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1.
Biochem Biophys Res Commun ; 521(4): 997-1002, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31727364

RESUMO

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize various substrates, and those which are tolerant to and even activated by salts have attracted a lot of attention due to their application potential in certain industries. The mechanism of the salt activation of laccases is awaiting to be elucidated yet. Our previous study (Li, Xie et al. 2018) supposed that the salt activation of marine laccase Lac15 might be attributed to Cl- ion specifically binding to some local sites to interfere substrate binding and/or electron transfer. In this study, we found two sites whose mutations resulted in elimination of the salt activation of Lac15's activity towards catechol and dopamine respectively, and revealed that the mutations affected the activity by altering both Em and kcat, demonstrating the supposed mechanism. A model for the salt activation of laccases was accordingly proposed, albeit some details are to be elucidated.

2.
J Cell Mol Med ; 24(3): 2252-2259, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31880394

RESUMO

As promising biomarkers and therapy targets, microRNAs (miRNAs) are involved in various physiological and tumorigenic processes. Genetic variants in miRNA-binding sites can lead to dysfunction of miRNAs and contribute to disease. However, systematic investigation of the miRNA-related single nucleotide polymorphisms (SNPs) for pancreatic cancer (PC) risk remains elusive. We performed integrative bioinformatics analyses to select 31 SNPs located in miRNA-target binding sites using the miRNASNP v2.0, a solid database providing miRNA-related SNPs for genetic research, and investigated their associations with risk of PC in two large case-control studies totally including 1847 cases and 5713 controls. We observed that the SNP rs3802266 is significantly associated with increased risk of PC (odds ratio (OR) = 1.21, 95% confidence intervals (CI) = 1.11-1.31, P = 1.29E-05). Following luciferase reporter gene assays show that rs3802266-G creates a stronger binding site for miR-181a-2-3p in 3' untranslated region (3'UTR) of the gene ZHX2. Expression quantitative trait loci (eQTL) analysis suggests that ZHX2 expression is lower in individuals carrying rs3802266-G with increased PC risk. In conclusion, our findings highlight the involvement of miRNA-binding SNPs in PC susceptibility and provide new clues for PC carcinogenesis.

3.
Biochem Biophys Res Commun ; 519(4): 894-900, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31563321

RESUMO

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize wide range of compounds thus have great application potential in diverse industries. The catalytic mechanisms of laccases have been extensively studied, while the details of proton transfer remain to be fully elucidated. In this study, we tried to uncover the sites that are crucial for the proton transfer of microbial laccase Lac15. A residue near the trinuclear copper center, D396, was indicated by statistical coupling analysis (SCA) and structural alignment to be an important site like D93, which is conserved in laccases and believed crucial for the catalysis by facilitating proton transfer. A representative mutant at this site, D396A, similar to D93A, exhibited significantly impaired catalysis with the global structure and substrate binding slightly perturbed. The mutation resulted in stay of the intermediate I, which would accept a proton to proceed to next catalysis stage, suggesting D396 might play a critical role in the proton transfer. Our finding may help to completely elucidate the proton transfer mechanism in laccases.

