Iterative Threading Assembly Refinement Research Articles

Cross-species molecular docking method to support predictions of species susceptibility to chemical effects

The advancement of protein structural prediction tools, exemplified by AlphaFold and Iterative Threading ASSEmbly Refinement, has enabled the prediction of protein structures across species based on available protein sequence and structural data. In this study, we introduce an innovative molecular docking method that capitalizes on this wealth of structural data to enhance predictions of chemical susceptibility across species. We demonstrated this method using the androgen receptor as a pertinent modulator of endocrine function. By using protein structures, this method contextualizes species susceptibility within a functional framework and helps to integrate molecular docking into the repertoire of New Approach Methodologies (NAMs) that support the Next-Generation Risk Assessment (NGRA) paradigm through the novel integration of various open-source tools.

A novel COL4A5 splicing mutation causes alport syndrome in a Chinese family

BackgroundAlport syndrome (AS) is characterised by haematuria, proteinuria, a gradual decline in kidney function, hearing loss, and eye abnormalities. The disease is caused by mutations in COL4An (n = 3, 4, 5) that encodes 3–5 chains of type IV collagen in the glomerular basement membrane. AS has three genetic models: X-linked, autosomal recessive, and autosomal dominant. The most common type of AS is X-linked AS, which is caused by COL4A5.MethodsWe enrolled children with renal insufficiency and a family history of kidney disorders. The proband was identified using whole-exome sequencing. Sanger sequencing was performed to verify the mutation site. Minigene technology was used to analyse the influence of mutant genes on pre-mRNA shearing, and the Iterative Threading ASSEmbly Refinement (I-TASSER) server was used to analyse the protein structure changes.ResultsThe proband, together with her mother and younger brother, displayed microscopic haematuria and proteinuria, Pathological examination revealed mesangial hyperplasia and sclerosis. A novel mutation (NM_000495.5 c.4298-8G > A) in the intron of the COL4A5 gene in the proband was discovered, which was also present in the proband’s mother, brother, and grandmother. In vitro minigene expression experiments verified that the c.4298-8G > A mutation caused abnormal splicing, leading to the retention of six base pairs at the end of intron 46. The I-TASSER software predicted that the mutation affected the hydrogen-bonding structure of COL4A5 and the electrostatic potential on the surface of the protein molecules.ConclusionsBased on the patient’s clinical history and genetic traits, we conclude that the mutation at the splicing site c.4298-8G > A of the COL4A5 gene is highly probable to be the underlying cause within this particular family. This discovery expands the genetic spectrum and deepens our understanding of the molecular mechanisms underlying AS.

Investigation of the Molecular Interactions of Vaccine Adjuvants: Can a Strategic Trio of Toll-like Receptor Agonists Enhance Efficacy in a Multifaceted Approach?

Background: The imperative need for effective vaccines against viral diseases has intensified research on adjuvants to enhance immune responses. Toll-like receptor (TLR) agonists, such as Imiquimod, Resiquimod, and CpG oligodeoxynucleotides (CpG ODN), are some crucial components in vaccine formulations. This study investigated the molecular interactions and binding affinities of TLR4, TLR7/8, and TLR9 agonists, through in silico techniques. Methods: The three-dimensional structure of human TLR9 was predicted using Iterative Threading ASSEmbly Refinement. Homology modeling was employed using a multi-step approach to generate the human TLR9 model including template identification, ab initio modeling, iterative refinement, and final model generation of the crystallographic structure. Molecular docking and simulation studies were performed using AutoDock Vina, HDOCK, and GROningen MAchine for Chemical Simulations tools. The molecular docking study revealed binding sites and binding affinities of adjuvants in the binding regions of target TLRs. Molecular dynamics simulations and Molecular Mechanics/Poisson-Boltzmann Surface Area calculations ascertain the stability and binding energies of the TLR-adjuvant complexes. Results: Results indicate distinct interactions, with Resiquimod showing superior affinity toward TLR7. QS21, on the other hand, emerged as a potent TLR4 agonist, while CpG ODN 2006 binds specifically to TLR9. The study proposes a strategic combination of QS21, Resiquimod, and CpG ODN 2006 as a potential adjuvant system, offering a multifaceted approach to enhance vaccine efficacy. Conclusions: This study furnishes preliminary data and establishes the foundational framework for subsequent inquiries encompassing both in vitro and in vivo studies concerning vaccine adjuvant systems.

