RESUMO
Protein-carbohydrate interactions are involved in several cellular and biological functions. Integrating structure and function of carbohydrate-binding proteins with disease-causing mutations help to understand the molecular basis of diseases. Although databases are available for protein-carbohydrate complexes based on structure, binding affinity and function, no specific database for mutations in human carbohydrate-binding proteins is reported in the literature. We have developed a novel database, CarbDisMut, a comprehensive integrated resource for disease-causing mutations with sequence and structural features. It has 1.17 million disease-associated mutations and 38,636 neutral mutations from 7,187 human carbohydrate-binding proteins. The database is freely available at https://web.iitm.ac.in/bioinfo2/carbdismut. The web-site is implemented using HTML, PHP and JavaScript and supports recent versions of all major browsers, such as Firefox, Chrome and Opera.
Assuntos
Carboidratos , Humanos , Bases de Dados Factuais , MutaçãoRESUMO
Alcohol use disorder (AUD) is a multifactorial psychiatric behavior disorder. Disulfiram is the first approved drug by the Food and Drug Administration for alcohol-dependent patients, which targets the ALDH2 enzyme. Several genes are known to be involved in alcohol metabolism; mutations in any of these genes are known to be associated with AUD. The E504K mutation in the ALDH2 of the precursor protein or the E487K of the mature protein (E504K/E487K; ALDH2*2 allele) is carried by approximately 8% of the world population. In this study, we aimed to test the known inactive allele ALDH2*2, to validate the use of our extensive computational pipeline (in silico tools, molecular modeling, and molecular docking) for testing the interaction between the ALDH2*2 allele, NAD+, and Disulfiram. In silico predictions showed that the E504K variant of ALDH2 to be pathogenic and destabilizing with the maximum number of prediction in silico tools. Consequently, we studied the effect of this mutation mainly on the interaction between NAD+ -E504K and Disulfiram-E504K complexes using molecular docking technique, and molecular dynamics (MD) analysis. From the molecular docking analysis with NAD+ , we observed that the interaction affinity of the NAD+ decreases with the impact of E504K variant. On the other hand, the drug Disulfiram showed similar interaction in both the native and mutant ALDH2 proteins. Further, the comprehensive MD analysis predicted that the E504K destabilizes the protein and influences the NAD+ and Disulfiram interactions. Our findings reveal that the interaction of NAD+ to the protein is disturbed by the E504K/E487K variant whereas the drug Disulfiram has a similar effect as both native ALDH2 and ALDH2 bearing E504K/E487K variant. This study provides a platform to understand the effect of E504K/E487K on the molecular interaction with NAD+ and Disulfiram.
Assuntos
Inibidores de Acetaldeído Desidrogenases/química , Aldeído-Desidrogenase Mitocondrial/química , Dissulfiram/química , Simulação de Acoplamento Molecular , Mutação , NAD/química , Inibidores de Acetaldeído Desidrogenases/metabolismo , Aldeído-Desidrogenase Mitocondrial/antagonistas & inibidores , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Biologia Computacional/métodos , Dissulfiram/metabolismo , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , NAD/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Galactosemia type 2 is an autosomal recessive disorder characterized by the deficiency of galactokinase (GALK) enzyme due to missense mutations in GALK1 gene, which is associated with various manifestations such as hyper galactosemia and formation of cataracts. GALK enzyme catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of α-d-galactose to galactose-1-phosphate. We searched 4 different literature databases (Google Scholar, PubMed, PubMed Central, and Science Direct) and 3 gene-variant databases (Online Mendelian Inheritance in Man, Human Gene Mutation Database, and UniProt) to collect all the reported missense mutations associated with GALK deficiency. Our search strategy yielded 32 missense mutations. We used several computational tools (pathogenicity and stability, biophysical characterization, and physiochemical analyses) to prioritize the most significant mutations for further analyses. On the basis of the pathogenicity and stability predictions, 3 mutations (P28T, A198V, and L139P) were chosen to be tested further for physicochemical characterization, molecular docking, and simulation analyses. Molecular docking analysis revealed a decrease in interaction between the protein and ATP in all the 3 mutations, and molecular dynamic simulations of 50 ns showed a loss of stability and compactness in the mutant proteins. As the next step, comparative physicochemical changes of the native and the mutant proteins were carried out using essential dynamics. Overall, P28T and A198V were predicted to alter the structure and function of GALK protein when compared to the mutant L139P. This study demonstrates the power of computational analysis in variant classification and interpretation and provides a platform for developing targeted therapeutics.
