and β-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTTs). The function of this region in cilia and flagella is still unclear, except that glutamates in CTTs act as the sites for post-translational modifications that affect ciliary motility. The unicellular alga Chlamydomonas possesses only two α-tubulin and two β-tubulin genes, each pair encoding an identical protein. This simple gene organization might enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutant strains expressing tubulins with modified CTTs. We found that the mutant strain in which four glutamate residues in the α-tubulin CTT had been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant strain lacking the glutamate-rich region of the β-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to mutant strains harboring a mutation in a subunit of katanin, the function of which has been shown to depend on the β-tubulin CTT. Therefore, our study reveals distinct and important roles of α- and β-tubulin CTTs in the formation and function of cilia.

An in-silico analysis predicting the impact of coding single nucleotide polymorphisms (SNPs) in the human multidrug pump ABCG2

ATP-Binding Cassette transporter G2 (ABCG2) is a multidrug resistance pump involved in pathophysiology and pharmacoresistance in various neurological, and neurogenerative disorders. Single nucleotide polymorphisms (SNPs) of ABCG2 affecting expression or function may be involved in the pharmacokinetics of its substrate drugs, influencing the bioavailability leading to disease progression. Thus, pathophysiology and pharmacogenetics linked to ABCG2 can be comprehended by understanding the functions of its SNPs. Identification of these SNPs predictive of pharmacoresistance and disease progression is still a major challenge. Here, we have prioritized ABCG2 SNPs for the identification and validation of deleterious/damaging coding SNPs (cSNPs) using an in-silico approach. Out of 965 cSNPs, only 6 were considered to be highly deleterious by five in-silico computational tools. These cSNPs were submitted to MutPred and PMut for further validation followed by subjected to I-Mutant 2.0 for protein stability analysis. We found 5 cSNPs with a DDG score less than −0.5, suggesting a damaging effect. Based on the conservation of amino acid residues, post-translational modification sites, and structural superimposition, rs3116448 (S248A) and rs41282401 (D296H) were predicted as high-risk cSNPs. Molecular dynamics (MD) simulations revealed that the substitution of the residues S248A in the ABCG2 model altered the structural conformation of the protein and may influence the functional activity as per previously reported in vitro studies. Thus, findings from our study, for the first time, identified clinically important ABCG2 variations utilizing a substantial bioinformatics pipeline that not only provides important potential targets in the field of pharmacogenomics but also curtails study costs.

Identification genetic variations in some heat shock protein genes of Tali goat breed and study their structural and functional effects on relevant proteins.

Animals of different regions have adapted to adverse environmental conditions by modifying their phenotypic and genotypic characteristics in the long run. In this study, the effect of genetic variations of 10 heat shock protein (HSP) genes (HSP70A4, HSP70A9, HSP40C17, HSP40C27, HSP90AA1, HSP90AB1, HSPB7, HSPB11, HSPD1 and HSPE1) on the three-dimensional protein structure and function of proteins in Tali goat (a tropical breed) were studied and were compared with Saanen goat (as a sensitive breed). A pooled DNA of 15 samples from blood was sequenced and mapped to the goat reference sequence. The bioinformatics analysis was used to identify nsSNPs in the Tali breed and was compared with the Saanen goat. Four online bioinformatics tools (Sorting Intolerant from Tolerant, Protein Variation Effect Analyzer, Polymorphism Phenotyping version2 and Single Nucleotide Polymorphism Database and Gene Ontology) showed three deleterious missense nsSNPs and seven natural missense SNPs in these HSPs genes of Tali goat. Out of 10 reported nsSNPs, 5 nsSNPs in HSP70A4, 1 nsSNP inHSP70A9, 2 nsSNPs in HSP40C17, 1 nsSNP in HSP40C27 and 1 nsSNP in HSPD1 were detected. ConSurf tools showed that the majority of the predicted nsSNPs occur in conserved sites. Moreover, several post-translational modification (PTM) predictors computed the probability of post-translation change of nsSNPs. The putative phosphorylation and glycosylation sites in HSPs proteins were substitutions rs669769139 and rs666336692 of the Tali goat breed. These results on the effect of type of genetic variants on the function of HSP proteins will assist to predict the resistance to hard conditions in goat breeds. Considering that the identified SNPid rs669769139 (S248) which is located on the N-terminal ATPase domain of HSP70A4 is a PTM site with a highly conserved score and a natural substitution on changing the stability and benign protein that can affect the functional and structural characterization of HSPs protein for adaptation to the local climate.