Ab initio modeling and ligand docking of quercetin and the MC-LR transporter protein Oatp1b2/OATP1B3

Trans-membrane proteins (TMPs) play a crucial role in the translocation of organic and inorganic molecules. Unlike other proteins, TMPs are difficult to model structurally because of their location within the amphipathic plasma membrane. In this study, we focused on examining the transport of the cyanotoxin microcystin-LR (MC-LR) through organic ion transporting polypeptides (OATPs) and whether the bioactive phytoconstituent quercetin can function as a barrier to the transportation of MC-LR. To test this hypothesis, we first modeled the transporters OATP1B3 and Oatp1b2 localized in the human and mouse liver, respectively, by ab initio modeling with the Iterative Threading ASSEmbly Refinement server and refined the generated model using the refinement tool of the ModLoop server. Using different tools and servers, the structural quality of the transmembrane helices was validated and found to be an accurate structure of a TMP. Docking analysis was performed with the ligands MC-LR and quercetin with both OATPs using the PatchDock and FireDock online servers. The results, in the form of the global energy of both docked structures, were based on predictions made earlier. The Oatp1b2 global energy for quercetin was −36.4 ​kcal/mol, compared with the corresponding value at the MC-LR location which was only −5.59 ​kcal/mol. Similarly, in the case of OATP1B3 with quercetin, the global energy was found to be −39.0 ​kcal/mol, whereas with MC-LR it was −15.6 ​kcal/mol. These results clearly show that quercetin competitively inhibits the binding of MC-LR to its respective targets.

Comparison, Analysis, and Molecular Dynamics Simulations of Structures of a Viral Protein Modeled Using Various Computational Tools.

The structural analysis of proteins is a major domain of biomedical research. Such analysis requires resolved three-dimensional structures of proteins. Advancements in computer technology have led to progress in biomedical research. In silico prediction and modeling approaches have facilitated the construction of protein structures, with or without structural templates. In this study, we used three neural network-based de novo modeling approaches-AlphaFold2 (AF2), Robetta-RoseTTAFold (Robetta), and transform-restrained Rosetta (trRosetta)-and two template-based tools-the Molecular Operating Environment (MOE) and iterative threading assembly refinement (I-TASSER)-to construct the structure of a viral capsid protein, hepatitis C virus core protein (HCVcp), whose structure have not been fully resolved by laboratory techniques. Templates with sufficient sequence identity for the homology modeling of complete HCVcp are currently unavailable. Therefore, we performed domain-based homology modeling for MOE simulations. The templates for each domain were obtained through sequence-based searches on NCBI and the Protein Data Bank. Then, the modeled domains were assembled to construct the complete structure of HCVcp. The full-length structure and two truncated forms modeled using various computational tools were compared. Molecular dynamics (MD) simulations were performed to refine the structures. The root mean square deviation of backbone atoms, root mean square fluctuation of Cα atoms, and radius of gyration were calculated to monitor structural changes and convergence in the simulations. The model quality was evaluated through ERRAT and phi-psi plot analysis. In terms of the initial prediction for protein modeling, Robetta and trRosetta outperformed AF2. Regarding template-based tools, MOE outperformed I-TASSER. MD simulations resulted in compactly folded protein structures, which were of good quality and theoretically accurate. Thus, the predicted structures of certain proteins must be refined to obtain reliable structural models. MD simulation is a promising tool for this purpose.