4.
AMB Express ; 9(1): 151, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31535295

RESUMO

Engineering of fungal laccases with optimum catalytic activity at alkaline pH has been a long-lasting challenge. In this study, a mutant library containing 3000 clones was obtained by error-prone PCR to adapt the optimum pH of a fungal laccase Lcc9 from the basidiomycete Coprinopsis cinerea. After three rounds of functional screening, a mutant with three amino acid changes (E116K, N229D, I393T) named PIE5 was selected. PIE5 showed an optimum pH of 8.5 and 8.0 against guaiacol and 2,6-DMP when expressed in Pichia pastoris, representing the first fungal laccase that possesses an optimum pH at an alkaline condition. Site directed mutagenesis disclosed that N229D contributed the most to the optimum pH increment. A single N229D mutation caused an increase in optimum pH by 1.5 units. When used in indigo dye decolorization, PIE5 efficiently decolorized 87.1 ± 1.1% and 90.9 ± 0.3% indigo dye at the optimum conditions of pH 7.0-7.5 and 60 °C, and with either methyl 3,5-dimethoxy-4-hydroxybenzoate or 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate) as the mediator. In comparison, the commercially available fungal laccase TvLac from Trametes villosa decolorized 84.3 ± 1.8% of indigo dye under its optimum conditions (opt. pH 5.0 and 60 °C). The properties of an alkaline-dependent activity and the high indigo dye decolorization ability (1.3-fold better than the parental Lcc9) make the new fungal laccase PIE5 an alternative for specific industrial applications.

5.
Clin Epigenetics ; 11(1): 112, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370883

RESUMO

SET (Suppressor of variegation, Enhancer of Zeste, Trithorax) and MYND (Myeloid-Nervy-DEAF1) domain-containing protein 2 (SMYD2) is a protein methyltransferase that methylates histone H3 at lysine 4 (H3K4) or lysine 36 (H3K36) and diverse nonhistone proteins. SMYD2 activity is required for normal organismal development and the regulation of a series of pathophysiological processes. Since aberrant SMYD2 expression and its dysfunction are often closely related to multiple diseases, SMYD2 is a promising candidate for the treatment of these diseases, such as cardiovascular disease and cancer. Here, we present an overview of the complex biology of SMYD2 and its family members and their context-dependent nature. Then, we discuss the discovery, structure, inhibitors, roles, and molecular mechanisms of SMYD2 in distinct diseases, with a focus on cardiovascular disease and cancer.

6.
Int J Syst Evol Microbiol ; 69(11): 3414-3419, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31380736

RESUMO

A Gram-negative bacterium, namely strain ANRC-JHZ47T, was isolated from a seawater sample collected at Biological Bay, Fildes Peninsula, Antarctica. Cells of strain ANRC-JHZ47T were rod-shaped and motile by a single polar flagellum. Strain ANRC-JHZ47T was aerobic, oxidase-negative, and catalase-positive. The strain grew at 4-37 °C (optimum, 25 °C), pH at 3.5-10.0 (optimum, pH 5.5) and in NaCl at 1-7.0 % (w/v; optimum, 2-3 %). Strain ANRC-JHZ47T used Q-8 as the predominant respiratory quinone. Its predominant fatty acids were C16 : 0 (21.9 %), C12 : 0 (12.6 %), C19 : 0cyclo ω8c (12.4 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 13.1 %), C10 : 0 3-OH (11.3 %) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 6.0 %). Its major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified aminolipid and five unknown polar lipids. The DNA G+C content was 42.6 mol%. Strain ANRC-JHZ47T showed the highest 16S rRNA gene sequence similarity to Marinomonas arenicola KMM 3893T (97.9 %), followed by Marinomonas primoryensis KMM 3633T (97.6 %), Marinomonas profundimaris D104T (97.2 %) and Marinomonas pollencensis IVIA-Po-185T (97.0 %). Furthermore, the average nucleotide identity values between strain ANRC-JHZ47T and M. arenicola KMM 3893T, M. primoryensis KMM 3633T and M. profundimaris D104T were 79.8, 74.0, and 74.1 %, respectively. The in silico DNA-DNA hybridization values between them were 22.5±2.5, 20.4±2.3 and 19.9±2.3 %, respectively. Based on the results of phenotypic and phylogenetic analyses, strain ANRC-JHZ47T is considered to represent a novel species of the genus Marinomonas, for which the name Marinomonas flavescens sp. nov. is proposed. The type strain is ANRC-JHZ47T (=MCCC 1K03604T=KCTC 72113T).