Class III peroxidase genes in the moss Dicranum scoparium: Identification and abiotic stress induced expression analysis

Class III peroxidases (POD: EC 1.11.1.7) are classical secretory plant peroxidases belonging to a large multigene family with diverse functions. Members of the POD family have been well-studied and characterized in many plants, including three species of bryophytes, but not from the moss Dicranum scoparium Hedw. Ecologically, D. scoparium is a very important species, which has a widespread distribution throughout the Holarctic. Here we present the first comprehensive report on the POD gene family in D. scoparium, identifying 22 genes encoding PODs (DsPODs), two of which were cloned for verification. All genes were deposited to GenBank under the third-party annotation (Accession numbers TPA: BK061169 – BK061190). Here, we present an in silico study of the physicochemical properties of these proteins. Analyses of conserved domains and subcellular localization suggested that DsPODs have classical peroxidase domain structure; they are secretory proteins and most of them are extracellular. Eight DsPODs highly homologous to Class III peroxidases from the mosses Pohlia nutans and Physcomitrium patens were further microcharacterized. All eight DsPODs possess a haem ligand and active sites necessary for enzymatic activity; they also contained sites for posttranslational modifications. Prediction of secondary structure indicated that these proteins mainly consist of α-helices and random coils. Experiments involving the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) showed that DsPOD1, DsPOD2, DsPOD6, and DsPOD8 are differentially upregulated in response to stress. The stresses applied here included CdCl2, paraquat, unfavorable temperatures, and a hydration-desiccation-rehydration cycle. Our results indicate that Class III PODs contribute to the abiotic stress tolerance of D. scoparium, and specific DsPOD genes may play diverse roles in the response of the moss to stress.

Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden.

Aspen mosaic-associated virus (AsMaV) is a newly identified Emaravirus, in the family Fimoviridae, Bunyavirales, associated with mosaic symptoms in aspen trees (Populus tremula). Aspen trees are widely distributed in Europe and understanding the population structure of AsMaV may aid in the development of better management strategies. The virus genome consists of five negative-sense single-stranded RNA (-ssRNA) molecules. To investigate the genetic diversity and population parameters of AsMaV, different regions of the genome were amplified and analyzed and full-length sequence of the divergent isolates were cloned and sequenced. The results show that RNA3 or nucleoprotein is a good representative for studying genetic diversity in AsMaV. Developed RT-PCR-RFLP was able to identify areas with a higher number of haplotypes and could be applied for screening the large number of samples. In general, AsMaV has a conserved genome and based on the phylogenetic studies, geographical structuring was observed in AsMaV isolates from Sweden and Finland, which could be attributed to founder effects. The genome of AsMaV is under purifying selection but not distributed uniformly on genomic RNAs. Distant AsMaV isolates displayed amino acid sequence variations compared to other isolates, and bioinformatic analysis predicted potential post-translational modification sites in some viral proteins.

The interplay of post-translational protein modifications in Arabidopsis leaves during photosynthesis induction.

Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light-dependent post-translational modifications (PTM) and have been studied at depth at the level of individual proteins. In contrast, a global picture of the light-dependent PTMome dynamics is lacking, leaving the response of a large proportion of cellular function undefined. Here, we investigated the light-dependent metabolome and proteome changes in Arabidopsis rosettes in a time resolved manner to dissect their kinetic interplay, focusing on phosphorylation, lysine acetylation, and cysteine-based redox switches. Of over 24 000 PTM sites that were detected, more than 1700 were changed during the transition from dark to light. While the first changes, as measured 5 min after onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin-Benson cycle and the synthesis of sugars at later timepoints. Our data reveal connections between metabolism and PTM-based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insight into the extent by which photosynthesis reprograms global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function.

