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1.
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
2.
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
3.
Int J Mol Sci ; 20(9)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075847

RESUMO

A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2ΔCBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2ΔCBM had an identical optimum temperature at 70 °C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2ΔCBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s-1, but this was reduced by 40% for XynRA2ΔCBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.


Assuntos
Bactérias/enzimologia , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Variação Genética , Proteínas Mutantes/metabolismo , Tolerância ao Sal , Sequência de Aminoácidos , Endo-1,4-beta-Xilanases/genética , Cinética , Proteínas Mutantes/química , Filogenia , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Especificidade por Substrato , Xilanos/metabolismo
4.
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
5.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934824

RESUMO

Interferon-induced viperin (VP) was identified as playing an important role in the innate immune response against Zika virus (ZIKV). The 361 amino acid long human VP protein comprises of a highly conserved C-terminal region, which has been associated with VP antiviral properties against ZIKV. In the present study, we sought to determine whether the very last C-terminal amino-acid residues of VP might play a role in VP-mediated ZIKV inhibition. To address this issue, a recombinant human viperin (rVPwt) was overexpressed by transfection in human epithelial A549 cells. We confirmed that transient overexpression of rVPwt prior to ZIKV infection dramatically reduced viral replication in A549 cells. Deletion of the last 17 C-terminal amino acids of VP resulted in a higher expression level of mutant protein compared to wild-type VP. Mutational analysis revealed that residue substitution at positions 356 to 360 with five alanine led to the same phenotype. The charged residues Asp356, Lys358, and Asp360 were then identified to play a role in the weak level of VPwt protein in A549 cells. Mutant VP bearing the D360A substitution partially rescued ZIKV growth in A549 cells. Remarkably, a single Lys-to-Arg substitution at position 358 was sufficient to abrogate VP antiviral activity against ZIKV. In conclusion, our study showed that Asp356, Lys358, and Asp360 may have an influence on biochemical properties of VP. Our major finding was that Lys358 was a key amino-acid in VP antiviral properties against ZIKV.


Assuntos
Substituição de Aminoácidos , Antivirais/farmacologia , Proteínas Mutantes/metabolismo , Proteínas/genética , Zika virus/efeitos dos fármacos , Células A549 , Sequência de Aminoácidos , Animais , Cercopithecus aethiops , Humanos , Proteínas Mutantes/química , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacos
6.
MBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940700

RESUMO

The flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. The Salmonella FliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCE The bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Salmonella typhimurium/fisiologia , Movimento (Física) , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Supressão Genética
7.
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
8.
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
9.
Int J Mol Sci ; 20(7)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934684

RESUMO

Modern sequencing technologies provide an unprecedented amount of data of single-nucleotide variations occurring in coding regions and leading to changes in the expressed protein sequences. A significant fraction of these single-residue variations is linked to disease onset and collected in public databases. In recent years, many scientific studies have been focusing on the dissection of salient features of disease-related variations from different perspectives. In this work, we complement previous analyses by updating a dataset of disease-related variations occurring in proteins with 3D structure. Within this dataset, we describe functional and structural features that can be of interest for characterizing disease-related variations, including major chemico-physical properties, the strength of association to disease of variation types, their effect on protein stability, their location on the protein structure, and their distribution in Pfam structural/functional protein models. Our results support previous findings obtained in different data sets and introduce Pfam models as possible fingerprints of patterns of disease related single-nucleotide variations.


Assuntos
Doença/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Bases de Dados de Proteínas , Humanos , Domínios Proteicos , Solventes
10.
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
11.
Horm Metab Res ; 51(4): 248-255, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31022740

RESUMO

The objective of the study is the functional characterization of a novel POU1F1 c.605delC mutation in combined pituitary hormone deficiency (CPHD) and to report the clinical and genetic details of 160 growth hormone deficiency patients. Screening of GH1, GHRHR, POU1F1, PROP1, and HESX1 genes by Sanger sequencing was carried out in 160 trios and 100 controls followed by characterization of the POU1F1 c.605delC mutation by expression studies including site directed mutagenesis, co-transfection, protein degradation, and luciferase assays to compare the wild type and mutant POU1F1. In vitro studies showed that the POU1F1 c.605delC mutation codes for a truncated protein with reduced transactivation capacity on its downstream effectors, viz., growth hormone (GH) and prolactin (PRL) causing severe CPHD. Experiments using different protease inhibitors reveal rescue of the protein upon blockage of the lysosomal pathway that might be useful in novel drug designing using targeted approach thereby maintaining the milieu and preventing/delaying the disease. The study provides an insight into the disease causing mechanism of POU1F1 c.605delC mutation identified in a CPHD child with severe short stature and failure to thrive. It also shows mutation effect on the expression, function and turnover of protein and highlights mechanistic details by which these potent regulators may operate.