Designing and cloning of fusion protein CpsA-CpsC-L-ACAN

BackgroundCervical cancer is the fourth most common cause of cancer and the fourth most common cause of cancer deaths in women. Some reports have shown the effect of Streptococcus agalactiae proteins and capsule products against cancer cell lines. MethodsThis study aimed to design and produce a fusion of recombinant protein (containing the capsules of Streptococcus agalactiaes with a linker and Antti cancer sequences) CpsA-CpsC-L-ACAN in the plasmid pET-22b (+) vector. Construct pET-22b (+) was designed by researchers, optimized with bioinformatics software, and synthesized by a Biometrics company. For the confirmation of recombinant protein, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed. Antibacterial activity was performed according to protocol CLSI2020. Based on bioinformatics analysis, the recombinant protein had good spatial structure and stability and half-life. Iterative Threading ASSEmbly Refinement (I-TASSER) results predicted a surfing topology with C-score values (−3.60). ResultsThe results of Gastro-Oesophageal Reflux 4 (GOR4) analysis showed a little extended strand (30.63%), a random coil with a percentage (50.18%), and an alpha helix with a percentage (19.19%). The recombinant protein was confirmed by SDS-PAGE and western blotting with Anti-his tag. ConclusionThe purpose of peptide fusion design in this article is to help in the development of anti-cervical cancer medicine. Of course, this issue needs to be investigated in animal and human phases. In this article, only bioinformatics investigations and validation of the results in the laboratory have been discussed.

From Protein Sequence to Structure: The Next Frontier in Cross-Species Extrapolation for Chemical Safety Evaluations.

Computational screening for potentially bioactive molecules using advanced molecular modeling approaches including molecular docking and molecular dynamic simulation is mainstream in certain fields like drug discovery. Significant advances in computationally predicting protein structures from sequence information have also expanded the availability of structures for nonmodel species. Therefore, the objective of the present study was to develop an analysis pipeline to harness the power of these bioinformatics approaches for cross-species extrapolation for evaluating chemical safety. The Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool compares protein-sequence similarity across species for conservation of known chemical targets, providing an initial line of evidence for extrapolation of toxicity knowledge. However, with the development of structural models from tools like the Iterative Threading ASSEmbly Refinement (ITASSER), analyses of protein structural conservation can be included to add further lines of evidence and generate protein models across species. Models generated through such a pipeline could then be used for advanced molecular modeling approaches in the context of species extrapolation. Two case examples illustrating this pipeline from SeqAPASS sequences to I-TASSER-generated protein structures were created for human liver fatty acid-binding protein (LFABP) and androgen receptor (AR). Ninety-nine LFABP and 268 AR protein models representing diverse species were generated and analyzed for conservation using template modeling (TM)-align. The results from the structural comparisons were in line with the sequence-based SeqAPASS workflow, adding further evidence of LFABL and AR conservation across vertebrate species. The present study lays the foundation for expanding the capabilities of the web-based SeqAPASS tool to include structural comparisons for species extrapolation, facilitating more rapid and efficient toxicological assessments among species with limited or no existing toxicity data. Environ Toxicol Chem 2023;42:463-474. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Expression and in silico Analysis of CIDRα1 Recombinant Protein from Plasmodium Falciparum as a Malaria Subunit Vaccine Candidate