Characterization of Adeno-Associated Virus Capsid Proteins by Microflow Liquid Chromatography Coupled with Mass Spectrometry.

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC-MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance.

Characterizing proteomic and transcriptomic features of missense variants in amyotrophic lateral sclerosis genes.

Within recent years, there has been a growing number of genes associated with amyotrophic lateral sclerosis (ALS), resulting in an increasing number of novel variants, particularly missense variants, many of which are of unknown clinical significance. Here, we leverage the sequencing efforts of the ALS Knowledge Portal (3,864 individuals with ALS and 7,839 controls) and Project MinE ALS Sequencing Consortium (4,366 individuals with ALS and 1,832 controls) to perform proteomic and transcriptomic characterization of missense variants in 24 ALS-associated genes. The two sequencing datasets were interrogated for missense variants in the 24 genes, and variants were annotated with genomic database minor allele frequencies, ClinVar pathogenicity classifications, protein sequence features including Uniprot functional site annotations and PhosphoSitePlus post-translational modification (PTM) site annotations, structural features from AlphaFold predicted monomeric 3D structures, and transcriptomic expression levels from Genotype-Tissue Expression (GTEx). We then applied missense variant enrichment and gene-burden testing following binning of variation based on the selected proteomic and transcriptomic features to identify those most relevant to pathogenicity in ALS-associated genes. Using predicted human protein structures from AlphaFold, we determined that missense variants carried by individuals with ALS were significantly enriched in β-sheets and α-helices, as well as in core, buried, or moderately buried regions. At the same time, we identified that hydrophobic amino acid residues, compositionally biased protein regions and protein-protein interaction regions are predominantly enriched in missense variants carried by individuals with ALS. Assessment of expression level based on transcriptomics also revealed enrichment of variants of high and medium expression across all tissues and within the brain. We further explored enriched features of interest using burden analyses and identified individual genes were indeed driving certain enrichment signals. A case study is presented for SOD1 to demonstrate proof of concept of how enriched features may aid in defining variant pathogenicity. Our results present proteomic and transcriptomic features that are important indicators of missense variant pathogenicity in ALS and are distinct from features associated with neurodevelopmental disorders.

Bioinformatics-based prediction and screening of immunogenic epitopes of Toxoplasma gondii rhoptry proteins 7, 21 and 22 as candidate vaccine target

IntroductionToxoplasmosis is a well-known zoonotic disease caused by Toxoplasma gondii. The main causes of the disease range from eating undercooked or contaminated meat and shellfish to cleaning litter trays into which cats that excreted toxoplasma via faeces. This pathogen can live for a very long time, possibly a lifetime, within the bodies of humans and other animals. Aims and objectivesThis study aimed to predict and analyse candidate immunogenic epitopes for vaccine development by evaluating the physio-chemical properties, multiple sequence alignment, secondary and tertiary structures, phosphorylation sites, transmembrane domains, and signal peptides, of T. gondii rhoptry proteins ROP7, ROP21, and ROP22 using bioinformatics tools. MethodsTo find immunogenic epitopes of rhoptry proteins, numerous bioinformatics web servers were used containing multiple sequence alignment, physiochemical properties, antigenicity and allergenicity, post-translational modification sites (PTMs), signal peptides, transmembrane domains, secondary and tertiary structures, and screening of predicted epitopes. We evaluated immunogenic linear B-cell epitopes as candidate proteins for vaccine development. ResultsNine epitopes were identified for each protein, and analysis of immunogenicity, revealed three candidate epitopes for ROP7, one for ROP21, and four for ROP22. Among all candidate epitopes, ROP22 contained the most immunogenic epitopes with immunogenicity score of 0.50575. ConclusionWe acquired detailed information on predicted immunogenic epitopes using in-silico methods. The results provide a foundation for further experimental analysis of toxoplasmosis, and potential vaccine development.