Assuntos
Marinomonas/classificação , Filogenia , Água do Mar/microbiologia , Regiões Antárticas , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Marinomonas/isolamento & purificação , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química
7.
J Biosci Bioeng ; 128(5): 518-524, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31196789

RESUMO

Laccase Lcc9 from Coprinopsis cinerea heterologously expressed in Pichia pastoris (rLcc9) displayed different molecular weight and specific activity from the native laccase (nLcc9). Glycosylation may play a role in regulating the Lcc9 specific activity. To elucidate this hypothesis, in this study, firstly we demonstrated that rLcc9 and nLcc9 were glycoproteins, and then enzymatically deglycosylated them. The obtained drLcc9 and dnLcc9 showed an apparent decrease in their specific activities. Three putative N-glycosylation sites (N293, N313, and N454) were then predicted in Lcc9 and substituted to evaluate their roles in its specific activity. Molecular weight analysis on those mutants suggested that glycosylation should have occurred on N313 and N454 whereas not on N293 in rLcc9. Comparison of catalytic properties of those mutants revealed that glycosylation at N313 and N454 in rLcc9 could affect the binding affinity to substrates and the catalytic rate, respectively. In addition, the glycosylation could also affect the thermal stability of rLcc9 and nLcc9 since deglycosylation of those Lcc9s resulted in decreases in their thermal stability to some extent. These results will help us to understand the effect of glycosylation on biochemical characteristics of fungal laccases, and provide us support for the improvement of fungal laccase activity based on N-linked glycosylation modification.


Assuntos
Lacase/metabolismo , Pichia/metabolismo , Biocatálise , Glicosilação , Lacase/química , Lacase/genética , Pichia/genética , Proteínas Recombinantes/metabolismo
8.
Biotechnol Biofuels ; 12: 95, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31044008

RESUMO

Background: Starch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch. Results: A novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli, the recombinant AmyZ1 showed high activity at pH 6.0-7.5 and 25-50 °C. Its optimal pH and temperature were 7.0 and 35 °C, respectively. Similar to most α-amylases, AmyZ1 activity was enhanced (2.4-fold) by 1.0 mM Ca2+. Its half-life time at 35 °C was also extended from about 10 min to 100 min. In comparison, AmyZ1 showed a broad substrate specificity toward raw starches, including those derived from rice, corn, and wheat. The specific activity of AmyZ1 towards raw rice starch was 12,621 ± 196 U/mg, much higher than other reported raw starch hydrolases. When used in raw starch hydrolyzing process, AmyZ1 hydrolyzed 52%, 47% and 38% of 30% (w/v) rice, corn, and wheat starch after 4 h incubation. It can also hydrolyze marine raw starch derived from Chlorella pyrenoidosa, resulting in 50.9 mg/g DW (dry weight of the biomass) of reducing sugars after 4 h incubation at 35 °C. Furthermore, when hydrolyzing raw corn starch using the combination of AmyZ1 and commercial glucoamylase, the hydrolysis rate reached 75% after 4.5 h reaction, notably higher than that obtained in existing starch-processing industries. Conclusions: As a novel raw starch-digesting α-amylase with high specific activity, AmyZ1 efficiently hydrolyzed raw starches derived from both terrestrial and marine environments at near ambient temperature, suggesting its application potential in starch-based industrial processes.