Malaria vaccination is an essential approach to combat malaria. One major protein studied for vaccine development is Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1). It contains several important domains for malaria pathogenesis. The binding of Cysteine-rich interdomain region α1 (CIDRα1) of PfEMP1 to endothelial protein C receptor (EPCR) is associated with cerebral malaria, while CIDRα1 binding to CD36 has been correlated with uncomplicated malaria. The vital function of CIDRα1 of PfEMP1 makes it a potential vaccine candidate to prevent clinical features of malaria. A long journey of vaccine development can be shortened by the advancement of bioinformatics and biotechnology techniques. This study aimed to express the recombinant CIDRα1 of PfEMP1 and investigate its potency as a malaria subunit vaccine candidate by in silico analysis. Constructed CIDRα1-PfEMP1 was expressed in E. coli BL21(DE3) after induction with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and purified using Ni-NTA column. In silico analysis on CIDRα1 of PfEMP1 sequence was conducted using ProtParam Tool for its physicochemical properties, Iterative Threading ASSEmbly Refinement (I-TASSER) server and JPred4 program to predict secondary structure, 3D modelling, and ligand-binding site, BepiPred 2.0 and Kolaskar-Tangaonkar to predict B-cell epitope, NetCTL server to determine T-cell epitope, and Vaxijen v2.0 server to predict its antigenicity. The chimeric CIDRα1 of PfEMP1 protein had a 27 kDa molecular weight and was classified as a stable protein. The secondary structure consisted of 6 helices connected with loops. It revealed similarity to CD36-binding protein, EPCR-binding domain, and protein involved in rosetting. The 3D structure modelling demonstrated conserved ligand-binding sites and accessible surface area, which are vital for receptor binding. It had B-cell and T-cell epitopes and was non-allergenic. The properties of the chimeric CIDRα of PfEMP1 indicated its potential as a malaria subunit vaccine candidate. HIGHLIGHTS The binding capacity of CIDRα1 of PfEMP1 to endothelial protein C receptor (EPCR) and CD36 makes it a potential vaccine candidate to prevent clinical malaria The chimeric CIDRα1 of PfEMP1 protein was a stable protein and showed similarity to CD36-binding protein, EPCR-binding domain, and protein involved in rosetting, which demonstrated conserved ligand-binding sites and accessible surface area, which are vital for receptor binding The chimeric CIDRα1 of PfEMP1 protein had B-cell and T-cell epitopes and was non-allergenic in in silico analysis, indicating its potential as a malaria vaccine candidate GRAPHICAL ABSTRACT

In silico structural and functional characterization of Antheraea mylitta cocoonase

BackgroundCocoonase is a serine protease present in sericigenous insects and majorly involved in dissolving of sericin protein allowing moth to escape. Cocoon structure is made up of sericin protein which holds fibroin filaments together. Cocoonase enzyme hydrolyzes sericin protein without harming the fibroin. However, until date, no detailed characterization of cocoonase enzyme and its presence in wild silk moth Antheraea mylitta has been carried out. Therefore, current study aimed for detailed characterization of amplified cocoonase enzyme, secondary and tertiary structure prediction, sequence and structural alignment, phylogenetic analysis, and computational validation. Several computational tools such as ProtParam, Iterative Threading Assembly Refinement (I-TASSER), PROCHECK, SAVES v6.0, TM-align, Molecular Evolutionary Genetics Analysis (MEGA) X, and Figtree were employed for characterization of cocoonase protein. ResultsThe present study elucidates about the isolation of RNA, cDNA preparation, PCR amplification, and in silico characterization of cocoonase from Antheraea mylitta. Here, total RNA was isolated from head region of A. mylitta, and gene-specific primers were designed using Primer3 followed by PCR-based amplification and sequencing. The newly constructed 377-bp length sequence of cocoonase was subjected to in silico characterization. In silico study of A. mylitta cocoonase showed 26% similarity to A. pernyi strain Qing-6 cocoonase using Blastp and belongs to member of chymotrypsin-like serine protease superfamily. From phylogenetic study, it was found that A. mylitta cocoonase sequence is closely related to A. pernyi cocoonase sequence. ConclusionsThe present study revealed about the detailed in silico characterization of cocoonase gene and encoded protein obtained from A. mylitta head region. The results obtained infer the presence of cocoonase enzyme in the wild silkworm A. mylitta and can be used for cocoon degumming which will be a valuable and cost-effective strategy in silk industry.

Ex silico engineering of cystine-dense peptides yielding a potent bispecific T cell engager.

