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1.
Gene ; 720: 144082, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476406

RESUMO

The enzyme ß-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115-147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Proteínas de Bactérias/química , Isoenzimas/química , Proteínas Mutantes/química , Mutação , Mycobacterium tuberculosis/enzimologia , Tuberculose/microbiologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Proteínas de Bactérias/genética , Isoenzimas/genética , Simulação de Dinâmica Molecular , Proteínas Mutantes/genética , Conformação Proteica , Tuberculose/genética , Tuberculose/metabolismo
2.
J Cancer Res Clin Oncol ; 145(9): 2273-2283, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31428934

RESUMO

OBJECTIVES: Recent research has classified lung adenocarcinoma patients with KRAS mutation into three subtypes by co-occurring genetic events in TP53 (KP subgroup), STK11/LKB1 (KL subgroup) and CDKN2A/B inactivation plus TTF-1 low expression (KC subgroup). The aim of this study was to identify valuable biomarkers by searching the candidate molecules that contribute to lung adenocarcinoma pathogenesis, especially KC subtype. MATERIALS AND METHODS: We analyzed the publicly available database and identified the candidate REG4 using the E-GEOD-31210 dataset, and then confirmed by TCGA dataset. In addition, an independent cohort of 55 clinical samples was analyzed by quantitative real-time PCR analysis. Functional studies and RNA sequencing were performed after silencing the REG4 expression. RESULTS: REG4, an important regulator of gastro-intestinal carcinogenesis, was highly expressed in KRAS mutant lung adenocarcinoma with low expression of TTF-1 (KC subtype). The results were validated both by gene expression analysis and immunohistochemistry study in an independent 55 clinical samples from Fudan University Shanghai Cancer Center. Further in vitro and in vivo functional assays revealed silencing REG4 expression significantly reduces cancer cell proliferation and tumorigenesis. Moreover, RNA sequencing and GSEA analysis displayed that REG4 knockdown might induce cell cycle arrest by regulating G2/M checkpoint and E2F targets. CONCLUSION: Our results indicate that REG4 plays an important role in KRAS-driven lung cancer pathogenesis and is a novel biomarker of lung adenocarcinoma subtype. Future studies are required to clarify the underlying mechanisms of REG4 in the division and proliferation of KC tumors and its potential therapeutic value.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/diagnóstico , Proteínas Associadas a Pancreatite/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Fatores de Transcrição/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Estudos de Coortes , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/metabolismo
3.
Chem Biol Interact ; 310: 108756, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325422

RESUMO

Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37°C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.


Assuntos
Butirilcolinesterase/metabolismo , Engenharia de Proteínas/métodos , Butirilcolinesterase/genética , Substâncias para a Guerra Química/metabolismo , Dimerização , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Inseticidas/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Compostos Organofosforados/metabolismo
4.
Enzyme Microb Technol ; 128: 67-71, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31186112

RESUMO

The substrate selectivity of the Trp416Gly mutant of Methanothermobacter thermautotrophicus acetyl-CoA synthetase (Trp416Gly MT-ACS1) was explored. The goal was to identify its substrate range, particularly for functionalized carboxylic acid substrates that would allow post-synthesis functionalization of CoA thioesters or downstream products using metathesis or Click chemistry. Relative activities were determined by in situ formation of acyl-hydroxamate iron (III) complexes. Trp416Gly MT-ACS1 showed good activities for saturated straight chain carboxylic acids from C2 to C8, for ω-alkenyl straight chain carboxylic acids from C4 to C7 and for ω-alkynyl straight chain carboxylic acids from C5 to C7. Carboxylic acids showing ≥20% conversion in screening reactions were used in preparative conversions that completely consumed the added CoASH.