9.
Metabolomics ; 15(4): 57, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30937548

RESUMO

INTRODUCTION: Mitral valve disease (MVD), including mitral valve regurgitation (MR) and mitral valve stenosis (MS), is a chronic and progressive cardiac malady. However, the metabolic alterations in MVD is not well-understood till now. The current gold standard diagnostic test, transthoracic echocardiography, has limitations on high-throughput measurement and lacks molecular information for early diagnosis of the disease. OBJECTIVE: The present study aimed to investigate the biochemical alterations and to explore their diagnostic potential for MVD. METHODS: Plasma metabolic profile derangements and their diagnostic potential were non-invasively explored in 34 MR and 20 MS patients against their corresponding controls, using high-throughput NMR-based untargeted metabolomics. RESULTS: Eighteen differential metabolites were identified for MR and MS patients respectively, on the basis of multivariate and univariate data analysis, which were mainly involved in energy metabolism, amino acid metabolism, calcium metabolism and inflammation. These differential metabolites, notably the significantly down-regulated formate and lactate, showed high diagnostic potential for MVD by using Spearman's rank-order correlation analysis and ROC analysis. CONCLUSIONS: To the best of our knowledge, the present study is the first one that explores the metabolic derangements and their diagnostic values in MVD patients using metabolomics. The findings indicated that metabolic disturbance occurred in MVD patients, with plasma formate and lactate emerged as important candidate biomarkers for MVD.

10.
Int J Syst Evol Microbiol ; 69(6): 1585-1590, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30912740

RESUMO

A taxonomic study was carried out on a Gram-stain-negative bacterium, namely strain ANRC-JH13T, isolated from a sediment sample collected at Jasper beach, adjacent to Fildes Peninsula, Antarctica. Cells of strain ANRC-JH13T were non-spore-forming rods and motile by the way of flagellum. Strain ANRC-JH13T was facultatively anaerobic, oxidase-positive, and catalase-positive. Growth of strain ANRC-JH13T occurred at 10-42 °C (optimum, 28 °C), pH 4.0-11.0 (pH 7.0) and 0-12.0 % (w/v) NaCl (1.0-2.0 %). Its predominant fatty acids were C16 : 0 (21.7 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 38.3 %), and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 20.1 %). Isoprenoid quinone Q-8 was the major respiratory quinone. Its major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, and four unknown polar lipids. The DNA G+C content was 48 mol%. Strain ANRC-JH13T showed the highest 16S rRNA gene sequence similarity to Amphritea balenae JAMM 1525T (97.9 %), followed by Amphritea atlantica M41T (97.8 %) and Amphritea japonica JAMM 1866T (97.3 %), and formed a lineage within the genus Amphritea on the phylogenetic trees. However, the in silico average nucleotide identity values between strain ANRC-JH13T and A. balenae JAMM 1525T, A. atlantica M41T, and A. japonica JAMM 1866T were 74.0, 76.7, and 74.9 %, respectively. The in silico DNA-DNA hybridization values between them were 19.8, 20.6, and 19.4 %, respectively. Based on the results from phenotypic, chemotaxonomic, and phylogenetic analyses, strain ANRC-JH13T is considered to represent a novel species of the genus Amphritea, for which the name Amphriteaopalescens sp. nov. is proposed. The type strain is ANRC-JH13T (=MCCC 1K03512T=KCTC 62532T).


Assuntos
Sedimentos Geológicos/microbiologia , Oceanospirillaceae/classificação , Filogenia , Regiões Antárticas , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Oceanospirillaceae/isolamento & purificação , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química
11.
Mol Med ; 25(1): 10, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30925865

RESUMO

BACKGROUND: The pathological features of aortic dissection (AD) include vascular smooth muscle cell (VSMC) loss, elastic fiber fraction, and inflammatory responses in the aorta. However, little is known about the post-translational modification mechanisms responsible for these biological processes. METHODS: A total of 72 aorta samples, used for protein detection, were collected from 36 coronary artery disease (CAD, served as the control) patients and 36 type A AD (TAAD) patients. Chromatin immunoprecipitation (ChIP)-PCR was used to identify the genes regulated by H3K23ac, and tubastatin A, an inhibitor of HDAC6, was utilized to clarify the downstream mechanisms regulated by HDAC6. RESULTS: We found that the protein level of histone deacetylase HDAC6 was reduced in the aortas of patients suffering from TAAD and that the protein levels of H4K12ac, and H3K23ac significantly increased, while H3K18ac, H4K8ac, and H4K5ac dramatically decreased when compared with CAD patients. Although H3K23ac, H3K18ac, and H4K8ac increased in the human VSMCs after treatment with the HDAC6 inhibitor tubastatin A, only H3K23ac showed the same results in human tissues. Notably, the results of ChIP-PCR demonstrated that H3K23ac was enriched in extracellular matrix (ECM)-related genes, including Col1A2, Col3A1, CTGF, POSTN, MMP2, TIMP2, and ACTA2, in the aortic samples of TAAD patients. In addition, our results showed that HDAC6 regulates H4K20me2 and p-MEK1/2 in the pathological process of TAAD. CONCLUSIONS: These results indicate that HDAC6 is involved in human TAAD formation by regulating H3K23ac, H4K20me2 and p-MEK1/2, thus, providing a strategy for the treatment of TAAD by targeting protein post-translational modifications (PTMs), chiefly histone PTMs.


