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It is desired to design and construct more efficient enzymes with better performance to catalyze carbene N-H insertions for the synthesis of bioactive molecules. To this end, we exploited and designed a series of human neuroglobin (Ngb) mutants. As shown in this study, a double mutant, A15C/H64G Ngb, with an additional disulfide bond and a modified heme active site, exhibited yields up to >99% and total turnover numbers up to 33000 in catalyzing the carbene N-H insertions for aromatic amine derivatives, including those with a large size such as 1-aminopyrene. Moreover, for o-phenylenediamine derivatives, they underwent two cycles of N-H insertions, followed by cyclization to form quinoxalinones, as confirmed by the X-ray crystal structures. This study suggests that Ngb can be designed into a functional carbene transferase for efficiently catalyzing carbene N-H insertion reactions with a range of substrates. It also represents the first example of the formation of quinoxalinones catalyzed by an engineered heme enzyme.
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The design of functional metalloenzymes is attractive for the biosynthesis of biologically important compounds, such as phenoxazinones and phenazines catalyzed by native phenoxazinone synthase (PHS). To design functional heme enzymes, we used myoglobin (Mb) as a model protein and introduced an artificial CXXC motif into the heme distal pocket by F46C and L49C mutations, which forms a de novo disulfide bond, as confirmed by the X-ray crystal structure. We further introduced a catalytic Tyr43 into the heme distal pocket and found that the F43Y/F46C/L49C Mb triple mutant and the previously designed F43Y/F46S Mb exhibit PHS-like activity (80-98% yields in 5-15 min), with the catalytic efficiency exceeding those of natural metalloenzymes, including o-aminophenol oxidase, laccase, and dye-decolorizing peroxidase. Moreover, we showed that the oxidative coupling product of 1,6-disulfonic-2,7-diaminophenazine is a potential pH indicator, with the orange-magenta color change at pH 4-5 (pKa = 4.40). Therefore, this study indicates that functional heme enzymes can be rationally designed by structural modifications of Mb, exhibiting the functionality of the native PHS for green biosynthesis.
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Metaloproteínas , Mioglobina , Mioglobina/química , Heme/química , Oxazinas , Óxido Nítrico SintaseRESUMO
Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy.
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Neoplasias da Mama , Mioglobina , Humanos , Feminino , Mioglobina/genética , Mioglobina/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Espécies Reativas de Oxigênio , Neoplasias da Mama/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Nitrito Redutases/genética , Nitrito Redutases/metabolismo , NitrogênioRESUMO
Backgrounds: Long noncoding RNAs (lncRNAs) are strongly associated with the development of breast cancer (BC). As yet, the function of LINC01234 in BC remains unknown. Methods: Using biological information, the potential lncRNA, miRNA, and target gene were predicted. LINC01234 and miR-525-5p expression in BC tissues was detected using quantitative real-time reverse transcription polymerase chain reaction. Fluorescence in situ hybridization was used to determine the distribution of LINC01234. Cell proliferation was analyzed using CCK-8 assay, colony formation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and apoptosis evaluated using flow cytometry. Western blotting was used to evaluate protein expression. Dual-luciferase® reporter, RNA pull-down, and RNA immunoprecipitation assays were performed to analyze the binding relationships among LINC01234, miR-525-5p, and cold shock domain-containing E1 (CSDE1). Results: We screened out LINC01234, found to be significantly increased in BC tissues, associated with a poor prognosis, and positively correlated with tumor size of BC. Knockdown of LINC01234 suppressed BC cell growth and facilitated apoptosis. Dual-luciferase reporter®, RNA pull-down, and RNA immunoprecipitation assays confirmed that LINC01234 and CSDE1 directly interacted with miR-525-5p. Upregulation of miR-525-5p and suppression of CSDE1 inhibited BC cell growth and induced cell apoptosis. Conclusion: Upregulation of LINC01234 contributes to the development of BC through the miR-525-5p/CSDE1 axis. LINC01234 may be one of the potential diagnostic and treatment targets for BC.