Assuntos
Galactoquinase/genética , Galactosemias/genética , Simulação de Acoplamento Molecular , Mutação de Sentido Incorreto , Trifosfato de Adenosina/metabolismo , Galactoquinase/metabolismo , Galactosemias/metabolismo , Humanos , Ligação Proteica , Conformação ProteicaRESUMO
X-linked Charcot-Marie-Tooth type 1 X (CMTX1) disease is a subtype of Charcot-Marie-Tooth (CMT), which is mainly caused by mutations in the GJB1 gene. It is also known as connexin 32 (Cx32) that leads to Schwann cell abnormalities and peripheral neuropathy. CMTX1 is considered as the second most common form of CMT disease. The aim of this study is to computationally predict the potential impact of different single amino acid substitutions at position 75 of Cx32, from arginine (R) to proline (P), glutamine (Q) and tryptophan (W). This position is known to be highly conserved among the family of connexin. To understand the structural and functional changes due to these single amino acid substitutions, we employed a homology-modeling technique to build the three-dimensional structure models for the native and mutant proteins. The protein structures were further embedded into a POPC lipid bilayer, inserted into a water box, and subjected to molecular dynamics simulation for 50â¯ns. Our results show that the mutants R75P, R75Q and R75W display variable structural conformation and dynamic behavior compared to the native protein. Our data proves useful in predicting the potential pathogenicity of the mutant proteins and is expected to serve as a platform for drug discovery for patients with CMT.
Assuntos
Substituição de Aminoácidos , Arginina/genética , Doença de Charcot-Marie-Tooth/genética , Conexinas/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Sítios de Ligação/genética , Biologia Computacional/métodos , Conexinas/química , Conexinas/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Proteína beta-1 de Junções ComunicantesRESUMO
The ß-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Clostridium perfringens (CpeCA) was recently characterised kinetically and for its anion inhibition profile. In the search of effective CpeCA inhibitors, possibly useful to inhibit the growth/pathogenicity of this bacterium, we report here an inhibition study of this enzyme with a panel of aromatic, heterocyclic and sugar sulphonamides/sulphamates. Some sulphonamides, such as acetazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, sulthiame and 4-(2-hydroxymethyl-4-nitrophenyl-sulphonamido)ethylbenzenesulphonamide were effective CpeCA inhibitors, with KIs in the range of 37.4-71.6 nM. Zonisamide and saccharin were the least effective such inhibitors, whereas many other aromatic and heterocyclic sulphonamides were moderate - weak inhibitors with KIs ranging between 113 and 8755 nM. Thus, this study provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Clostridium perfringens/enzimologia , Sulfonamidas/farmacologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
Larsen syndrome (LRS) is a rare genetic disease associated with variable manifestations including skeletal malformations, dislocations of the large joints, and notable changes in facial and limb features. Genetic variants in the Filamin B (FLNB) gene are associated with the development of LRS. We searched two literature databases (OMIM and PubMed) and three gene variant databases (HGMD, UniProt, & dbSNP) to capture all the possible variants associated with LRS phenotype, which may have an impact on the FLNB function. Our search yielded 77 variants that might impact the FLNB protein function in patients with LRS. We performed rigorous computational analysis such as conservational, biochemical, pathogenicity, and structural computational analyses to understand the deleterious effect of the G1691S variant. Further, the structural changes of the G1691S variant was compared with a null variant (G1691A) and the native protein through a molecular dynamic simulation study of 50 ns. We found that the variant G1691S was highly deleterious and destabilize the protein when compared to the native and variant G1691A. This might be due to the physicochemical changes in the variant G1691S when compared to the native and variant G1691A. The destabilization was further supported by transformation of bend to coil in variant G1691S whereas bend was retained in native and variant G1691A through molecular dynamics analysis. Our study shed light on the importance of computational methods to understand the molecular nature of genetic variants and structural insights on the function of the FLNB protein. J. Cell. Biochem. 118: 1900-1910, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Biologia Computacional/métodos , Filaminas/metabolismo , Osteocondrodisplasias/metabolismo , Bases de Dados Genéticas , Filaminas/química , Filaminas/genética , Humanos , Mutação/genética , Osteocondrodisplasias/genética , Estabilidade ProteicaRESUMO