Assuntos
Aneurisma Dissecante/metabolismo , Aorta/metabolismo , Aneurisma Aórtico/metabolismo , Desacetilase 6 de Histona/metabolismo , Idoso , Animais , Células Cultivadas , Doença da Artéria Coronariana/metabolismo , Feminino , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/metabolismo , Processamento de Proteína Pós-Traducional , Coelhos
12.
Appl Microbiol Biotechnol ; 103(1): 411-425, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30406450

RESUMO

When sucrose was used as the carbon source, the Basidiomycete Coprinopsis cinerea showed poor growth and low laccase activity in pure culture, but greatly enhanced the level of laccase activity (>1800 U/L) during coculture with the Mucoromycete Gongronella sp. w5. As a result, the mechanism of laccase overproduction in coculture was investigated by starting from clarifying the function of sucrose. Results demonstrated that Gongronella sp. w5 in the coculture system hydrolyzed sucrose to glucose and fructose by an intracellular invertase. Fructose rather than glucose was supplied by Gongronella sp. w5  as the readily available carbon source for C. cinerea, and contributed to an alteration of its growth behavior and a basal laccase secretion of 110.6 ± 3.3 U/L. On the other hand, separating Gongronella sp. w5 of C. cinerea by transfer into dialysis tubes yielded the same level of laccase activity as without separation, indicating that enhanced laccase production probably resulted from the metabolites in the fermentation broth. Further investigation showed that the ethyl acetate-extracted metabolites generated by Gongronella sp. w5 induced C. cinerea laccase production. One of the laccase-inducing compounds namely p-hydroxybenzoic acid (HBA) was purified and identified from the extract. When using HBA as the inducer and fructose as the carbon source in monoculture, C. cinerea observed similar high laccase activity to that in coculture, and zymograms revealed the same expression of laccase Lcc9 as the main and Lcc1 and Lcc5 as the minor enzymes. Overall, our experiments verified that Gongronella sp. w5 elevates Coprinopsis cinerea laccase production by carbon source syntrophism and secondary metabolite induction.


Assuntos
Agaricales/metabolismo , Carbono/metabolismo , Lacase/metabolismo , Mucorales/fisiologia , Agaricales/crescimento & desenvolvimento , Técnicas de Cocultura , Frutose/metabolismo , Glucose/metabolismo , Hidroxibenzoatos/isolamento & purificação , Hidroxibenzoatos/metabolismo , Hidroxibenzoatos/farmacologia , Mucorales/metabolismo , Sacarose/metabolismo , beta-Frutofuranosidase/metabolismo
13.
Protein Expr Purif ; 154: 16-24, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30248451

