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1.
Protein & Cell ; (12): 877-888, 2021.
Article in English | WPRIM | ID: wpr-922482

ABSTRACT

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M


Subject(s)
Antiviral Agents/therapeutic use , Binding Sites , COVID-19/virology , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Drug Evaluation, Preclinical , Drug Repositioning , High-Throughput Screening Assays/methods , Humans , Imidazoles/therapeutic use , Inhibitory Concentration 50 , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Naphthoquinones/therapeutic use , Protease Inhibitors/therapeutic use , Protein Structure, Tertiary , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification
2.
Chinese Journal of Biotechnology ; (12): 1232-1240, 2020.
Article in Chinese | WPRIM | ID: wpr-826854

ABSTRACT

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.


Subject(s)
Base Sequence , Cloning, Molecular , Genetic Vectors , Genetics , Mutagenesis, Site-Directed , Methods , Nucleic Acid Amplification Techniques , Plasmids , Polymerase Chain Reaction
3.
Chinese Journal of Biotechnology ; (12): 801-809, 2020.
Article in Chinese | WPRIM | ID: wpr-826896

ABSTRACT

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.


Subject(s)
DNA Primers , Genetics , Escherichia coli , Mutagenesis, Site-Directed , Methods , Plasmids , Genetics , Polymerase Chain Reaction
4.
Electron. j. biotechnol ; 42: 49-55, Nov. 2019. tab, ilus, graf
Article in English | LILACS | ID: biblio-1087461

ABSTRACT

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.


Subject(s)
Bacillus licheniformis/enzymology , Glutaminase/metabolism , Arginine , Plasmids , Prostaglandins A/chemistry , Bacillus subtilis , Protein Sorting Signals , Base Sequence , Mutagenesis, Site-Directed , Aspartic Acid , Escherichia coli , Bacillus licheniformis/genetics , Glutaminase/genetics
5.
Article in English | WPRIM | ID: wpr-761784

ABSTRACT

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.


Subject(s)
Calcium , Carbachol , Humans , Hydrogen-Ion Concentration , Ion Channels , Kidney , Mass Screening , Mutagenesis, Site-Directed , Mutant Proteins , Protein Kinase C , Receptors, Muscarinic
6.
Chinese Journal of Biotechnology ; (12): 921-930, 2019.
Article in Chinese | WPRIM | ID: wpr-771834

ABSTRACT

Genome editing is a genetic engineering technique that uses site-directed cleavage activity of specific artificial nucleases and endogenous DNA damage repair activity to generate insertions, deletions or substitutions in the targeted genomic loci. As the accuracy and efficiency of genome editing is improving and the operation is simple, the application of genome editing is expanding. This article provides an overview of the three major genome editing technologies and genome editing types, and the regulatory frameworks for genome-edited products were summarized in the United States, the European Union, and other countries. At the same time, based on the Chinese safety management principles and systems for genetically modified organisms (GMOs), the authors proposed a regulatory framework for genome-edited products. Genome-edited products should first be classified according to whether containing exogenous genetic components such as Cas9 editing enzymes or not. They should be regulated as traditional genetically modified organisms if they do. Otherwise, the regulation of genome-edited products depends on targeted modifications.


Subject(s)
CRISPR-Cas Systems , Endonucleases , Gene Editing , Genome , Mutagenesis, Site-Directed
7.
Braz. j. med. biol. res ; 51(2): e6546, 2018. graf
Article in English | LILACS | ID: biblio-889018

ABSTRACT

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).


Subject(s)
Humans , Gene Expression Regulation, Neoplastic/genetics , Cyclooxygenase 2/genetics , Haplotypes , Up-Regulation , Mutagenesis, Site-Directed , Polymorphism, Single Nucleotide , Cell Line, Tumor , Cyclooxygenase 2/metabolism , MCF-7 Cells , Genotype , Luciferases/metabolism
8.
Rio de Janeiro; s.n; 2018. xiv,119 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-909411