Glutaredoxins (GRXs) are thiol-disulfide oxidoreductases abundant in prokaryotes, although little is understood of these enzymes from the domain Archaea. The numerous characterized GRXs from the domain Bacteria utilize a diversity of low-molecular-weight thiols in addition to glutathione as reductants. We report here the biochemical and structural properties of a GRX-like protein named methanoredoxin (MRX) from Methanosarcina acetivorans of the domain Archaea. MRX utilizes coenzyme M (CoMSH) as reductant for insulin disulfide reductase activity, which adds to the diversity of thiol protectants in prokaryotes. Cell-free extracts of M. acetivorans displayed CoMS-SCoM reductase activity that complements the CoMSH-dependent activity of MRX. The crystal structure exhibits a classic thioredoxin-glutaredoxin fold comprising three α-helices surrounding four antiparallel ß-sheets. A pocket on the surface contains a CVWC motif, identifying the active site with architecture similar to GRXs. Although it is a monomer in solution, the crystal lattice has four monomers in a dimer of dimers arrangement. A cadmium ion is found within the active site of each monomer. Two such ions stabilize the N-terminal tails and dimer interfaces. Our modeling studies indicate that CoMSH and glutathione (GSH) bind to the active site of MRX similar to the binding of GSH in GRXs, although there are differences in the amino acid composition of the binding motifs. The results, combined with our bioinformatic analyses, show that MRX represents a class of GRX-like enzymes present in a diversity of methane-producing Archaea.
Assuntos
Proteínas Arqueais/metabolismo , Glutarredoxinas/metabolismo , Mesna/metabolismo , Methanosarcina/metabolismo , Proteínas Arqueais/química , Glutarredoxinas/química , Proteína Dissulfeto Redutase (Glutationa)/química , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Estrutura Secundária de ProteínaRESUMO
Bioinformatics analyses predict the distribution in nature of several classes of diverse disulfide reductases that evolved from an ancestral plant-type ferredoxin:thioredoxin reductase (FTR) catalytic subunit to meet a variety of ecological needs. Methanosarcina acetivorans is a methane-producing species from the domain Archaea predicted to encode an FTR-like enzyme with two domains, one resembling the FTR catalytic subunit and the other containing a rubredoxin-like domain replacing the variable subunit of present-day FTR enzymes. M. acetivorans is of special interest as it was recently proposed to have evolved at the time of the end-Permian extinction and to be largely responsible for the most severe biotic crisis in the fossil record by converting acetate to methane. The crystal structure and biochemical characteristics were determined for the FTR-like enzyme from M. acetivorans, here named FDR (ferredoxin disulfide reductase). The results support a role for the rubredoxin-like center of FDR in transfer of electrons from ferredoxin to the active-site [Fe4S4] cluster adjacent to a pair of redox-active cysteines participating in reduction of disulfide substrates. A mechanism is proposed for disulfide reduction similar to one of two mechanisms previously proposed for the plant-type FTR. Overall, the results advance the biochemical and evolutionary understanding of the FTR-like family of enzymes and the conversion of acetate to methane that is an essential link in the global carbon cycle and presently accounts for most of this greenhouse gas that is biologically generated.
Assuntos
Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Methanosarcina/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Estrutura Secundária de ProteínaRESUMO
Carbonic anhydrase is a metalloenzyme catalyzing the reversible hydration of carbon dioxide to bicarbonate. Five independently evolved classes have been described for which one or more are found in nearly every cell type underscoring the general importance of this ubiquitous enzyme in Nature. The bulk of research to date has centered on the enzymes from mammals and plants with less emphasis on prokaryotes. Prokaryotic carbonic anhydrases play important roles in the ecology of Earth's biosphere including acquisition of CO2 for photosynthesis and the physiology of aerobic and anaerobic prokaryotes decomposing the photosynthate back to CO2 thereby closing the global carbon cycle. This review focuses on the physiology and biochemistry of carbonic anhydrases from prokaryotes belonging to the domains Bacteria and Archaea that play key roles in the ecology of Earth's biosphere.