Roles of post-translational modifications of C-type lectin receptor-induced signaling cascades in innate immune responses against Candida albicans

Candida albicans (C. albicans), a conditional pathogenic fungus, is widespread in nature and can live in symbiosis with organisms in small quantities. When the normal microflora is imbalanced, the epithelial barrier is disrupted or the immune system becomes dysfunctional, C. albicans can change from commensal to pathogenic pathogen, causing both superficial and life-threatening systemic infections with no effective treatment. The morbidity and mortality of invasive Candida infections in perioperative patients are high due to underlying chronic diseases, immune deficiencies, and pathophysiological disorders. C-type lectin receptors (CLRs) are the main pattern-recognition receptors for fungal activation of innate immunity and host defense. Upon binding to ligands, CLRs induce multiple signal transduction cascades followed by activation of nuclear factor kappa B through spleen tyrosine kinase - and caspase recruitment domain containing protein 9-dependent pathways. Analyzing the effects of regulatory CLR-induced signaling cascades on host immune cells is critical for understanding the molecular mechanism in regulating antifungal immunity. As one of the core factors in host innate immune regulation, protein post-translational modifications regulate the strength of immune effects by modulating protein conformation, stability, affinity, subcellular localization, etc. This makes the post-translational modification sites promising as potential targets for modulating antifungal immunity. This review primarily described the study progress of post-translational modifications in controlling CLR-induced signaling cascades throughout the process of innate immunity against C. albicans. We aim to provide better understanding of these mechanisms and aid in the identification and development of biomarkers and drug targets for invasive candidiasis.

Comparative analysis of adipokinetic hormones and their receptors in Blattodea reveals novel patterns of gene evolution.

Adipokinetic hormone (AKH) is a neuropeptide produced in the insect corpora cardiaca that plays an essential role in mobilising carbohydrates and lipids from the fat body to the haemolymph. AKH acts by binding to a rhodopsin-like G protein-coupled receptor (GPCR), the adipokinetic hormone receptor (AKHR). In this study, we tackle AKH ligand and receptor gene evolution as well as the evolutionary origins of AKH gene paralogues from the order Blattodea (termites and cockroaches). Phylogenetic analyses of AKH precursor sequences point to an ancient AKH gene duplication event in the common ancestor of Blaberoidea, yielding a new group of putative decapeptides. In total, 16 different AKH peptides from 90 species were obtained. Two octapeptides and seven putatively novel decapeptides are predicted for the first time. AKH receptor sequences from 18 species, spanning solitary cockroaches and subsocial wood roaches as well as lower and higher termites, were subsequently acquired using classical molecular methods and in silico approaches employing transcriptomic data. Aligned AKHR open reading frames revealed 7 highly conserved transmembrane regions, a typical arrangement for GPCRs. Phylogenetic analyses based on AKHR sequences support accepted relationships among termite, subsocial (Cryptocercus spp.) and solitary cockroach lineages to a large extent, while putative post-translational modification sites do not greatly differ between solitary and subsocial roaches and social termites. Our study provides important information not only for AKH and AKHR functional research but also for further analyses interested in their development as potential candidates for biorational pest control agents against invasive termites and cockroaches.

In Silico Analysis to Predict the Pathogenic Variants of CANT1 Gene Causing Desbuquios Dysplasia (DBQD) Type 1

Desbuquois dysplasia (DBQD) is an autosomal recessive chondrodysplasia that belongs to the multiple dislocation group and causes parental and afterbirth growth retardation, hand and proximal femur abnormalities, joint laxity, and scoliosis. Several missense and splice site mutations in CANT1 gene are linked with the development of DBQD. In silico approaches can predict the pathogenic variations causing hereditary diseases. Hence, in the current study, in silico analysis was used to forecast the variants of CANT1 gene that harm the functionality of calcium-dependent nucleotidase. A total of 281 variants with uncertain significance, retrieved from the gnomAD, dbSNP, ClinVar, and Variation Viewer databases, were analyzed using CADD, Meta SNP, CAPiCE, and Condel to predict 61 highly pathogenic variants. Stability change predicting computational tools were applied to filter 19 highly pathogenic amino acid variants that impact protein dynamics via sample conformation or during vibrational entropy. UCSF Chimera was used for interactive visualization and analysis of unwanted interaction among 5 variants in the molecular structure of the protein. Ligand binding computational tools were used to interpret the protein-ligand interactions. A total of three (3) post-translational modification sites were also predictably disrupted by 16 variants. Spice and HSF 3.1 tools were applied to 95 variants to check their disease-causing potential. The variants of the gene were analyzed using computational tools based on different algorithms. The most damaging variants of CANT1 gene that can affect the functionality and stability of the protein were predicted. It was determined that an extensive in silico analysis can determine the likely pathogenic variations for further in vitro experimental analysis.