RESUMO

The fungal laccase Lcc9 from Coprinopsis cinerea is a promising candidate for biotechnological applications due to its distinct biochemical properties. In the present work, Lcc9 cDNA was cloned from C. cinerea using reverse transcription polymerase chain reaction and heterologously expressed in Pichia pastoris GS115. The recombinant laccase was found to be a heavily hyperglycoprotein, with the molecular weight of 60.2 kDa as determined by MALDI-TOF. Laccase activity in the culture supernatant was 1750 ±â€¯83 U/L and reached 3138 ±â€¯62 U/L after expression condition optimization using orthogonal experiment. The biochemical property of the purified recombinant Lcc9 (rLcc9) was compared to that of wild-type Lcc9. rLcc9 shows a higher specific activity (315.3 U/mg) than Lcc9 (92.9 U/mg) when using ABTS (2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate)) as the substrate. Although rLcc9 and Lcc9 showed comparable optimal pH (6.5) and temperature (70 °C) toward syringaldazine, rLcc9 displayed higher activity and stability in the pH range of 6.5-8.5. rLcc9 showed improved ability to oxidize indigo carmine and 5 azo dyes when methyl syringate was used as the mediator, with the decolorization rate range from 71.9 ±â€¯3.2% to 99.1 ±â€¯1.6% for different dyes in a wide pH (4.5-9.0) and temperature (4-70 °C) ranges. In comparison, Lcc9 decolorized 50.3 ±â€¯2.1% to 98.2 ±â€¯2.0% of the dyes used. The improved activity and stability in alkaline pH of rLcc9 relative to Lcc9, and improved dye decolorization ability towards 6 dyes suggested greater application potential of rLcc9 in biotechnologies such as wastewater treatment.


Assuntos
Agaricales , Proteínas Fúngicas , Expressão Gênica , Lacase , Agaricales/enzimologia , Agaricales/genética , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Lacase/biossíntese , Lacase/química , Lacase/genética , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
14.
J Biotechnol ; 286: 1-4, 2018 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-30194967

RESUMO

Gongronella sp. w5 (w5) is a soil fungus isolated from Anhui, China. Here we report the high-quality genome sequence of w5 and its phenotypic characteristics based on genomic information. The genome of w5 consists of 34,723,828 bp assembled into 149 scaffolds and 11,302 predicted protein-coding genes. Genome analysis suggested that w5 may possess host cell infection capacity and maybe a biotrophic fungus that relies on plant sucrose as carbon source. W5 shows the ability of rapid invasion into the plant root cells based on CAZymes analysis. Further results evidenced that w5 can use sucrose as the carbon source. Plant inoculation revealed that w5 penetrates the root cells of Actinidia chinensis with its hypha, and simultaneously promotes plant growth. It may promote plant growth by secreting organic acid and facilitating phosphate acquisition. The new genomic data and phenotype features will facilitate future applications of this strain in biotechnology.


Assuntos
Cunninghamella/fisiologia , Genoma Fúngico , Raízes de Plantas/crescimento & desenvolvimento , Análise de Sequência de DNA/métodos , China , Cunninghamella/genética , Tamanho do Genoma , Desenvolvimento Vegetal , Raízes de Plantas/microbiologia , Microbiologia do Solo , Sacarose/metabolismo
16.
Sheng Wu Gong Cheng Xue Bao ; 34(3): 379-388, 2018 Mar 25.
Artigo em Chinês | MEDLINE | ID: mdl-29577688

RESUMO

In producing recombinant ß-glucosidase in Escherichia coli by high-cell density cultivation (HCDC), insufficient soluble oxygen is always a problem. To address it, Vitreoscilla hemoglobin (VHb) was introduced into Escherichia coli by the bicistron and T7 promoter expression systems, to improve soluble oxygen by bacterial cells and thereby to enhance the biomass and recombinant ß-glucosidase production. In the case of bicistron expression system, cell density in shaking flask reached OD600=(4.24±0.29), 35.03% higher than that of the control without VHb. Correspondingly, the maximum activity of ß-glucosidase co-expressed with VHb was (9.78±0.55) U/mL, 25.38% higher than that of the control. In a 3-L fermentor, the maximum activity of ß-glucosidase was 141.23 U/mL, 35.57% higher than that of the control. In contrast, the activity of ß-glucosidase co-expressed with VHb under T7 promoter was lower than that of the control, either in flask or in fermentor. Co-expressing ß-glucosidase with VHb using the bicistron expression system may improve the tolerance of E. coli to insufficient soluble oxygen and thus promote the bacterial biomass and the enzyme yield.