Assuntos
Acetato-CoA Ligase/metabolismo , Substituição de Aminoácidos , Methanobacteriaceae/enzimologia , Proteínas Mutantes/metabolismo , Acetato-CoA Ligase/genética , Ácidos Carboxílicos/metabolismo , Proteínas Mutantes/genética , Mutação Puntual , Especificidade por Substrato
5.
Biochimie ; 163: 21-32, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31075282

RESUMO

Huntington's diseases (HD) is a very devastating disease caused by r(CAG) expansion in HTT gene, encoding the huntingtin protein. r(CAG) expansion causes disease via multiple pathways including, 1) loss of normal protein function like sequestration of RNA binding protein such as Muscleblind-like (MBNL) and nucleolin, 2) Gain of function for mutant proteins and 3) repeat-associated non-ATG (RAN) translation; in which expanded r(CAG) translates into toxic poly glu, poly ser, or poly ala without the use of any canonical start codon. Herein, we have rationally designed and synthesized a unique class of pyridocoumarin derivatives that target the r(CAG)exp involved in HD and spinocerebellar ataxia (SCA) pathogenesis. Notably, compounds 3 and 15 showed higher affinity (nanomolar Kd) and selectivity for diseased r(CAG)exp RNA compared to regular duplex AU-paired RNA. Interestingly, both scaffolds are cell permeable, exhibit low toxicity to healthy fibroblast cells and are also capable of reducing the level of poly Q aggregation in cellular models. Indeed, our current study offers promising facet for selectively targeting repeats containing RNAs that cause severe diseases like HD and SCAs.


Assuntos
Cumarínicos/química , Doença de Huntington/tratamento farmacológico , Proteínas Mutantes/genética , RNA Mensageiro/química , Ataxias Espinocerebelares/tratamento farmacológico , Células Cultivadas , Cumarínicos/farmacologia , Cumarínicos/uso terapêutico , Desenho de Drogas , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Cinética , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/efeitos dos fármacos , Ataxias Espinocerebelares/metabolismo , Expansão das Repetições de Trinucleotídeos
6.
Biomed Res Int ; 2019: 1425281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058184

RESUMO

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.


Assuntos
Leishmania major/genética , RNA Polimerase III/genética , Fator de Transcrição TFIIIB/genética , Transcrição Genética , Simulação por Computador , Sequência Conservada/genética , Regulação da Expressão Gênica/genética , Recombinação Homóloga/genética , Proteínas Mutantes/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Subunidades Proteicas/genética , RNA Ribossômico 5S/biossíntese , RNA Nuclear Pequeno/biossíntese , RNA de Transferência/biossíntese
7.
Enzyme Microb Technol ; 127: 22-31, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088613

RESUMO

The recombinant rAgaZC-1 was a family GH50 ß-agarase from Vibrio sp. ZC-1 (CICC 24670). In this paper, the mutant D622G (i.e., mutate the aspartic acid at position 622 to glycine) had better thermo-stability than rAgaZC-1, showing 1.5℃ higher T5010 (the temperature at which the half-time is 10 min) and 4-folds of half-time at 41℃, while they had almost same optimum temperature (38.5℃), optimum pH (pH6.0) and catalytic efficiency. Thermal deactivation kinetical analysis showed that D622G had higher activation energy for deactivation, enthalpy and Gibbs free energy than rAgaZC-1, indicating that more energy is required by D622G for deactivation. Substrate can protect agarase against thermal inactivation, especially D622G. Hence the yield of agarose hydrolysis catalyzed by D622G was higher than that by rAgaZC-1. The models of D622G and rAgaZC-1 predicted by homology modeling were compared to find that it is the improved distribution of surface electrostatic potential, great symmetric positive potential and more hydrophobic interactions of D622G that enhance the thermo-stability.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Mutagênese , Vibrio/enzimologia , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Conformação Proteica , Estabilidade Proteica , Sefarose/metabolismo
8.
MBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040246