Implications of specific lysine residues within ataxin-3 for the molecular pathogenesis of Machado-Joseph disease.

Lysine residues are one of the main sites for posttranslational modifications of proteins, and lysine ubiquitination of the Machado-Joseph disease protein ataxin-3 is implicated in its cellular function and polyglutamine expansion-dependent toxicity. Despite previously undertaken efforts, the individual roles of specific lysine residues of the ataxin-3 sequence are not entirely understood and demand further analysis. By retaining single lysine residues of otherwise lysine-free wild-type and polyglutamine-expanded ataxin-3, we assessed the effects of a site-limited modifiability on ataxin-3 protein levels, aggregation propensity, localization, and stability. We confirmed earlier findings that levels of lysine-free ataxin-3 are reduced due to its decreased stability, which led to a diminished load of SDS-insoluble species of its polyglutamine-expanded form. The isolated presence of several single lysine residues within the N-terminus of polyglutamine-expanded ataxin-3 significantly restored its aggregate levels, with highest fold changes induced by the presence of lysine 8 or lysine 85, respectively. Ataxin-3 lacking all lysine residues presented a slightly increased nuclear localization, which was counteracted by the reintroduction of lysine 85, whereas presence of either lysine 8 or lysine 85 led to a significantly higher ataxin-3 stability. Moreover, lysine-free ataxin-3 showed increased toxicity and binding to K48-linked polyubiquitin chains, whereas the reintroduction of lysine 85, located between the ubiquitin-binding sites 1 and 2 of ataxin-3, normalized its binding affinity. Overall, our data highlight the relevance of lysine residues 8 and 85 of ataxin-3 and encourage further analyses, to evaluate the potential of modulating posttranslational modifications of these sites for influencing pathophysiological characteristics of the Machado-Joseph disease protein.

Comprehensive In Silico Characterization of the Coding and Non-Coding SNPs in Human Dectin-1 Gene with the Potential of High-Risk Pathogenicity Associated with Fungal Infections.

The human C-type lectin domain family 7 member A (CLEC7A) gene encodes a Dectin-1 protein that recognizes beta-1,3-linked and beta-1,6-linked glucans, which form the cell walls of pathogenic bacteria and fungi. It plays a role in immunity against fungal infections through pathogen recognition and immune signaling. This study aimed to explore the impact of nsSNPs in the human CLEC7A gene through computational tools (MAPP, PhD-SNP, PolyPhen-1, PolyPhen-2, SIFT, SNAP, and PredictSNP) to identify the most deleterious and damaging nsSNPs. Further, their effect on protein stability was checked along with conservation and solvent accessibility analysis by I-Mutant 2.0, ConSurf, and Project HOPE and post-translational modification analysis using MusiteDEEP. Out of the 28 nsSNPs that were found to be deleterious, 25 nsSNPs affected protein stability. Some SNPs were finalized for structural analysis with Missense 3D. Seven nsSNPs affected protein stability. Results from this study predicted that C54R, L64P, C120G, C120S, S135C, W141R, W141S, C148G, L155P, L155V, I158M, I158T, D159G, D159R, I167T, W180R, L183F, W192R, G197E, G197V, C220S, C233Y, I240T, E242G, and Y3D were the most structurally and functionally significant nsSNPs in the human CLEC7A gene. No nsSNPs were found in the predicted sites for post-translational modifications. In the 5' untranslated region, two SNPs, rs536465890 and rs527258220, showed possible miRNA target sites and DNA binding sites. The present study identified structurally and functionally significant nsSNPs in the CLEC7A gene. These nsSNPs may potentially be used for further evaluation as diagnostic and prognostic biomarkers.

Genomic study of TEX15 variants: prevalence and allelic heterogeneity in men with spermatogenic failure.