Assuntos
Dióxido de Carbono/metabolismo , Anidrases Carbônicas/química , Ecologia , Meio Ambiente , Aerobiose , Anaerobiose , Archaea/enzimologia , Bactérias/enzimologia , Dióxido de Carbono/química , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismoRESUMO
Dental caries is a common human oral disease worldwide, caused by an acid-producing bacteria Streptococcus mutans. The use of synthetic drugs and antibiotics to prevent dental caries has been increasing, but this can lead to severe side effects. To solve this issue, developing and developed countries have resorted to herbal medicines as an alternative to synthetic drugs for the treatment and prevention of dental caries. Therefore, there is an urgent need for plant-derived products to treat such diseases. Bacopa monnieri, a well-documented medicinal plant, contains 52 phytocompounds, including the pentacyclic triterpenoid metabolite known as asiatic acid (ASTA). Hence, this study aimed to demonstrate, for the first time, the antibacterial activity of phytocompound ASTA against S. mutans. The findings revealed that ASTA significantly inhibited the growth of S. mutans and the production of virulence factors such as acidurity, acidogenicity, and eDNA synthesis. Molecular docking analysis evaluated the potential activity of ASTA against S. mutans virulence genes, including VicR and GtfC. Furthermore, toxicity assessment of ASTA in human buccal epithelial cells was performed, and no morphological changes were observed. An in vivo analysis using Danio rerio (zebrafish) confirmed that the ASTA treatment significantly increased the survival rates of infected fish by hindering the intestinal colonization of S. mutans. Furthermore, the disease protection potential of ASTA against the pathognomonic symptom of S. mutans infection was proven by the histopathological examination of the gills, gut, and kidney. Overall, these findings suggest that ASTA may be a promising therapeutic and alternative drug for the treatment and prevention of oral infection imposed by S. mutans.
RESUMO
The carbonic anhydrase (Cpb) from Clostridium perfringens strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the ß class, the first ß class enzyme investigated from a strictly anaerobic species of the domain Bacteria. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with Km and kcat/Km values of 3.1 mM CO2 and 4.8 × 106 s⻹ M⻹, respectively. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO2. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO2 in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO2 fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Anidrases Carbônicas/metabolismo , Clostridium perfringens/enzimologia , Dióxido de Carbono , Anidrases Carbônicas/classificação , Anidrases Carbônicas/genética , Modelos Teóricos , FilogeniaRESUMO
A ß-carbonic anhydrases (CAs, EC 4.2.1.1) was recently cloned, purified and characterized kinetically in the pathogen Clostridium perfringens. We report here the first inhibition study of this enzyme (CpeCA). CpeCA was poorly inhibited by iodide and bromide, and was inhibited with KIs in the range of 1-10mM by a range of anions such as (thio)cyanate, azide, bicarbonate, nitrate, nitrite, hydrogensulfite, hydrogensulfide, stannate, tellurate, pyrophosphate, divanadate, tetraborate, peroxydisulfate, sulfate, iminodisulfonate and fluorosulfonate. Better inhibitory power, with K(I)s of 0.36-1.0 mM, was observed for cyanide, carbonate, selenate, selenocyanide, trithiocarbonate and diethyldithiocarbamate, whereas the best CpeCA inhibitors were sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, which had KIs in the range of 7-75 µM. This study thus provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.