Cystine-dense peptides (CDPs) are a miniprotein class that can drug difficult targets with high affinity and low immunogenicity. Tools for their design, however, are not as developed as those for small-molecule and antibody drugs. CDPs have diverse taxonomic origins, but structural characterization is lacking. Here, we adapted Iterative Threading ASSEmbly Refinement (I-TASSER) and Rosetta protein modeling software for structural prediction of 4298 CDP scaffolds and performed in silico prescreening for CDP binders to targets of interest. Mammalian display screening of a library of docking-enriched, methionine and tyrosine scanned (DEMYS) CDPs against PD-L1 yielded binders from four distinct CDP scaffolds. One was affinity-matured, and cocrystallography yielded a high-affinity (KD = 202 pM) PD-L1-binding CDP that competes with PD-1 for PD-L1 binding. Its subsequent incorporation into a CD3-binding bispecific T cell engager produced a molecule with pM-range in vitro T cell killing potency and which substantially extends survival in two different xenograft tumor-bearing mouse models. Both in vitro and in vivo, the CDP-incorporating bispecific molecule outperformed a comparator antibody-based molecule. This CDP modeling and DEMYS technique can accelerate CDP therapeutic development.

CR-I-TASSER: assemble protein structures from cryo-EM density maps using deep convolutional neural networks.

Cryo-electron microscopy (cryo-EM) has become a leading approach for protein structure determination, but it remains challenging to accurately model atomic structures with cryo-EM density maps. We propose a hybrid method, CR-I-TASSER, which integrates deep neural-network learning with I-TASSER assembly simulations for automated cryo-EM structure determination. The method is benchmarked on 778 proteins with simulated and experimental density maps, where CR-I-TASSER constructs models with a correct fold (TM-score>0.5) for 643 targets that is 64% higher than the best of other de novo and refinement-based approaches on high-resolution data samples. Detailed data analyses showed that the major advantage of CR-I-TASSER lies in the deep-learning based position prediction, which significantly improves the threading template quality and therefore boosts the accuracy of final models through optimized fragment assembly simulations. These results demonstrate a new avenue to determine cryo-EM protein structures with high accuracy and robustness covering various target types and density-map resolutions.

GPU-I-TASSER: a GPU accelerated I-TASSER protein structure prediction tool.

Accurate and efficient predictions of protein structures play an important role in understanding their functions. Iterative Threading Assembly Refinement (I-TASSER) is one of the most successful and widely used protein structure prediction methods in the recent community-wide CASP experiments. Yet, the computational efficiency of I-TASSER is one of the limiting factors that prevent its application for large-scale structure modeling. We present I-TASSER for Graphics Processing Units (GPU-I-TASSER), a GPU accelerated I-TASSER protein structure prediction tool for fast and accurate protein structure prediction. Our implementation is based on OpenACC parallelization of the replica-exchange Monte Carlo simulations to enhance the speed of I-TASSER by extending its capabilities to the GPU architecture. On a benchmark dataset of 71 protein structures, GPU-I-TASSER achieves on average a 10× speedup with comparable structure prediction accuracy compared to the CPU version of the I-TASSER. The complete source code for GPU-I-TASSER can be downloaded and used without restriction from https://zhanggroup.org/GPU-I-TASSER/. Supplementary data are available at Bioinformatics online.

Conjugation of Silver Nanoparticles With De Novo-Engineered Cationic Antimicrobial Peptides: Exploratory Proposal.