RESUMO

The global spread of Plasmodium falciparum chloroquine resistance transporter (PfCRT) variant haplotypes earlier caused the widespread loss of chloroquine (CQ) efficacy. In Asia, novel PfCRT mutations that emerged on the Dd2 allelic background have recently been implicated in high-level resistance to piperaquine, and N326S and I356T have been associated with genetic backgrounds in which resistance emerged to artemisinin derivatives. By analyzing large-scale genome sequencing data, we report that the predominant Asian CQ-resistant Dd2 haplotype is undetectable in Africa. Instead, the GB4 and previously unexplored Cam783 haplotypes predominate, along with wild-type, drug-sensitive PfCRT that has reemerged as the major haplotype. To interrogate how these alleles impact drug susceptibility, we generated pfcrt-modified isogenic parasite lines spanning the mutational interval between GB4 and Dd2, which includes Cam783 and involves amino acid substitutions at residues 326 and 356. Relative to Dd2, the GB4 and Cam783 alleles were observed to mediate lower degrees of resistance to CQ and the first-line drug amodiaquine, while resulting in higher growth rates. These findings suggest that differences in growth rates, a surrogate of parasite fitness, influence selection in the context of African infections that are frequently characterized by high transmission rates, mixed infections, increased immunity, and less recourse to treatment. We also observe that the Asian Dd2 allele affords partial protection against piperaquine yet does not directly impact artemisinin efficacy. Our results can help inform the regional recommendations of antimalarials, whose activity is influenced by and, in certain cases, enhanced against select PfCRT variant haplotypes.IMPORTANCE Our study defines the allelic distribution of pfcrt, an important mediator of multidrug resistance in Plasmodium falciparum, in Africa and Asia. We leveraged whole-genome sequence analysis and gene editing to demonstrate how current drug combinations can select different allelic variants of this gene and shape region-specific parasite population structures. We document the ability of PfCRT mutations to modulate parasite susceptibility to current antimalarials in dissimilar, pfcrt allele-specific ways. This study underscores the importance of actively monitoring pfcrt genotypes to identify emerging patterns of multidrug resistance and help guide region-specific treatment options.


Assuntos
Resistência a Múltiplos Medicamentos , Aptidão Genética , Genótipo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , África/epidemiologia , Ásia/epidemiologia , Frequência do Gene , Genética Populacional , Malária Falciparum/epidemiologia , Proteínas Mutantes/genética , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação
9.
Appl Microbiol Biotechnol ; 103(11): 4539-4548, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997553

RESUMO

The MtrA-MtrB two-component regulatory system is highly conserved in Actinobacteria and plays crucial roles in cell cycle progression, cell morphology, antibiotic resistance, and osmoprotection. Previously, we revealed that the MtrA protein of Saccharopolyspora erythraea E3 strain (a high erythromycin-producing strain) had a two amino acid (H197 and V198) deletion in the DNA recognition helices of the C-terminal domain compared to the wild type S. erythraea strain NRRL2338. Here, we identified mepA (encoding a membrane protein related to metalloendopeptidases) as an MtrA target gene, and found that deleting the two amino acids in MtrA (MtrAdel) resulted in the loss of its DNA-binding activity for the mepA gene. The mutant MtrAdel lost its regulatory activity and affected various physiological functions consistent with mtrA deletion, including increased erythromycin biosynthesis, enhanced antibiotic resistance, deregulated osmoprotection, and improved transport of substances. The introduction of the wild type mtrA gene into the S. erythraea E3 strain with the mtrAdel gene decreased the erythromycin yield by approximately 50%, confirming that MtrA repressed erythromycin production. These findings demonstrate that MtrA is an important pleiotropic regulator of erythromycin biosynthesis, antibiotic resistance, osmoprotection, and substance transport in S. erythraea and provide new insights for improving erythromycin production. Future studies linking the molecular effects of MtrA to these phenotypes will improve our understanding of the MtrA-MtrB two-component regulatory system in Actinobacteria.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eritromicina/biossíntese , Saccharopolyspora/enzimologia , Saccharopolyspora/metabolismo , Deleção de Sequência , Transporte Biológico , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fenótipo , Saccharopolyspora/crescimento & desenvolvimento
10.
Appl Microbiol Biotechnol ; 103(10): 4053-4064, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937498

RESUMO

D-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic D-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic D-amino acids and a neutral D-amino acid, D-Gln, with the highest activity toward D-Glu. The Km and kcat values for D-Glu were 2.16 mM and 217 s-1, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and D-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.