Introduction: Human spermatogenesis is a highly intricate process that requires the input of thousands of testis-specific genes. Defects in any of them at any stage of the process can have detrimental effects on sperm production and/or viability. In particular, the function of many meiotic proteins encoded by germ cell specific genes is critical for maturation of haploid spermatids and viable spermatozoa, necessary for fertilization, and is also extremely sensitive to even the slightest change in coding DNA. Methods: Here, using whole exome and genome approaches, we identified and reported novel, clinically significant variants in testis-expressed gene 15 (TEX15), in unrelated men with spermatogenic failure (SPGF). Results: TEX15 mediates double strand break repair during meiosis. Recessive loss-of-function (LOF) TEX15 mutations are associated with SPGF in humans and knockout male mice are infertile. We expand earlier reports documenting heterogeneous allelic pathogenic TEX15 variants that cause a range of SPGF phenotypes from oligozoospermia (low sperm) to nonobstructive azoospermia (no sperm) with meiotic arrest and report the prevalence of 0.6% of TEX15 variants in our patient cohort. Among identified possible LOF variants, one homozygous missense substitution c.6835G>A (p.Ala2279Thr) co-segregated with cryptozoospermia in a family with SPGF. Additionally, we observed numerous cases of inferred in trans compound heterozygous variants in TEX15 among unrelated individuals with varying degrees of SPGF. Variants included splice site, insertions/deletions (indels), and missense substitutions, many of which resulted in LOF effects (i.e., frameshift, premature stop, alternative splicing, or potentially altered posttranslational modification sites). Conclusion: In conclusion, we performed an extensive genomic study of familial and sporadic SPGF and identified potentially damaging TEX15 variants in 7 of 1097 individuals of our combined cohorts. We hypothesize that SPGF phenotype severity is dictated by individual TEX15 variant's impact on structure and function. Resultant LOFs likely have deleterious effects on crossover/recombination in meiosis. Our findings support the notion of increased gene variant frequency in SPGF and its genetic and allelic heterogeneity as it relates to complex disease such as male infertility.

An In silico Approach towards Finding the Cancer-Causing Mutations in Human MET Gene.

Mesenchymal-epithelial transition (MET) factor is a proto-oncogene encoding tyrosine kinase receptor with hepatocyte growth factor (HGF) or scatter factor (SF). It is found on the human chromosome number 7 and regulates the diverse cellular mechanisms of the human body. The impact of mutations occurring in the MET gene is demonstrated by their detrimental effects on normal cellular functions. These mutations can change the structure and function of MET leading to different diseases such as lung cancer, neck cancer, colorectal cancer, and many other complex syndromes. Hence, the current study focused on finding deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) and their subsequent impact on the protein's structure and functions, which may contribute to the emergence of cancers. These nsSNPs were first identified utilizing computational tools like SIFT, PROVEAN, PANTHER-PSEP, PolyPhen-2, I-Mutant 2.0, and MUpro. A total of 45359 SNPs of MET gene were accumulated from the database of dbSNP, and among them, 1306 SNPs were identified as non-synonymous or missense variants. Out of all 1306 nsSNPs, 18 were found to be the most deleterious. Moreover, these nsSNPs exhibited substantial effects on structure, binding affinity with ligand, phylogenetic conservation, secondary structure, and post-translational modification sites of MET, which were evaluated using MutPred2, RaptorX, ConSurf, PSIPRED, and MusiteDeep, respectively. Also, these deleterious nsSNPs were accompanied by changes in properties of MET like residue charge, size, and hydrophobicity. These findings along with the docking results are indicating the potency of the identified SNPs to alter the structure and function of the protein, which may lead to the development of cancers. Nonetheless, Genome-wide association study (GWAS) studies and experimental research are required to confirm the analysis of these nsSNPs.