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Neoplasias da Mama , MicroRNAs , RNA Longo não Codificante , Apoptose/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Resposta ao Choque Frio , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Malachite green (MG)-contaminated wastewater resulting from industrialization causes a global problem because of its toxicity and widespread usage. Compared with traditional physical and chemical approaches, biodegradation provides a new route for the degradation of MG. As promising candidates for native enzymes, artificial enzymes have received tremendous attention for potential applications due to unlimited possibilities based on precise design. In this study, we rationally engineered artificial enzymes based on myoglobin (Mb) and neuroglobin (Ngb). We introduced an aspartic acid (H64D mutation) in the heme pocket of Mb. A distal histidine (F43H mutation) was further introduced into H64D Mb to obtain a double mutant of F43H/H64D Mb. Moreover, we used A15C/H64D Ngb as designed recently for comparison studies. The H64D Mb, F43H/H64D Mb, and A15C/H64D Ngb were found to catalyze MG degradation efficiently, with activities much higher than those of native enzymes, such as dye-decolorizing peroxidase and laccase (83-205-fold). The crystal structure of H64D Mb was solved and the interactions of MG and H64D Mb and A15C/H64D Ngb were investigated by using both experimental and molecular docking studies. The biodegradation products of MG were also revealed by ESI-MS analysis. Therefore, these artificial enzymes have potential applications in the biodegradation of MG in textile industries and fisheries.
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Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b5 (Cyt b5) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b5 content. Based on the low content of Cyt b5 in MDA-MB-231 cells, the overexpression of Cyt b5 was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b5 MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H2O2, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b5 most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b5 has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b5 and provides clues for clinical treatment.
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Neoplasias da Mama , Citocromos b5 , Neoplasias da Mama/genética , Citocromos b5/genética , Citocromos b5/metabolismo , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
An artificial disulfide bond (Cys46-Cys61) was designed in the heme distal site of myoglobin, which regulates the conformation of the heme distal His64 and the protein reactivity, as confirmed by X-ray crystallography, EPR, and kinetic UV-vis studies. This study shows the successful design of a disulfide bond with suitable positions in globins, conferring a structure and function like those of the native human neuroglobin.
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Dissulfetos , Mioglobina , Dissulfetos/química , Globinas/química , Heme/química , Humanos , Mioglobina/química , Neuroglobina , Conformação ProteicaRESUMO
Triple-negative breast cancers (TNBCs) are now acknowledged as a collection of diseases encompassing distinct histological plasticity, multi-tier molecular heterogeneity, as well as different outcomes. Despite decades of efforts, the molecular subtyping strategy has been theoretical, and target therapies based on molecular alternations barely improve survival rates of TNBC patients, and thus traditional chemotherapy remains the standard of care in clinic. The Wnt signaling is an evolutionarily conserved signaling pathway, playing critical roles in embryogenesis and neoplastic disease. The dysregulation of Wnt signaling pathway endows cancer cells with stem cell-like capacities of self-renewal, cell proliferation and differentiation, thus exerting crucial roles in tumorigenesis and therapy responses. Recently, the gene expression assays and genomic sequencing have demonstrated that the dysregulation of Wnt signaling is associated with progression of TNBCs, particularly with metastasis, relapse and therapy resistance. In this review, we highlight the dysregulation of Wnt signaling in TNBCs and its potential biological roles in molecular subtyping and stemness traits of specific subtypes, as well as its crosstalk with ncRNAs in regulation of the biological features of TNBCs, aiming to update this important oncogenic signaling pathway in TNBCs.
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Neoplasias de Mama Triplo Negativas , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Recidiva Local de Neoplasia/genética , RNA não Traduzido , Neoplasias de Mama Triplo Negativas/patologia , Via de Sinalização Wnt/genéticaRESUMO
Human cytochrome c (hCyt c) is a crucial heme protein and plays an indispensable role in energy conversion and intrinsic apoptosis pathways. The sequence and structure of Cyt c were evolutionarily conserved and only a few naturally occurring mutants were detected in humans. Among those variable sites, position 81 was proposed to act as a peroxidase switch in the initiation stages of apoptosis. In this study, we show that Ile81 not only suppresses the intrinsic peroxidase activity but also is essential for Cyt c to interact with neuroglobin (Ngb), a potential protein partner. The kinetic assays showed that the peroxidase activity of the naturally occurring variant I81N was enhanced up to threefold under pH 5. The local stability of the Ω-loop D (residues 70-85) in the I81N variant was decreased. Moreover, the Alphafold2 program predicted that Ile81 forms stable contact with human Ngb. Meanwhile, the Ile81 to Asn81 missense mutation abolishes the interaction interface, resulting in a â¼40-fold decrease in binding affinity. These observations provide an insight into the structure-function relationship of the conserved Ile81 in vertebrate Cyt c.