BackgroundCationic antimicrobial peptides have broad antimicrobial activity and provide a novel way of targeting multidrug-resistant bacteria in the era of increasing antimicrobial resistance. Current developments show positive prospects for antimicrobial peptides and silver nanoparticles (AgNPs) individually.ObjectiveThe primary objective is to propose another method for enhancing antimicrobial activity by conjugating AgNPs with cationic antimicrobial peptides, with a subsequent preliminary assessment of the minimum inhibitory concentration of multidrug-resistant bacteria. The secondary objective is to evaluate the safety of the conjugated compound and assess its viability for in vivo use.MethodsThe proposal involves 3 stages. First, WLBU2C, a modified version of the antimicrobial peptide WLBU2 with an added cysteine group, needs to be synthesized using a standard Fmoc procedure. It can then be stably conjugated with AgNPs ideally through photochemical means. Second, the WLBU2C-AgNP conjugate should be tested for antimicrobial activity according to the Clinical & Laboratory Standards Institute manual on standard minimum inhibitory concentration testing. Third, the cytotoxicity of the conjugate should be tested using cell lysis assays if the above stages are completed.ResultsI-TASSER (iterative threading assembly refinement) simulation revealed that the modified peptide WLBU2C has a secondary structure similar to that of the original WLBU2 peptide. No other results have been obtained at this time.ConclusionsThe addition of AgNPs to already developed de novo–engineered antimicrobial peptides provides an opportunity for the development of potent antimicrobials. Future prospects include emergency last-line therapy and treatment for current difficult-to-eradicate bacterial colonization, such as in cystic fibrosis, implantable medical devices, cancer, and immunotherapy. As I do not anticipate funding at this time, I hope this proposal provides inspiration to other researchers.International Registered Report Identifier (IRRID)PRR1-10.2196/28307

Identification and Structure Prediction of Human Septin-4 as a Biomarker for Diagnosis of Asthenozoospermic Infertile Patients-Critical Finding Toward Personalized Medicine.

Semen parameters are been found as a key factor to evaluate the count and morphology in the given semen sample. The deep knowledge of male infertility will unravel with semen parameters correlated with molecular and biochemical parameters. The current research study is to identify the motility associated protein and its structure through the in-silico approach. Semen samples were collected and initial analysis including semen parameters was analyzed by using the World Health Organization protocol. Semen biochemical parameters, namely, seminal plasma protein concentration, fructose content, and glucosidase content were calculated and evaluated for correlation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) were carried out for identification of Septin-4 presence in the semen sample. Mascot search was done for protein conformation and in-silico characterization of Septin-4 by structural modeling in Iterative Threading Assembly Refinement (I-TASSER). Twenty-five nanoseconds molecular dynamics (MD) simulations results showed the stable nature of Septin-4 in the dynamic system. Overall, our results showed the presence of motility-associated protein in normospermia and control samples and not in the case of asthenospermia and oligoasthenospermia. Molecular techniques characterized the presence of Septin-4 and as a novel biomarker for infertility diagnosis.

Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain.

Ecarin is one of the most widely used drug compounds in blood clotting experiments and is used to monitor and treat many diseases such as cancer, liver, lupus, and cardiovascular disease. The metalloproteinase domain is known as the active site of ecarin. In this study, an ecarin metalloproteinase cassette was designed and synthesized in the pUC57 vector. The gene fragment was released and cloned into the pET-28a vector and expressed in Escherichia coli. The recombinant protein was confirmed by western blotting. Enzyme activity was estimated by a laboratory coagulation test, and prothrombin time and tertiary structure were determined by using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Data from blood clotting tests for the produced ecarin activity were analyzed using an independent t test. As per I-TASSER server prediction, model 1 with the highest confidence score 0.95, template modeling score (0.84 ± 0.08), and root mean square deviation (3.5 ± 2.4Å) was considered as the best model, and the 2e3xA enzyme was more similar to the target protein. The predictive results helped to better understand the relationship between the structure and function of the ecarin metalloproteinase domain. Also, the production of this active site in the prokaryotic expression system, which is simpler and more cost-effective than the production of the eukaryotic system, showed that this recombinant ecarin could be used as a substitute for the raw snake venom of Echis carinatus because it converts prothrombin into thrombin, and its activity, as estimated using the prothrombin time test, was found to be faster than normal ecarin.

Whole Exome Sequencing Identifies a Novel Homozygous Missense Mutation in the CSB Protein-Encoding ERCC6 Gene in a Taiwanese Boy with Cockayne Syndrome.