Assuntos
Ácido Aspártico/metabolismo , D-Aspartato Oxidase/metabolismo , Eurotiales/enzimologia , Clonagem Molecular , D-Aspartato Oxidase/química , D-Aspartato Oxidase/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
11.
MBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940706

RESUMO

Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections each year. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis, both as frontline and as salvage therapy. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide, and a large proportion of this resistance remains unexplained. In this work, we characterize the contributions of previously identified mechanisms of triazole resistance, including mutations in the sterol-demethylase-encoding gene cyp51A, overexpression of sterol-demethylase genes, and overexpression of the efflux pump-encoding gene abcC, among a large collection of highly triazole-resistant clinical A. fumigatus isolates. Upon revealing that these mechanisms alone cannot substantiate the majority of triazole resistance exhibited by this collection, we subsequently describe the identification and characterization of a novel genetic determinant of triazole resistance. Mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase-encoding gene, hmg1, were identified in a majority of triazole-resistant clinical isolates in our collection. Introduction of three different hmg1 mutations, predicted to encode residue alterations in the conserved sterol sensing domain of Hmg1, resulted in significantly increased resistance to the triazole class of agents. Additionally, correction of a hmg1 mutation in a pan-triazole-resistant clinical isolate of A. fumigatus with a novel Cas9-ribonucleoprotein-mediated system was shown to restore clinical susceptibility to all triazole agents. Mutations in hmg1 were also shown to lead to the accumulation of ergosterol precursors, such as eburicol, by sterol profiling, while not altering the expression of sterol-demethylase genes.IMPORTANCE Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections annually. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide. In this work, we challenge the current paradigm of clinical triazole resistance in A. fumigatus, by first demonstrating that previously characterized mechanisms of resistance have nominal impact on triazole susceptibility and subsequently identifying a novel mechanism of resistance with a profound impact on clinical triazole susceptibility. We demonstrate that mutations in the HMG-CoA reductase gene, hmg1, are common among resistant clinical isolates and that hmg1 mutations confer resistance to all clinically available triazole antifungals.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Farmacorresistência Fúngica , Hidroximetilglutaril-CoA Redutases/metabolismo , Proteínas Mutantes/metabolismo , Triazóis/farmacologia , Aspergilose/microbiologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Proteínas Mutantes/genética
12.
Chem Biol Interact ; 306: 138-146, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31009643

RESUMO

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 µs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.


Assuntos
Biocatálise , Butirilcolinesterase/genética , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Projeto Auxiliado por Computador , Iodeto de Ecotiofato/farmacologia , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Butirilcolinesterase/química , Inibidores da Colinesterase/química , Iodeto de Ecotiofato/química , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Proteínas Mutantes/genética , Teoria Quântica
13.
Protein Pept Lett ; 26(6): 449-457, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-30919764

RESUMO

BACKGROUND: The significance of multi-site phosphorylation of BCL-2 protein in the flexible loop domain remains controversial, in part due to the lack of structural biology studies of phosphorylated BCL-2. OBJECTIVE: The purpose of the study is to explore the phosphorylation induced structural changes of BCL-2 protein. METHODS: We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEEBCL- 2-EK (62-206)), in which the BH4 domain and the part of loop region was truncated (residues 2-61) to enable a backbone resonance assignment. The phosphorylation-induced structural change was visualized by overlapping a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy between EEE-BCL-2-EK (62-206) and BCL-2. RESULTS: The EEE-BCL-2-EK (62-206) protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from non-phosphorylated BCL-2 (npBCL-2) protein. Some residues in BH3 binding groove occurred chemical shift in the EEEBCL- 2-EK (62-206) spectrum, indicating that the phosphorylation in the loop region induces a structural change of active site. CONCLUSION: The phosphorylation of BCL-2 induced structural change in BH3 binding groove.