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Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications.
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Membrana Celular/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Cristalografia por Raios X , Fluoresceína-5-Isotiocianato/química , Humanos , Lisina/química , Células MCF-7 , Mutação , Mioglobina/genética , Mioglobina/farmacocinética , Espectrofotometria Ultravioleta , CachaloteRESUMO
Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.
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Globinas , Proteínas do Tecido Nervoso , Sítios de Ligação , Dissulfetos/química , Globinas/química , Globinas/genética , Globinas/metabolismo , Heme/química , Humanos , Ligantes , Proteínas do Tecido Nervoso/química , Neuroglobina , Raios XRESUMO
Exosomes are lipid bilayer extracellular vesicles with a size of 30-150 nm, which can be released by various types of cells including breast cancer cells. Exosomes are enriched with multiple nucleic acids, lipids, proteins and play critical biological roles by binding to recipient cells and transmitting various biological cargos. Studies have reported that tumor-derived exosomes are involved in cancer initiation and progression, such as promoting cancer invasion and metastasis, accelerating angiogenesis, contributing to epithelial-mesenchymal transition, and enhancing drug resistance in tumors. Recently the dysregulating of exosomes has been found in triple-negative breast cancer (TNBC), relating to the clinicopathological characteristics and prognosis of TNBC patients. Considering the poor prognosis and lack of adequate response to conventional therapy of TNBC, the discovery of certain exosomes as a new target for diagnosis and treatment of TNBC may be a good choice that provides new opportunities for the early diagnosis, clinical treatment of TNBC. Here, we first discuss the innovative prognostic and predictive effects of exosomes on TNBC, as well as the practical clinical problems. Secondly, we focus on the new therapeutic areas represented by exosomes, especially the impact of introducing exosomes in TNBC treatment in the future.
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Exossomos/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Feminino , HumanosRESUMO
Protein glycation is an important protein post-translational modification and is one of the main pathogenesis of diabetic angiopathy. Other than glycated hemoglobin, the protein glycation of other globins such as myoglobin (Mb) is less studied. The protein glycation of human Mb with ribose has not been reported, and the glycation sites in the Mb remain unknown. This article reports that d-ribose undergoes rapid protein glycation of human myoglobin (HMb) at lysine residues (K34, K87, K56, and K147) on the protein surface, as identified by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). Moreover, glycation by d-ribose at these sites slightly decreased the rate of the met heme (FeIII) in reaction with H2O2 to form a ferryl heme (FeIV=O). This study provides valuable insight into the protein glycation by d-ribose and provides a foundation for studying the structure and function of glycated heme proteins.
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Compostos Férricos/química , Heme/química , Peróxido de Hidrogênio/química , Mioglobina/química , Ribose/química , Cromatografia Líquida , Glicosilação , Humanos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Triple-negative breast cancer (TNBC) is the most aggressive subtype among breast cancers with high recurrence and this condition is partly due to chemoresistance. Therefore, fully understanding the mechanism of TNBC-resistance is the key to overcoming chemoresistance, which will be an effective strategy for TNBC therapy. Various potential mechanisms involved in the chemoresistance of TNBC have been investigated and indicated that noncoding RNAs (ncRNAs) especially microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) take part in most TNBC resistance. The ncRNA-induced chemoresistance process is involved in the alteration of many activities. here, we mainly summarize the mechanisms of ncRNAs in the chemoresistance of TNBC and discuss the potential clinical application of ncRNAs in the treatment of TNBC, indicating that targeting ncRNAs might be a promising strategy for resensitization to chemotherapies.