Assuntos
Proteínas de Bactérias/biossíntese , Escherichia coli/metabolismo , Hemoglobinas Truncadas/biossíntese , beta-Glucosidase/biossíntese , Reatores Biológicos , Microbiologia Industrial , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese
17.
Cell Death Dis ; 9(2): 180, 2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29416002

RESUMO

Enhancer of zeste homolog 2 (EZH2), a methyltransferase that di- and tri-methylates lysine-27 of histone H3, largely functions as a transcriptional repressor, and plays a critical role in various kinds of cancers. Here we report a novel function of EZH2 in regulating autophagic cell death (ACD) of vascular smooth muscle cells (VSMCs) that affect aortic dissection (AD). Inhibition of EZH2 activity by UNC1999 or knockdown EZH2 resulted in VSMC loss, while overexpression of EZH2 facilitated VSMC growth, and these effects of EZH2 on VSMCs were independent of proliferation and apoptosis. Interestingly, more autophagic vacuoles and increased LC3II protein levels were identified in VSMCs with EZH2 inhibition or deficiency. Moreover, when compared with counterparts, chloroquine alone, or chloroquine with rapamycin treatment led to more LC3II accumulation in EZH2 inhibited or knockdown VSMCs, which indicated that EZH2 negatively regulated autophagosome formation. In conjunction to this, ATG5 and ATG7 protein levels were remarkably increased in EZH2 inhibited or deficient VSMCs, and ATG5 or ATG7 knockdown virtually rescued VSMC loss induced by EZH2 inhibition or knockdown. In addition, we found that the MEK-ERK1/2 signaling pathway, but not AMPKα, mTOR, or AKT pathway, is responsible for the impact of EZH2 on ACD of VSMCs. Additionally, the adverse effects of EZH2 inhibition or knockdown on VSMCs were largely reversed by PD98059, an inhibitor of MEK1. More importantly, decreased EZH2 expression levels in the aortic wall of patients with AD indicated its contribution to VSMC loss and AD occurrence. Overall, these findings revealed that EZH2 affects ACD of VSMCs and the pathologic process of AD via regulating ATG5 and ATG7 expression and MEK-ERK1/2 signaling. Our hitherto unrecognized findings indicate that EZH2 activation has therapeutic or preventive potential for AD.


Assuntos
Aneurisma Dissecante/enzimologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Músculo Liso Vascular/enzimologia , Aneurisma Dissecante/patologia , Animais , Autofagossomos/metabolismo , Autofagia/fisiologia , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Músculo Liso Vascular/patologia , Piridonas/farmacologia , Transdução de Sinais
18.
J Biosci Bioeng ; 125(2): 185-191, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29046264

RESUMO

Complicated purification steps, together with the fact that ß-glucosidase has to be tolerant to ethanol restricts the application of ß-glucosidase in isoflavone aglycone hydrolyzing process. ß-Glucosidase Bgl1A(A24S/F297Y) is a promising enzyme in hydrolyzing isoflavones. In this work, six different carbohydrate-binding modules (CBMs), which were from 3 families, were fused to the C-terminal of Bgl1A(A24S/F297Y), respectively, to simplify the enzyme preparation process. The fusion proteins were expressed in Escherichia coli and adsorbed onto cellulose. The Bgl-CBM24 was found to have the highest immobilization efficiency at room temperature within 1 h adsorption. Notably, 1-g cellulose absorbs up to 254.9±5.7 U of Bgl-CBM24. Interestingly, the immobilized Bgl-CBM24 showed improved ethanol tolerance ability, with the IC50 of 35% (v/v) ethanol. Bgl-CBM24 effectively hydrolyze soybean isoflavone glycosides. The hydrolysis rate of daidzin and gemistin was 85.22±3.24% and 82.14±3.82% within 10 min, with the concentrations of daidzein and genistein increased by 6.36±0.18 mM and 3.98±0.22 mM, respectively. In the repetitive hydrolytic cycles, the concentrations of daidzein and genistein still increased by 3.07±0.24 mM and 1.94±0.34 mM in the fourth cycle with 20% (v/v) ethanol. These results suggest that the immobilized Bgl-CBM24 has excellent potential in the preparation of isoflavone aglycones.