Assuntos
Ânions/química , Ânions/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Clostridium perfringens/enzimologia , Sequência de Aminoácidos , Animais , Ânions/metabolismo , Proteínas de Bactérias/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Anidrases Carbônicas/metabolismo , Clostridium perfringens/classificação , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Dados de Sequência Molecular , Filogenia , Ligação Proteica/efeitos dos fármacos , Alinhamento de SequênciaRESUMO
Breast cancer biomarkers that detect marginally advanced stages are still challenging. The detection of specific abnormalities, targeted therapy selection, prognosis, and monitoring of treatment effectiveness over time are all made possible by circulating free DNA (cfDNA) analysis. The proposed study will detect specific genetic abnormalities from the plasma cfDNA of a female breast cancer patient by sequencing a cancer-related gene panel (MGM455 - Oncotrack Ultima), including 56 theranostic genes (SNVs and small INDELs). Initially, we determined the pathogenicity of the observed mutations using PredictSNP, iStable, Align-GVGD, and ConSurf servers. As a next step, molecular dynamics (MD) was implemented to determine the functional significance of SMAD4 mutation (V465M). Lastly, the mutant gene relationships were examined using the Cytoscape plug-in GeneMANIA. Using ClueGO, we determined the gene's functional enrichment and integrative analysis. The structural characteristics of SMAD4 V465M protein by MD simulation analysis further demonstrated that the mutation was deleterious. The simulation showed that the native structure was more significantly altered by the SMAD4 (V465M) mutation. Our findings suggest that SMAD4 V465M mutation might be significantly associated with breast cancer, and other patient-found mutations (AKT1-E17K and TP53-R175H) are synergistically involved in the process of SMAD4 translocate to nuclease, which affects the target gene translation. Therefore, this combination of gene mutations could alter the TGF-ß signaling pathway in BC. We further proposed that the SMAD4 protein loss may contribute to an aggressive phenotype by inhibiting the TGF-ß signaling pathway. Thus, breast cancer's SMAD4 (V465M) mutation might increase their invasive and metastatic capabilities.Communicated by Ramaswamy H. Sarma.
Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Biomarcadores Tumorais/genética , Proteína Smad4/genética , Mutação , Fator de Crescimento Transformador beta/genética , Proteína Supressora de Tumor p53/genética , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
BACKGROUND: Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer. NSCLC accounts for 84% of all lung cancer cases. In recent years, advances in pathway understanding, methods for discovering novel genetic biomarkers, and new drugs designed to inhibit the signaling cascades have enabled clinicians to personalize therapy for NSCLC. OBJECTIVES: The primary aim of this study is to identify the genes associated with NSCLC that harbor pathogenic variants that could be causative for NSCLC. The second aim is to investigate their roles in different pathways that lead to NSCLC. METHODS: We examined exome-sequencing datasets from 54 NSCLC patients to characterize the variants associated with NSCLC. RESULTS: Our findings revealed that 17 variants in 14 genes were considered highly pathogenic, including CDKN2A, ERBB2, FOXP1, IDH1, JAK3, KMT2D, K-Ras, MSH3, MSH6, POLE, RNF43, TCF7L2, TP53, and TSC1. Gene set enrichment analysis revealed the involvement of transmembrane receptor protein tyrosine kinase activity, protein binding, ATP binding, phosphatidylinositol-4,5-bisphosphate 3-kinase, and Ras guanyl-nucleotide exchange factor activity. Pathway analysis of these genes yielded different cancer-related pathways, including colorectal, prostate, endometrial, pancreatic, PI3K-Akt signaling pathways, and signaling pathways regulating pluripotency of stem cells. Module 1 from protein-protein interactions (PPIs) identified genes that harbor pathogenic SNPs. Three of the most deleterious SNPs are ERBB2 (rs1196929947), K-Ras (rs121913529), and POLE (rs751425952). Interestingly, one patient has a pathogenic K-Ras variant (rs121913529) co-occurred with the missense variant (rs752054698) inTSC1 gene. CONCLUSION: This study maps highly pathogenic variants associated with NSCLC and investigates their contributions to the pathogenesis of NSCLC. This study sheds light on the potential applications of precision medicine in patients with NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Fatores de Transcrição Forkhead , Humanos , Masculino , Fosfatidilinositol 3-Quinases , Polimorfismo de Nucleotídeo Único , Proteínas Repressoras , Sequenciamento do ExomaRESUMO
Type I galactosemia is a very rare autosomal recessive genetic metabolic disorder that occurs because of the mutations present in the galactose-1-phosphate uridyl transferase (GALT) gene, resulting in a deficiency of the GALT enzyme. The action of the GALT enzyme is to convert galactose-1-phosphate and uridine diphosphate glucose into glucose-1-phosphate (G1P) and uridine diphosphate-galactose, a crucial second step of the Leloir pathway. A missense mutation in the GALT enzyme leads to variable galactosemia's clinical presentations, ranging from mild to severe. Our study aimed to employ a comprehensive computational pipeline to analyze the most prevalent missense mutations (p.S135L, p.K285 N, p.Q188R, and p.N314D) responsible for galactosemia; these genes could serve as potential targets for chaperone therapy. We analyzed the four mutations through different computational analyses, including amino acid conservation, in silico pathogenicity and stability predictions, and macromolecular simulations (MMS) at 50 ns The stability and pathogenicity predictors showed that the p.Q188R and p.S135L mutants are the most pathogenic and destabilizing. In agreement with these results, MMS analysis demonstrated that the p.Q188R and p.S135L mutants possess higher deviation patterns, reduced compactness, and intramolecular H-bonds of the protein. This could be due to the physicochemical modifications that occurred in the mutants p.S135L and p.Q188R compared to the native. Evolutionary conservation analysis revealed that the most prevalent mutations positions were conserved among different species except N314. The proposed research study is intended to provide a basis for the therapeutic development of drugs and future treatment of classical galactosemia and possibly other genetic diseases using chaperone therapy.