Background: Cockayne syndrome (CS) is a rare form of dwarfism that is characterized by progressive premature aging. CS is typically caused by mutations in the excision repair cross-complementing protein group 6 (ERCC6) gene that encodes the CS group B (CSB) protein. Using whole exome sequencing, we recently identified a novel homozygous missense mutation (Leu536Trp) in CSB in a Taiwanese boy with CS. Since the current database (Varsome) interprets this variant as likely pathogenic, we utilized a bioinformatic tool to investigate the impact of Leu536Trp as well as two other variants (Arg453Ter, Asp532Gly) in similar articles on the CSB protein structure stability. Methods: We used iterative threading assembly refinement (I-TASSER) to generate a predictive 3D structure of CSB. We calculated the change of mutation energy after residues substitution on the protein stability using I-TASSER as well as the artificial intelligence program Alphafold. Results: The Asp532Gly variant destabilized both modeled structures, while the Leu536Trp variant showed no effect on I-TASSER’s model but destabilized the Alphafold’s modeled structure. Conclusions: We propose here the first case of CS associated with a novel homozygous missense mutation (Leu536Trp) in CSB. Furthermore, we suggest that the Asp532Gly and Leu536Trp variants are both pathogenic after bioinformatic analysis of protein stability.

Whole-Exome Sequencing Identifies a Recurrent Small In-Frame Deletion in MYO15A Causing Autosomal Recessive Nonsyndromic Hearing Loss in 3 Iranian Pedigrees.

Hearing loss (HL) is the most prevalent and genetically heterogeneous sensory disabilities in humans throughout the world. In this study, we used whole-exome sequencing (WES) to determine the variant causing autosomal recessive nonsyndromic hearing loss (ARNSHL) segregating in 3 separate Iranian consanguineous families (with 3 different ethnicities: Azeri, Persian, and Lur), followed by cosegregation analysis, computational analysis, and structural modeling using the I-TASSER (Iterative Threading ASSEmbly Refinement) server. Also, we used speech-perception tests to measure cochlear implant (CI) performance in patients. One small in-frame deletion variant (MYO15A c.8309_8311del (p.Glu2770del)), resulting in deletion of a single amino-acid residue was identified. We found it to be cosegregating with the disease in the studied families. We provide some evidence suggesting the pathogenesis of this variant in HL based on the American College of Medical Genetics (ACMG) and Genomics guidelines. Evaluation of auditory and speech performance indicated favorable outcome after cochlear implantation in our patients. The findings of this study demonstrate the utility of WES in genetic diagnostics of HL.

Structural basis for the binding of a selective inverse agonist AF64394 with the human G-protein coupled receptor 3 (GPR3)

The orphan class A G-protein coupled receptor 3 (GPR3) is highly expressed in brain and linked with various neuronal functions, and therefore, expected to play a vital role in the progression of Alzheimer’s disease. In view of the lack of its experimental structure, we describe herein the three-dimensional structure and conformational dynamics of GPR3 complexed with the inverse agonist AF64394. The GPR3 model was predicted using the Iterative Threading ASSEmbly Refinement (I-TASSER) method. The Induced Fit Docking predicted two unique poses, Pose 1 and Pose 2, for AF64394, and then, molecular dynamics (MD) simulations followed by binding free-energy calculation revealed the Pose 1 as a very stable pose with the least fluctuation during the MD simulation while the Pose 2 underwent a significant fluctuation. The [1,2,4]triazolo[1,5-a]pyrimidine core was engaged in multiple hydrogen bonds (H-bonds), such as a water-mediated H-bond between the triazole nitrogen and T31, two direct H-bonds between the protonated triazole-ring nitrogen and V186 and T279, a direct H-bond between the secondary amine and V187. The phenyl substituent of AF64394 exhibited aromatic π-π stacking interactions with F97, F101, W43 and Y280. AF64394 showed a direct interaction with E28 and polar interactions with H96, T31 and T279. Throughout the MD simulation, the toggle switch residues, F120 and W260, remained in close contact, indicating that the GPR3 conformation represented an inactive state. The 4-(3-chloro-5-isopropoxyphenethyl) group resided near to the toggle switch residues. The insights gained here are expected to be useful in the structure-based design of new ligands targeting GPR3 modulation. Communicated by Ramaswamy H. Sarma