Assuntos
Metabolismo dos Carboidratos , Celulose/metabolismo , Glicosídeos/metabolismo , Isoflavonas/metabolismo , Soja/química , beta-Glucosidase/isolamento & purificação , beta-Glucosidase/metabolismo , Estabilidade Enzimática , Genisteína/metabolismo , Hidrólise , Cinética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , beta-Glucosidase/química
19.
Mol Neurobiol ; 55(7): 6007-6020, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29134514

RESUMO

Aggregation of amyloid-ß (Aß) peptides, which are the cleavage products of amyloid precursor protein (APP), is a major pathological hallmark in the brain of Alzheimer's disease (AD). Now, we know little about the roles of APP translation in the disease progression of AD. Here, we show that BC1, a long noncoding RNA (lncRNA), is expressed in the brain of AD mice. BC1 induces APP mRNA translation via association with a fragile X syndrome protein (FMRP). Inhibition of BC1 or BC1-FMRP association in AD mice blocks aggregation of Aß in the brain and protects against the spatial learning and memory deficits. Expression of exogenous BC1 in excitatory pyramidal neurons of mice induces Aß peptides accumulation and the spatial learning and memory impairments. This study provides a novel mechanism underlying aggregation of Aß peptides via BC1 induction of APP mRNA translation and hence warrants a promising target for AD therapy.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/metabolismo , Memória , Biossíntese de Proteínas , RNA Longo não Codificante/metabolismo , Aprendizagem Espacial , Precursor de Proteína beta-Amiloide/genética , Animais , Proteína do X Frágil de Retardo Mental/metabolismo , Transtornos da Memória/genética , Transtornos da Memória/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Eur Biophys J ; 47(3): 225-236, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28875401

RESUMO

Laccase (benzenediol: oxygen oxidoreductases, EC1.10.3.2) is a multi-copper oxidase capable of oxidizing a variety of phenolic and other aromatic organic compounds. The catalytic power of laccase makes it an attractive candidate for potential applications in many areas of industry including biodegradation of organic pollutants and synthesis of novel drugs. Most laccases are vulnerable to high salt and have limited applications. However, some laccases are not only tolerant to but also activated by certain concentrations of salt and thus have great application potential. The mechanisms of salt-induced activity enhancement of laccases are unclear as yet. In this study, we used dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, intrinsic fluorescence emission, circular dichroism, ultraviolet-visible light absorption, and an enzymatic assay to investigate the potential correlation between the structure and activity of the marine-derived laccase, Lac15, whose activity is promoted by low concentrations of NaCl. The results showed that low concentrations of NaCl exert little influence on the protein structure, which was partially folded in the absence of the salt; moreover, the partially folded rather than the fully folded state seemed to be favorable for enzyme activity, and this partially folded state was distinctive from the so-called 'molten globule' occasionally observed in active enzymes. More data indicated that salt might promote laccase activity through mechanisms involving perturbation of specific local sites rather than a change in global structure. Potential binding sites for chloride ions and their roles in enzyme activity promotion are proposed.


Assuntos
Cloretos/farmacologia , Haloferax volcanii/enzimologia , Lacase/metabolismo , Cobre/metabolismo , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Lacase/química , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Especificidade por Substrato
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