Assuntos
Galactosemias , UTP-Hexose-1-Fosfato Uridililtransferase , Galactosemias/genética , Galactosefosfatos , Humanos , Mutação/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , UTP-Hexose-1-Fosfato Uridililtransferase/genéticaRESUMO
CONTEXT: Homology modeling plays role in determining the therapeutic targets dreadful for condition such as neurodegenerative diseases (NDD), which pose challenge in achieving the effective managements. The structures of the serotonin transporter (SERT), aquaporin (AQP), and tropomyosin receptor kinase (TrkA) which are implicated in NDD pathology are still unknown for Lumbricus terrestris, but the three-dimensional (3D) structure of the human counterpart for modeling. AIM: This study aims to generate and evaluate the 3D structure of TrkA, SERT, and AQP proteins and their interaction with the ligands, namely Asiaticoside-D (AD) and levodopa (L-DOPA) the anti-NDD agents. SUBJECTS AND METHODS: Homology modeling for SERT, AQP, and TrkA proteins of Lumbricus terrestris using SWISS-MODEL Server and the modeled structure was validated using Rampage Server. Wet-lab analysis of their correspondent m-RNA levels was also done to validate the in silico data. RESULTS: It was found that TrkA had moderately high homology (67%) to human while SERT and AQP could exhibit 58% and 42%, respectively. The reliability of the model was assessed by Ramachandran plot analysis. Interactions of AD with the SERT, AQP-4, and TrkA showed the binding energies as -9.93, 8.88, and -7.58 of Kcal/mol, respectively, while for L-DOPA did show -3.93, -5.13, and -6.0 Kcal/mol, respectively. The levels of SERT, TrkA, and AQP-4 were significantly reduced (P < 0.001) on ROT induced when compared to those of control worms. On ROT + AD supplementation group (III), m-RNA levels were significantly increased (P < 0.05) when compared to those of ROT induced worms (group II). CONCLUSION: Our pioneering docking data propose the possible of target which is proved useful for therapeutic investigations against the unconquered better of NDD.
Assuntos
Aquaporinas/metabolismo , Levodopa/farmacologia , Modelos Moleculares , Fármacos Neuroprotetores/farmacologia , Receptor trkA/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Triterpenos/farmacologia , Animais , Aquaporinas/genética , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/lesões , Gânglios dos Invertebrados/metabolismo , Oligoquetos , Receptor trkA/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genéticaRESUMO
BACKGROUND: Protein-carbohydrate interactions play vital roles in several biological processes in living organisms. The comparative analysis of binding site residues along with stabilizing residues in protein-carbohydrate complexes provides ample insights to understand the structure, function and recognition mechanism. OBJECTIVE: The main objective of this study is to identify and analyze the residues, which are involved in both folding and binding of the protein-carbohydrate complexes. METHODS: We have identified the stabilizing residues using the knowledge of hydrophobicity, longrange interactions and conservation, as well as binding site residues using a distance cutoff of 3.5Å between any heavy atoms in protein and ligand. Residues, which are common in stabilizing and binding, are termed as key residues. These key resides are analyzed with various sequence and structure based parameters such as frequency of occurrence, surrounding hydrophobicity, longrange order and conservation score. RESULTS: In this work, we have identified 2.45% binding site residues in a non-redundant dataset of 1130 complexes using distance-based criteria and 7.07% stabilizing residues using the concepts of hydrophobicity, long-range interactions and conservation of residues. Further, 5.9% of binding and 2.04% of stabilizing residues are common to each other, which are termed as key residues. The key residues have been analysed based on protein classes, carbohydrate types, gene ontology functional classifications, amino acid preference and structure-based parameters. We found that all-ß, α+ß and α/ß have more key residues than other protein classes and most of the KRs are present in ß-strands, which shows their importance in stability and binding of complexes. On the ligand side, Lsaccharide has the highest number of key residues and it has a high percentage of KRs in SRs and BRs than other carbohydrate types. Further, polar and charged residues have a high tendency to serve as key residues. Classifications based on gene ontology terms revealed that Lys is preferred in all the three groups: molecular functions, biological processes and cellular components. Key residues have 6 to 9 contacts within the protein and make only one contact with the carbohydrate ligand. These contacts are dominant to form polar-nonpolar contacts followed by the contacts between charged atoms. Further, the influence of sequence and structural parameters such as surrounding hydrophobicity, solvent accessibility, secondary structure, long-range order and conservation score has been discussed. CONCLUSION: The results obtained in the present work provide deep insights for understanding the interplay between stability and binding in protein-carbohydrate complexes.