ABSTRACT

Resumo: A Febre Amarela (FA) é uma doença infecciosa não contagiosa, causada por um arbovírus e objeto de preocupação sanitária mundial. A principal medida de controle é a vacinação com o vírus atenuado da FA, cepa 17D, que é capaz de induzir resposta imune protetora a longo prazo com administração em dose única. Vírus atenuados são potentes vetores de expressão, pois disseminam o antígeno no hospedeiro e induzem resposta imune protetora, contribuindo para o desenvolvimento de vacinas recombinantes. Diversas estratégias foram desenvolvidas para expressão de genes heterólogos pelo vírus vacinal FA 17D. Entretanto, a inserção induz proliferação viral e imunogenicidade reduzidas. Neste trabalho foi utilizado o vírus vacinal FA 17D recombinante expressando a proteína repórter enhanced green fluorescent protein (EGFP) para avaliar o impacto de mutações específicas que possam modular a replicação viral. Mutações nas proteínas E, NS3 e NS4B foram descritas por aumentarem a proliferação viral em cultura de células e em camundongos, no genoma do vírus da dengue, sorotipos 1 e 2. As mutações em E400 (F→L), E403 (T→I), NS3439 (V→S) e NS4B54 (L→F) foram inseridas no genoma do vírus FA/EGFP, com a finalidade de caracterizar o seu efeito na proliferação viral e na indução de resposta imune humoral. O cDNA do genoma viral FA/EGFP foi utilizado para gerar vírus recombinantes carreando uma, duas ou três mutações. O estudo de proliferação viral foi realizado por cinética de infecção de células das linhagens Vero, Huh7 e C6/36. Os resultados mostram que os vírus da FA recombinantes se proliferam menos que o vírus vacinal FA 17DD. Além disso, a infectividade dos vírus mutantes em células de mamífero é diferente da infectividade em células de mosquitoOs vírus que carreiam as mutações em E400/NS3439 e E400/NS3439/NS4B54 tem a proliferação viral significativamente prejudicada em células de mamífero. Os vírus que carreiam a mutação em E400 apresentaram aumento de proliferação viral em comparação com o vírus FA/EGFP original, em células de mosquito. As diferenças entre os tipos celulares podem ter sido causadas pelas características fisiológicas das células durante a infecção viral e pelas diferenças de propriedades das proteínas virais ocasionadas pela inserção das mutações. Não foi possível recuperar partículas virais infecciosas carreando a mutação em E403. A modelagem molecular das proteínas virais mostrou diferenças discretas de carga, volume de superfície proteica e propriedade físico-química induzidas pelas mutações. Nenhuma das mutações influenciou nas interações intramoleculares. A imunogenicidade foi avaliada por imunização de camundongos das linhagens C57BL/6 e BALB/c com os vírus carreando mutações únicas e os soros foram analisados por PRNT e ELISA para obter os títulos de anticorpos neutralizantes para FA e anticorpos para GFP, respectivamente. Os soros dos camundongos imunizados com os vírus recombinantes apresentam menores títulos de anticorpos neutralizantes em comparação ao grupo imunizado com o vírus vacinal, porém não houveram alterações na indução de anticorpos para GFP. De maneira geral, as mutações em E400 e E403 produziram maiores efeitos sobre a proliferação viral e as mutações NS3439 e NS4B54, na imunogenicidade. (AU)


Subject(s)
Mice , Dengue Virus , Mutagenesis, Site-Directed , Yellow Fever , Yellow fever virus
9.
Article in English | WPRIM | ID: wpr-727934

ABSTRACT

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, ⁹²²FMDRLK⁹²⁷ , in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922–927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO₃⁻ transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO₃⁻ secretion in several types of epithelia.


Subject(s)
Amino Acids , Catalytic Domain , Consensus , Hydrogen-Ion Concentration , Immunoprecipitation , Mutagenesis, Site-Directed , Protein Phosphatase 1 , Signal Transduction
10.
Electron. j. biotechnol ; 26: 52-59, Mar. 2017. ilus, tab, graf
Article in English | LILACS | ID: biblio-1008980

ABSTRACT

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14­52 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59­149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.