Homology modelling and analysis of structure predictions of human tumour necrosis factor ligand superfamily member 8

BackgroundTumour necrosis factor ligand superfamily member 8 (TNFL8) is a cytokine that plays vital roles in immune activations and inflammatory responses through its interaction with the tumour necrosis factor superfamily member. Despite multiple studies on the involvement of its receptor in the inflammatory response, there is limited information on the molecular characterization and structural elucidation of the cytokine. Considering the significance of the cytokine, the three-dimensional structure of TNFL8 model was generated by homology modelling through the Iterative Threading ASSEmbly Refinement (I-TASSER) server and validated through PROCHECK and Qualitative Model Energy Analysis (QMEAN) servers.ResultsThe predicted structure has 90.00% of residues in the most favoured region of the Ramachandran plot while the QMEAN value gives − 3.06. The sequence and structural alignment between the generated model of the cytokine and template (1XU2) reveal that similar active site residues such as ILE-142, THR-175, GLU-178, and PHE-228 could be involved in binding pocket formation. However, docking studies of the 3D model of TNFL8 with eight phytochemicals from the extract of Xylopia aethiopica (Dunal) A. Rich revealed the phytochemical bound to two different binding sites which could be the active regions of the cytokine that could be essential for inhibition. More so, the docking analysis showed most of the phytochemicals have good binding affinity to the cytokine with ellagic acid showing the highest affinity with a binding energy of − 6.58 ± 0.18 kcal/mol.ConclusionThe proposed model may shed light on the mechanisms of TNFL8 binding and provide insights into the identification of potential molecular targets for the development of the novel compound for the regulation of the functional activities of the cytokine.

Computational-approach understanding the structure-function prophecy of Fibrinolytic Protease RFEA1 from Bacillus cereus RSA1.

Microbial fibrinolytic proteases are therapeutic enzymes responsible to ameliorate thrombosis, a fatal cardiac-disorder which effectuates due to excessive fibrin accumulation in blood vessels. Inadequacies such as low fibrin specificity, lethal after-effects and short life-span of available fibrinolytic enzymes stimulates an intensive hunt for novel, efficient and safe substitutes. Therefore, we herewith suggest a novel and potent fibrinolytic enzyme RFEA1 from Bacillus cereus RSA1 (MK288105). Although, attributes such as in-vitro purification, characterization and thrombolytic potential of RFEA1 were successfully accomplished in our previous study. However, it is known that structure-function traits and mode of action significantly aid to commercialization of an enzyme. Also, predicting structural model of a protein from its amino acid sequence is challenging in computational biology owing to intricacy of energy functions and inspection of vast conformational space. Our present study thus reports In-silico structural-functional analysis of RFEA1. Sequence based modelling approaches such as—Iterative threading ASSEmbly Refinement (I-TASSER), SWISS-MODEL, RaptorX and Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) were employed to model three-dimensional structure of RFEA1 and the modelled RFEA1 was validated by structural analysis and verification server (SAVES v6.0). The modelled crystal structure revealed the presence of high affinity Ca1 binding site, associated with hydrogen bonds at Asp147, Leu181, Ile185 and Val187residues. RFEA1 is structurally analogous to Subtilisin E from Bacillus subtilis 168. Molecular docking analysis using PATCH DOCK and FIRE DOCK servers was performed to understand the interaction of RFEA1 with substrate fibrin. Strong RFEA1-fibrin interaction was observed with high binding affinity (−21.36 kcal/mol), indicating significant fibrinolytic activity and specificity of enzyme RFEA1. Overall, the computational research suggests that RFEA1 is a subtilisin-like serine endopeptidase with proteolytic potential, involved in thrombus hydrolysis.