Computational Exploration of Functional and Structural Impact of Single Nucleotide Changes in DNMT3A Gene among Acute Myeloid Leukemia Patients

Acute myeloid leukemia (AML) is a blood cell malignancy of the myeloid line, characterized by fast proliferation of aberrant cells that build up in the bone marrow and blood, interfering with normal blood cell synthesis. DNMT3A is a DNA methyltransferase that plays a role in DNA methylation, an epigenetic modification associated with gene expression regulation. DNMT3A mutations are frequently found in AML and are associated with poor prognosis. Objective: To evaluate the impact of DNMT3A mutations on protein structure and function, specifically in the context of AML. Methods: SNPs of DNMT3A gene reported in AML (R882P, R882L, R882S, R882G, and R882C) were retrieved from National Centre for Biotechnology Information (NCBI) database and different in silico approaches were used to investigate how these mutations affect protein structure and function. Results: Prediction tools indicated that mutations are pathogenic affecting DNMT3A function and were found in evolutionarily conserved regions. Protein stability analysis showed that mutations reduce DNMT3A's structural stability, alter secondary structure of the protein, particularly helices, interacts with other proteins and reduce protein-protein affinity. RNA folding analysis revealed abnormal folding patterns caused by mutant, affecting protein translation. DNMT3A expression was reported to be considerably greater in AML compared to normal tissues, and mutations were associated with poor overall survival in AML patients. Methylation levels and post-translational modification sites of DNMT3A were also investigated. Conclusions: Overall, this research highlighted the negative impact of DNMT3A mutations on protein structure and function, emphasizing their importance in the development and prognosis of AML. Acute myeloid leukemia (AML) is a blood cell malignancy of the myeloid line, characterized by fast proliferation of aberrant cells that build up in the bone marrow and blood, interfering with normal blood cell synthesis. DNMT3A is a DNA methyltransferase that plays a role in DNA methylation, an epigenetic modification associated with gene expression regulation. DNMT3A mutations are frequently found in AML and are associated with poor prognosis. Objective: To evaluate the impact of DNMT3A mutations on protein structure and function, specifically in the context of AML. Methods: SNPs of DNMT3A gene reported in AML (R882P, R882L, R882S, R882G, and R882C) were retrieved from National Centre for Biotechnology Information (NCBI) database and different in silico approaches were used to investigate how these mutations affect protein structure and function. Results: Prediction tools indicated that mutations are pathogenic affecting DNMT3A function and were found in evolutionarily conserved regions. Protein stability analysis showed that mutations reduce DNMT3A's structural stability, alter secondary structure of the protein, particularly helices, interacts with other proteins and reduce protein-protein affinity. RNA folding analysis revealed abnormal folding patterns caused by mutant, affecting protein translation. DNMT3A expression was reported to be considerably greater in AML compared to normal tissues, and mutations were associated with poor overall survival in AML patients. Methylation levels and post-translational modification sites of DNMT3A were also investigated. Conclusions: Overall, this research highlighted the negative impact of DNMT3A mutations on protein structure and function, emphasizing their importance in the development and prognosis of AML.

An In Silico Functional Analysis of Non-Synonymous Single-Nucleotide Polymorphisms of Bovine CMAH Gene and Potential Implication in Pathogenesis.

The sugar molecule N-glycolylneuraminic acid (Neu5Gc) is one of the most common sialic acids discovered in mammals. Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) catalyses the conversion of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc, and it is encoded by the CMAH gene. On the one hand, food metabolic incorporation of Neu5Gc has been linked to specific human diseases. On the other hand, Neu5Gc has been shown to be highly preferred by some pathogens linked to certain bovine diseases. We used various computational techniques to perform an in silico functional analysis of five non-synonymous single-nucleotide polymorphisms (nsSNPs) of the bovine CMAH (bCMAH) gene identified from the 1000 Bull Genomes sequence data. The c.1271C>T (P424L) nsSNP was predicted to be pathogenic based on the consensus result from different computational tools. The nsSNP was also predicted to be critical based on sequence conservation, stability, and post-translational modification site analysis. According to the molecular dynamic simulation and stability analysis, all variations promoted stability of the bCMAH protein, but mutation A210S significantly promoted CMAH stability. In conclusion, c.1271C>T (P424L) is expected to be the most harmful nsSNP among the five detected nsSNPs based on the overall studies. This research could pave the way for more research associating pathogenic nsSNPs in the bCMAH gene with diseases.