Subject(s)
Trichoderma/enzymology , Xylosidases/metabolism , Disulfides/metabolism , Mass Spectrometry , Temperature , Trichoderma/genetics , Trichoderma/metabolism , Xylans/metabolism , Xylosidases/genetics , Enzyme Stability , Kinetics , Mutagenesis, Site-Directed , Hydrogen-Ion Concentration , Mutation
11.
Article in English | WPRIM | ID: wpr-158420

ABSTRACT

We evaluated the role of IL-10- in IL-33-mediated cholesterol reduction in macrophage-derived foam cells (MFCs) and the mechanism by which IL-33 upregulates IL-10. Serum IL-33 and IL-10 levels in coronary artery disease patients were measured. The effects of IL-33 on intra-MFC cholesterol level, IL-10, ABCA1 and CD36 expression, ERK 1/2, Sp1, STAT3 and STAT4 activation, and IL-10 promoter activity were determined. Core sequences were identified using bioinformatic analysis and site-specific mutagenesis. The serum IL-33 levels positively correlated with those of IL-10. IL-33 decreased cellular cholesterol level and upregulated IL-10 and ABCA1 but had no effect on CD36 expression. siRNA-IL-10 partially abolished cellular cholesterol reduction and ABCA1 elevation by IL-33 but did not reverse the decreased CD36 levels. IL-33 increased IL-10 mRNA production but had little effect on its stability. IL-33 induced ERK 1/2 phosphorylation and increased the luciferase expression driven by the IL-10 promoter, with the highest extent within the −2000 to −1752 bp segment of the 5′-flank of the transcription start site; these effects were counteracted by U0126. IL-33 activated Sp1, STAT3 and STAT4, but only the STAT3 binding site was predicted in the above segment. Site-directed mutagenesis of the predicted STAT3-binding sites (CTGCTTCCTGGCAGCAGAA→CTGCCTGGCAGCAGAA) reduced luciferase activity, and a STAT3 inhibitor blocked the regulatory effects of IL-33 on IL-10 expression. Chromatin immunoprecipitation (CHIP) confirmed the STAT3-binding sequences within the −1997 to −1700 and −1091 to −811 bp locus regions. IL-33 increased IL-10 expression in MFCs via activating ERK 1/2 and STAT3, which subsequently promoted IL-10 transcription and thus contributed to the beneficial effects of IL-33 on MFCs.


Subject(s)
Binding Sites , Cholesterol , Chromatin Immunoprecipitation , Computational Biology , Coronary Artery Disease , Foam Cells , Humans , Interleukin-10 , Interleukin-33 , Luciferases , Macrophages , Mutagenesis, Site-Directed , Phosphorylation , RNA, Messenger , Transcription Initiation Site
12.
Electron. j. biotechnol ; 19(6): 32-37, Nov. 2016. ilus
Article in English | LILACS | ID: biblio-840310

ABSTRACT

Background: To identify the critical amino acid residues that contribute to the high enzyme activity and good thermostability of Yersinia enterocolitica subsp. palearctica (Y. NSN), 15 mutants of Y. NSN were obtained by site-directed mutagenesis in this study. And their enzyme activity and thermostability were assayed. Effect of several factors on the enzyme activity and thermostability of Y. NSN, was also investigated. Results: The results showed that the I203F and D264E mutants retained approximately 75% and 70% enzyme activity, respectively, compared to the wild-type enzyme. In addition to the I203F and D264E mutants, the mutant E202A had an obvious influence on the thermostability of Y. NSN. According to the analysis of enzyme activity and thermostability of Y. NSN, we found that Glu202, Ile203 and Asp264 might be the key residues for its high enzyme activity and good thermostability. Conclusions: Among all factors affecting enzyme activity and thermostability of Y. NSN, they failed to explain the experimental results well. One reason might be that the enzyme activity and thermostability of Y. NSN were affected not only by a single factor but also by the entire environment.


Subject(s)
Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Yersinia enterocolitica/enzymology , Endonucleases/chemistry , Endonucleases/genetics , Enzyme Assays , Enzyme Stability , Hot Temperature , Mutagenesis, Site-Directed
13.
Electron. j. biotechnol ; 19(4): 26-30, July 2016. ilus
Article in English | LILACS | ID: lil-793949

ABSTRACT

Background: Glycine oxidase (GO), a type of D-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study, we have characterized a new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking. Results: The results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between -J-18 and a-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme. Conclusion: Asn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure-function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application.