Assuntos
Nanismo Hipofisário/genética , Testes Genéticos , Mutação/genética , Fator de Transcrição Pit-1/genética , Criança , Feminino , Hormônio do Crescimento Humano/genética , Humanos , Hipopituitarismo/genética , Masculino , Proteínas Mutantes/metabolismo , Taxa de Mutação , Prolactina/genética , Domínios Proteicos , Proteólise , Fator de Transcrição Pit-1/química , Ativação Transcricional/genética
12.
Genes Dev ; 33(11-12): 705-717, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30948432

RESUMO

The Ccr4-Not complex regulates essentially every aspect of gene expression, from mRNA synthesis to protein destruction. The Not4 subunit of the complex contains an E3 RING domain and targets proteins for ubiquitin-dependent proteolysis. Ccr4-Not associates with elongating RNA polymerase II (RNAPII), which raises the possibility that it controls the degradation of elongation complex components. Here, we demonstrate that Ccr4-Not controls the ubiquitylation and turnover of Rpb1, the largest subunit of RNAPII, during transcription arrest. Deleting NOT4 or mutating its RING domain strongly reduced the DNA damage-dependent ubiquitylation and destruction of Rpb1. Surprisingly, in vitro ubiquitylation assays indicate that Ccr4-Not does not directly ubiquitylate Rpb1 but instead promotes Rpb1 ubiquitylation by the HECT domain-containing ligase Rsp5. Genetic analyses suggest that Ccr4-Not acts upstream of RSP5, where it acts to initiate the destruction process. Ccr4-Not binds Rsp5 and forms a ternary complex with it and the RNAPII elongation complex. Analysis of mutant Ccr4-Not lacking the RING domain of Not4 suggests that it both recruits Rsp5 and delivers the E2 Ubc4/5 to RNAPII. Our work reveals a previously unknown function of Ccr4-Not and identifies an essential new regulator of RNAPII turnover during genotoxic stress.


Assuntos
RNA Polimerase II/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Mutantes/metabolismo , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
13.
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
14.
Molecules ; 24(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889828

RESUMO

The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal-a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.


Assuntos
Aldeídos/metabolismo , Engenharia Metabólica/métodos , Monoterpenos/metabolismo , NADPH Desidrogenase/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Modelos Moleculares , Mutagênese/genética , Proteínas Mutantes/metabolismo , NADPH Desidrogenase/metabolismo , Oxirredução , Estereoisomerismo
15.
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
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.
Gen Comp Endocrinol ; 276: 37-44, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30836102

RESUMO

Eel follicle-stimulating hormone (eelFSH) is composed of a common α-subunit and a hormone specific ß-subunit, both of which contain two N-linked carbohydrate residues. We characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelFSH ß-subunit. Expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using Chinese hamster ovary (CHO) K1 cells expressing the eelFSH receptor gene. Mutagenesis of the individual and double glycosylated sites was performed to determine the functions of the oligosaccharide chains on signal transduction. The absence of the Asn22 (eelFSHßΔ22/α) and Asn5.22 (eelFSHßΔ5.22/α) N-linked oligosaccharide chain in the eelFSH ß-subunit completely reduced the secretion level in the medium and cell lysate of CHO-K1 cells. The expression levels of eelFSHß/α wild-type in CHO suspension (CHO-S) cells was approximately 4-fold higher in CHO-k1 cells. The molecular weight of rec-eelFSHß/α wild-type by western blotting analysis was found to be 34 kDa. Mutants (ß/αΔ56, ß/αΔ79, and ßΔ5/α) lacking single oligosaccharide sites showed molecular weights that were reduced by approximately 10%. The digestion of N-linked oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants were reduced to 27-kDa. The oligosaccharide chains in rec-eelFSHß/α wild-type were modified to a molecular weight of approximately 7-10 kDa in CHO-K1 and CHO-S cells. Oligosaccharide site deletions at positions Asn56 and Asn79 on the α-subunit and Asn5 on the ß-subunit were found to play an essential role in cAMP signal transduction through the eelFSH receptor. The EC50 values of Asn56 and Asn5 resulted in a significant decrease in potency to 64% and 53% of the wild type, respectively. Specifically, the removal of the carbohydrates at Asn79 of the α-subunit (ß/αΔ79) was drastically reduced to 53.8% of the wild-type levels in maximum response. These results have allowed for the identification of the site-specific roles of carbohydrate residues in eel FSH. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelFSH receptor as suggested in similar studies of other mammalian FSH hormones.


Assuntos
Enguias/metabolismo , Hormônio Foliculoestimulante/metabolismo , Oligossacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Glicosilação , Humanos , Proteínas Mutantes/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Receptores do FSH/metabolismo
18.
MBio ; 10(1)2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723127

RESUMO

APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B Last, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells.IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5'-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.


Assuntos
Antígenos Virais de Tumores/metabolismo , Citidina Desaminase/biossíntese , Interações Hospedeiro-Patógeno , Antígenos de Histocompatibilidade Menor/biossíntese , Polyomavirus/crescimento & desenvolvimento , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Antígenos Virais de Tumores/genética , Sítios de Ligação , Células Cultivadas , Fatores de Transcrição E2F/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neoplasias/patologia , Proteínas de Ligação a Retinoblastoma/metabolismo
19.
Database (Oxford) ; 20192019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30805645

RESUMO

Ionizable residues play key roles in many biological phenomena including protein folding, enzyme catalysis and binding. We present PKAD, a database of experimentally measured pKas of protein residues reported in the literature or taken from existing databases. The database contains pKa data for 1350 residues in 157 wild-type proteins and for 232 residues in 45 mutant proteins. Most of these values are for Asp, Glu, His and Lys amino acids. The database is available as downloadable file as well as a web server (http://compbio.clemson.edu/pkad). The PKAD database can be used as a benchmarking source for development and improvement of pKa's prediction methods. The web server provides additional information taken from the corresponding structures and amino acid sequences, which allows for easy search and grouping of the experimental pKas according to various biophysical characteristics, amino acid type and others.


Assuntos
Bases de Dados de Proteínas , Proteínas/química , Concentração de Íons de Hidrogênio , Íons , Proteínas Mutantes/metabolismo , Solventes , Propriedades de Superfície
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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