Assuntos
Carboidratos/química , Modelos Moleculares , Proteínas/química , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Solventes/química , TermodinâmicaRESUMO
Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function.
Assuntos
DNA/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica/genética , Sequência de Aminoácidos , Sítios de Ligação , DNA/química , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Fator 1-alfa Nuclear de Hepatócito/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Análise de Componente Principal , Domínios Proteicos/genética , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Eletricidade EstáticaRESUMO
INTRODUCTION: Mangled upper limb injuries are at surge because of industrialization, modernization, and severe motor vehicle accidents. The utility of various scoring systems are meant for decision making in mangled lower limb injuries, and the same have been extrapolated for mangled upper limb injuries to make a decision of salvage or amputation for the lack of separate scoring system. MATERIALS AND METHODS: We applied mangled extremity severity score (MESS) and mangled extremity syndrome index (MESI) scoring systems to 10 cases of mangled upper limb injuries during the period of November 2010 to September 2012 presented at our tertiary trauma care center. Average MESS score was 7.7 and MESI score was 18.1. Above elbow, amputation was needed in three patients, and salvage procedure was done in rest of the seven patients. All the patients were subjected to salvage procedure initially unless life threatening because of mangled limb injury. MESS scores over 7, MESI score over 20 is accepted for amputation in lower limbs, but could not be justified in our study for MESS whereas MESI was more reliable. Functional outcome was assessed using visual analog scale score and short form-36 (SF-36) score for all patients, which was satisfactory, elderly and diabetic patients were relatively less satisfied. CONCLUSIONS: (a) Upper limb and lower limb mangled injuries cannot be considered same because of their anatomy. The upper limb has more rich vascularity and efficient collaterals, small muscle mass, so ischemia time is relatively more. Therefore, the different scoring systems are needed for both. (b) In our case series, MESI scoring was more reliable then MESS score, but this needs a large prospective study to validate it. (c) Salvage should be prime realistic aim influenced by several factors. Prosthesis for upper limb is very expensive and not well tolerated, so even a woody limb is well accepted in our patients.
RESUMO
PURPOSE: To determine whether the cif gene is present in pathogenic Pseudomonas aeruginosa isolates from patients with bacterial keratitis at Aravind Eye Hospital, a referral eye care center in southern India, and from corresponding environmental isolates. METHODS: Polymerase chain reaction amplification was performed on strains of P. aeruginosa isolated from ocular infections and environmental soil samples were collected from the area surrounding Aravind Eye Hospital. DNA sequencing of 16S ribosomal DNA amplicons was performed to verify strain identity. RESULTS: We determined that 45 of 48 patient isolates carry a genomic copy of cif. Analysis of a catalog of environmental strains previously isolated from the surrounding area revealed that only 4 of 10 P. aeruginosa strains and 1 of 14 strains of related species carry the cif gene. CONCLUSIONS: This is the first study to show that P. aeruginosa strains with ocular pathogenicity carry the cif gene and that the presence of this gene may be enriched over its prevalence in the environment. Taken together, these results suggest a potential role for Cif in acute bacterial keratitis.