Assuntos
Proteínas Mutantes/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Proto-Oncogênicas c-bcl-2/química , Apoptose , Linhagem Celular , Escherichia coli/genética , Humanos , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Fosforilação , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transfecção
14.
Biomed Khim ; 65(1): 41-50, 2019 Jan.
Artigo em Russo | MEDLINE | ID: mdl-30816096

RESUMO

Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.


Assuntos
Proteínas de Bactérias/genética , Quimiotaxia , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Escherichia coli , Proteínas Mutantes/genética , Proteínas Recombinantes
15.
Anaerobe ; 57: 39-44, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30878603

RESUMO

OBJECTIVES: Increasing incidence and severity of Clostridium difficile infection (CDI) in the last decades has been attributed to the emergence of hypervirulent C. difficile strain PCR-ribotype 027 (RT027). Commercial multiplex real-time PCR tests allow the presumptive identification of RT027 by detecting a single-base deletion at nt117 in the tcdC gene (tcdCΔ117). The clinical usefulness of the detection of tcdCΔ117 is unclear. Therefore, we evaluated test performance and clinical association of the detection of tcdCΔ117 in patients with CDI in a prospective observational study conducted in a tertiary care hospital in Germany. METHODS: From June to October 2015, stool from all patients with suspected CDI was tested for C. difficile according to ESCMID guidelines. C. difficile was cultured from positive samples and ribotyping was performed. Clinical data were collected prospectively from all C. difficile positive patients. RESULTS: From 1121 tested stool samples 107 patients with CDI were included in the study. TcdCΔ117 was detected in 18 (16.8%) of these patients. Multivariable logistic regression analysis revealed an independent association of detection of tcdCΔ117 with a further episode of CDI (OR 14.6; 95% CI 3.6-58.3; p < 0.001) and death within 30 days of the positive test (OR 5.1; 95% CI 1.0-25.7; p = 0.046). As follow up data are limited, it remains unclear, whether the further episode of CDI was due to tcdCΔ117 (recurrence) or another type. CONCLUSION: In our setting, PCR-based detection of tcdCΔ117 identified patients at risk for recurrent CDI and increased mortality and thus may guide therapeutic choices in CDI patients at the time of diagnosis.


Assuntos
Proteínas de Bactérias/genética , Infecções por Clostridium/diagnóstico , Clostridium difficile/genética , Técnicas de Genotipagem , Proteínas Mutantes/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Repressoras/genética , Deleção de Sequência , Adulto , Idoso , Infecções por Clostridium/microbiologia , Infecções por Clostridium/mortalidade , Clostridium difficile/classificação , Feminino , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Recidiva , Ribotipagem , Medição de Risco , Análise de Sobrevida , Centros de Atenção Terciária
16.
Malar J ; 18(1): 62, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30845961