Subject(s)
Bacillus cereus/metabolism , Amino Acid Oxidoreductases/metabolism , Glycine/analogs & derivatives , Substrate Specificity , Kinetics , Polymerase Chain Reaction/methods , Mutagenesis, Site-Directed , Amino Acid Oxidoreductases/genetics
14.
Protein & Cell ; (12): 417-433, 2016.
Article in English | WPRIM | ID: wpr-757122

ABSTRACT

Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.


Subject(s)
Alzheimer Disease , Metabolism , Pathology , Amyloid beta-Peptides , Metabolism , Amyloid beta-Protein Precursor , Genetics , Metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Chemistry , Genetics , Metabolism , Brain , Metabolism , Cells, Cultured , Chloride Channels , Genetics , Metabolism , Disease Models, Animal , HEK293 Cells , Humans , Lysosomes , Genetics , Metabolism , Mice , Mice, Transgenic , Microglia , Cell Biology , Metabolism , Mutagenesis, Site-Directed , Peptides , Chemistry , Protein Binding , RNA Interference , Sirtuin 1 , Genetics , Metabolism
15.
Article in English | WPRIM | ID: wpr-728688

ABSTRACT

Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.


Subject(s)
Animals , Carrier Proteins , Hydrogen-Ion Concentration , Mice , Mutagenesis, Site-Directed , Phosphorylation , S-Adenosylhomocysteine , Serine
16.
Chinese Journal of Biotechnology ; (12): 468-477, 2016.
Article in Chinese | WPRIM | ID: wpr-337450

ABSTRACT

3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.


Subject(s)
Electrophoresis, Polyacrylamide Gel , Escherichia coli , Escherichia coli Proteins , Genetics , Industrial Microbiology , Mutagenesis, Site-Directed , Phosphoglycerate Dehydrogenase , Genetics , Serine
17.
Chinese Journal of Biotechnology ; (12): 669-682, 2016.
Article in Chinese | WPRIM | ID: wpr-337432

ABSTRACT

Faldaprevir analogue molecule (FAM) has been reported to effectively inhibit the catalytic activity of HCV NS3/4A protease, making it a potential lead compound against HCV. A series of HCV NS3/4A protease crystal structures were analyzed by bioinformatics methods, and the FAM-HCV NS3/4A protease crystal structure was chosen for this study. A 20.4 ns molecular dynamics simulation of the complex consists of HCV NS3/4A protease and FAM was conducted. The key amino acid residues for interaction and the binding driving force for the molecular recognition between the protease and FAM were identified from the hydrogen bonds and binding free energy analyses. With the driving force of hydrogen bonds and van der Waals, FAM specifically bind to the active pocket of HCV NS3/4A protease, including V130-S137, F152-D166, D77-D79 and V55, which agreed with the experimental data. The effect of R155K, D168E/V and V170T site-directed mutagenesis on FAM molecular recognition was analyzed for their effect on drug resistance, which provided the possible molecular explanation of FAM resistance. Finally, the system conformational change was explored by using free energy landscape and conformational cluster. The result showed four kinds of dominant conformation, which provides theoretical basis for subsequent design of Faldaprevir analogue inhibitors based on the structure of HCV NS3/4A protease.


Subject(s)
Antiviral Agents , Chemistry , Carrier Proteins , Chemistry , Drug Resistance, Viral , Endopeptidases , Hepacivirus , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Oligopeptides , Chemistry , Protease Inhibitors , Chemistry , Serine Proteases , Thiazoles , Chemistry , Viral Nonstructural Proteins , Chemistry
18.
Chinese Journal of Biotechnology ; (12): 1233-1242, 2016.
Article in Chinese | WPRIM | ID: wpr-310544

ABSTRACT

Ethyl carbamate as a potential carcinogen commonly exists in traditional fermented foods and beverages. Enzymatic removal of ethyl carbamate from fermented foods and beverages is an efficient and safe method. In this study, we mutated urethanase from Lysinibacillus fusiformis SC02 on the Q328 site through computer aided design approaches. The half-life of resulting mutants Q328C and Q328V was detected to be 7.46 and 1.96 folds higher than that of the original enzyme, and Q328R presented better thermal-tolerance than the original urethanase when incubated at high temperature. The tolerance of Q328C to ethanol and acid also increased when compared with that of the original enzyme. The stability and tolerance to acid and ethanol of urethanase could be improved by modification on its Q328 site.