RESUMO

BACKGROUND: UDP-glycosyltransferase (UGT) is an important biotransformation superfamily of enzymes. They catalyze the transfer of glycosyl residues from activated nucleotide sugars to acceptor hydrophobic molecules, and function in several physiological processes, including detoxification, olfaction, cuticle formation, pigmentation. The diversity, classification, scaffold location, characteristics, phylogenetics, and evolution of the superfamily of genes at whole genome level, and their association and mutations associated with pyrethroid resistance are still little known. METHODS: The present study identified UGT genes in Anopheles sinensis genome, classified UGT genes in An. sinensis, Anopheles gambiae, Aedes aegypti and Drosophila melanogaster genomes, and analysed the scaffold location, characteristics, phylogenetics, and evolution of An. sinensis UGT genes using bioinformatics methods. The present study also identified the UGTs associated with pyrethroid resistance using three field pyrethroid-resistant populations with RNA-seq and RT-qPCR, and the mutations associated with pyrethroid resistance with genome re-sequencing in An. sinensis. RESULTS: There are 30 putative UGTs in An. sinensis genome, which are classified into 12 families (UGT301, UGT302, UGT306, UGT308, UGT309, UGT310, UGT313, UGT314, UGT315, UGT36, UGT49, UGT50) and further into 23 sub-families. The UGT308 is significantly expanded in gene number compared with other families. A total of 119 UGTs from An. sinensis, An. gambiae, Aedes aegypti and Drosophila melanogaster genomes are classified into 19 families, of which seven are specific for three mosquito species and seven are specific for Drosophila melanogaster. The UGT308 and UGT302 are proposed to main families involved in pyrethroid resistance. The AsUGT308D3 is proposed to be the essential UGT gene for the participation in biotransformation in pyrethroid detoxification process, which is possibly regulated by eight SNPs in its 3' flanking region. The UGT302A3 is also associated with pyrethroid resistance, and four amino acid mutations in its coding sequences might enhance its catalytic activity and further result in higher insecticide resistance. CONCLUSIONS: This study provides the diversity, phylogenetics and evolution of UGT genes, and potential UGT members and mutations involved in pyrethroid resistance in An. sinensis, and lays an important basis for the better understanding and further research on UGT function in defense against insecticide stress.


Assuntos
Anopheles/efeitos dos fármacos , Anopheles/enzimologia , Glicosiltransferases/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Proteínas Mutantes/genética , Piretrinas/farmacologia , Aedes/enzimologia , Aedes/genética , Animais , Anopheles/genética , Biologia Computacional , Drosophila/enzimologia , Drosophila/genética , Feminino , Perfilação da Expressão Gênica , Glicosiltransferases/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
17.
MBio ; 10(2)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862746

RESUMO

Clostridioides difficile infection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two major C. difficile toxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified a C. difficile regulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, and rstA transcription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds the rstA promoter via the predicted DNA-binding domain. Through mutational analysis of the rstA promoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genes tcdA and tcdB, as well as the promoters for the sigD and tcdR genes, which encode regulators of toxin gene expression. Complementation analyses with the Clostridium perfringens RstA ortholog and a multispecies chimeric RstA protein revealed that the C. difficile C-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCE Clostridioides difficile is an anaerobic, gastrointestinal pathogen of humans and other mammals. C. difficile produces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link between C. difficile sporulation and toxin production. Further, our data suggest that C. difficile toxin production is regulated through a direct, species-specific sensing mechanism.


Assuntos
Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Clostridium difficile/genética , Clostridium difficile/fisiologia , Enterotoxinas/biossíntese , Regulação Bacteriana da Expressão Gênica , Locomoção , Proteínas Repressoras/metabolismo , Clostridium perfringens/genética , Análise Mutacional de DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética
18.
BMC Evol Biol ; 19(1): 72, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30849938

RESUMO

BACKGROUND: Frizzled family members belong to G-protein coupled receptors and encode proteins accountable for cell signal transduction, cell proliferation and cell death. Members of Frizzled receptor family are considered to have critical roles in causing various forms of cancer, cardiac hypertrophy, familial exudative vitreoretinopathy (FEVR) and schizophrenia. RESULTS: This study investigates the evolutionary and structural aspects of Frizzled receptors, with particular focus on FEVR associated FZD4 gene. The phylogenetic tree topology suggests the diversification of Frizzled receptors at the root of metazoans history. Moreover, comparative structural data reveals that FEVR associated missense mutations in FZD4 effect the common protein region (amino acids 495-537) through a well-known phenomenon called epistasis. This critical protein region is present at the carboxyl-terminal domain and encompasses the K-T/S-XXX-W, a PDZ binding motif and S/T-X-V PDZ recognition motif. CONCLUSION: Taken together these results demonstrate that during the course of evolution, FZD4 has acquired new functions or epistasis via complex patter of gene duplications, sequence divergence and conformational remodeling. In particular, amino acids 495-537 at the C-terminus region of FZD4 protein might be crucial in its normal function and/or pathophysiology. This critical region of FZD4 protein may offer opportunities for the development of novel therapeutics approaches for human retinal vascular disease.