Subject(s)
Amidohydrolases , Genetics , Bacillaceae , Genetics , Bacterial Proteins , Genetics , Computer-Aided Design , Enzyme Stability , Ethanol , Mutagenesis, Site-Directed , Protein Engineering
19.
Chinese Journal of Biotechnology ; (12): 1243-1254, 2016.
Article in Chinese | WPRIM | ID: wpr-310543

ABSTRACT

In proteins of thermophilic bacteria, Gly is tend to be replaced by Ala and Lys is tend to be replaced by Arg to adapt the high temperature. In order to improve the thermal stability of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum, all the Gly on PAH were mutated to Ala and Lys to Arg. Positive mutant enzymes with improved thermal stability were selected, followed by combined mutation and characterization. The results revealed that half-lives of K94R and G221A mutants at 50 °C were 26.2 min and 16.8 min, which were increased by 1.9-times and 0.9-times than the parent enzyme (9.0 min). The residual activity of K94R/G221A mutant was improved to 65.6% after keeping at 50 °C for 1 h, which was 6.6 time higher than the parent enzyme (8.6%). Circular dichroism (CD) spectroscopy revealed that Tm values of the parent enzyme, K94R, G221A and K94R/G221A were 51.5 ℃, 53.8 ℃, 53.1 ℃ and 54.8 ℃, respectively. According to the protein structure simulation, the two mutations were located on flexible loop. In the K94R mutant, the mutated Arg94 on the surface of the enzyme formed an extra hydrogen bond with Ile95, which stabilized the located loop. In the G221A mutant, the mutated Ala221 formed hydrophobic interaction with Leu281, which could stabilize both the loop and flexible area of the C-terminus of G221A. The results not only provided a reference for protein modification on thermal stability, but also laid the foundation for application of phenylalanine hydroxylase in the field of functional foods.


Subject(s)
Bacterial Proteins , Genetics , Chromobacterium , Enzyme Stability , Hot Temperature , Kinetics , Mutagenesis, Site-Directed , Mutation , Phenylalanine Hydroxylase , Genetics , Protein Engineering
20.
Article in English | WPRIM | ID: wpr-176937

ABSTRACT

PURPOSE: The development of a genetically modified live rabies vaccine applicable to wild raccoon dogs is necessary for the eradication of rabies in Korea. Thus, we constructed a recombinant rabies virus (RABV) called the ERAGS strain, using a reverse genetic system and evaluated its safety and efficacy in mice and its safety and immunogenicity in raccoon dogs. MATERIALS AND METHODS: ERAGS, which has Asn194Ser and Arg333Glu substitutions in the glycoprotein, was constructed using site-directed mutagenesis. Mice were inoculated with the ERAGS strain (either 10(5.0) or 10(7.0) FAID(50)/mL) via intramuscular (IM) or intracranial injections and then challenged with a virulent RABV. Raccoon dogs were administered the ERAGS strain (10(8.0) FAID(50)/mL) either orally or via the IM route and the immunogenicity of the strain was evaluated using fluorescent antibody virus neutralization tests. RESULTS: The ERAGS strain inoculated into murine neuroblastoma cells reached 10(7.8) FAID(50)/mL at 96-hour post-inoculation. The virus was not pathogenic and induced complete protection from virulent RABV in immunized 4- and 6-week-old mice. Korean raccoon dogs immunized with the ERAGS strain via IM or oral route were also safe from the virus and developed high titer levels (26.4-32.8 IU/mL) of virus-neutralizing antibody (VNA) at 4 weeks post-inoculation. CONCLUSION: The ERAGS RABV strain was effectively protective against rabies in mice and produced a high VNA titer in raccoon dogs.


Subject(s)
Animals , Glycoproteins , Korea , Mice , Mouth , Mutagenesis, Site-Directed , Neuroblastoma , Neutralization Tests , Rabies Vaccines , Rabies virus , Rabies , Raccoon Dogs , Raccoons , Vaccines
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