Assuntos
Evolução Molecular , Oftalmopatias Hereditárias/genética , Receptores Frizzled/química , Receptores Frizzled/genética , Doenças Retinianas/genética , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação de Sentido Incorreto/genética , Filogenia , Domínios Proteicos
19.
J Biosci Bioeng ; 128(1): 22-27, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30803783

RESUMO

Nucleoside deoxyribosyltransferase II (NDT) catalyzes the transglycosylation reaction of the 2'-deoxyribose moiety between purine and/or pyrimidine bases and has been widely used in the synthesis of nucleoside analogs. The high specificity of NDT for 2'-deoxyribose limits its applications. Because 2'C- and/or 3'C-modified nucleosides have been widely used as antiviral or antitumour agents, improving the activity of NDT towards these modified nucleosides by protein engineering is an area of interest to the pharmaceutical industry. NDT engineering is hindered by a lack of effective screening methods. This study developed a high-throughput screening system, which was established by nucleoside deoxyribosyltransferase II-cytidine deaminase co-expression, indophenol colorimetric assay and whole-cell catalysis. A high-throughput screening system for NDT was established for the first time. This system can be applied to detect NDT-specific activity for a variety of cytidine analogs with glycosyl and base modifications, such as 5-aza-2'-deoxycytidine, 2',3'-dideoxycytidine, cytosine-ß-d-arabinofuranoside. In this study, we adopted the semi-rational design of NDT and constructed a mutant library of NDT from Lactobacillus helveticus (LhNDT) by site-saturation mutagenesis. Over 600 mutants were screened, and a variant with up to a 5.2-fold higher conversion rate of 2',3'-dideoxyinosine was obtained.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Lactobacillus helveticus/genética , Proteínas Mutantes/isolamento & purificação , Pentosiltransferases/genética , Pentosiltransferases/isolamento & purificação , Pentosiltransferases/metabolismo , Catálise , Domínio Catalítico/genética , Ensaios Enzimáticos/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Mutagênese Sítio-Dirigida/métodos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nucleosídeos , Pentosiltransferases/química , Engenharia de Proteínas/métodos , Purinas , Pirimidinas , Relação Estrutura-Atividade , Especificidade por Substrato/genética
20.
Fungal Biol ; 123(3): 209-217, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30798876

RESUMO

In nature, white-rot fungi efficiently degrade lignin present in wood biomass. Elucidation of molecular mechanisms underlying wood lignin biodegradation by white-rot fungi would contribute to the development of efficient and ecofriendly methods of producing valuable chemical products from wood biomass. Here, using forward genetics approach, we demonstrate that the mutant of a putative transcription factor gene, gat1-1, significantly decreases the ligninolytic activity of the white-rot fungus Pleurotus ostreatus, when grown on beech wood sawdust medium. We also show that this phenotype is dominant. In Schizophyllum commune, Gat1 was previously shown to be involved in fruiting body development. In this study, we reveal that the mutations in gat1 gene cause defects in fruiting body development in P. ostreatus. Unlike the previously reported recessive gene mutations that decrease the ligninolytic activity of P. ostreatus, the gat1-1 mutation and Δgat1 are dominant and would thus be useful for future studies on the functional role of the orthologs in other white-rot fungi.


Assuntos
Carpóforos/crescimento & desenvolvimento , Lignina/metabolismo , Proteínas Mutantes/metabolismo , Pleurotus/genética , Pleurotus/metabolismo , Fatores de Transcrição/metabolismo , Biotransformação , Proteínas Mutantes/genética , Pleurotus/crescimento & desenvolvimento , Fatores de Transcrição/genética
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