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RNA Longo não Codificante/fisiologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose , Autofagia/fisiologia , Reparo do DNA , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Humanos , MicroRNAs/fisiologia , RNA Longo não Codificante/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/genética , Microambiente TumoralRESUMO
The conversion of Kraft lignin in plant biomass into renewable chemicals, aiming at harvesting aromatic compounds, is a challenge process in biorefinery. Comparing to the traditional chemical methods, enzymatic catalysis provides a gentle way for the degradation of lignin. Alternative to natural enzymes, artificial enzymes have been received much attention for potential applications. We herein achieved the biodegradation of Kraft lignin using an artificial peroxidase rationally designed in myoglobin (Mb), F43Y/T67R Mb, with a covalently linked heme cofactor. The artificial enzyme of F43Y/T67R Mb has improved catalytic efficiencies at mild acidic pH for phenolic and aromatic amine substrates, including Kraft lignin and the model lignin dimer guaiacylglycerol-ß-guaiacyl ether (GGE). We proposed a possible catalytic mechanism for the biotransformation of lignin catalyzed by the enzyme, based on the results of kinetic UV-Vis studies and UPLC-ESI-MS analysis, as well as molecular modeling studies. With the advantages of F43Y/T67R Mb, such as the high-yield by overexpression in E. coli cells and the enhanced protein stability, this study suggests that the artificial enzyme has potential applications in the biodegradation of lignin to provide sustainable bioresource.
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Numerous human circular RNAs (circRNAs/circ) have been functionally characterized. However, the potential role of circ-protein kinase C iota (PRKCI) in hepatocellular carcinoma (HCC) remains unknown. The effects of each transfection and expression levels of circ-PRKCI, microRNA (miR)-1294, miR-186-5p and forkhead box K1 (FOXK1) in HCC cells were analyzed using reverse transcription-quantitative PCR analysis. The interactions between circ-PRKCI and miR-1294 or miR-186-5p, and miR-1294 or miR-186-5p and FOXK1 were validated using dual luciferase reporter assays. The viability, invasion and migration of HCC cells were determined using Cell Counting Kit-8, Transwell and wound healing assays, respectively. The expression levels of FOXK1, hexokinase-2 (HK2), glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA) in HCC cells were analyzed using western blotting. The levels of glucose and lactic acid in the cultured supernatant were detected using commercially available kits. The results of the present study revealed that miR-1294 and miR-186-5p expression levels were downregulated in the HCC cell line, HCCLM3, and were subsequently downregulated by circ-PRKCI overexpression and upregulated by the knockdown of circ-PRKCI. circ-PRKCI overexpression promoted the viability, invasion and migration of HCCLM3 cells, which was also reversed by the overexpression of miR-1294 and miR-186-5p. In addition, the overexpression of circ-PRKCI upregulated FOXK1 expression levels, while the overexpression of miR-1294 and miR-186-5p downregulated FOXK1 expression levels. Conversely, the knockdown of circ-PRKCI expression downregulated FOXK1 expression levels, while the knockdown of miR-1294 and miR-186-5p upregulated FOXK1 expression levels. Furthermore, circ-PRKCI was identified to target miR-1294 and miR-186-5p, and miR-1294 and miR-186-5p were subsequently found to target FOXK1. The overexpression of circ-PRKCI also increased glucose and lactic acid levels, while the knockdown of FOXK1 decreased glucose and lactic acid levels. The knockdown of circ-PRKCI decreased glucose and lactic acid levels, which were reversed by FOXK1 overexpression. In conclusion, the findings of the present study suggested that circ-PRKCI may promote the viability, invasion and migration of HCC cells by sponging miR-1294 and miR-186-5p to upregulate FOXK1 expression levels.
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Carcinoma Hepatocelular/tratamento farmacológico , Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Glicólise/efeitos dos fármacos , Isoenzimas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/farmacologia , Proteína Quinase C/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/metabolismo , Hexoquinase/metabolismo , Humanos , Isoenzimas/efeitos dos fármacos , Isoenzimas/genética , L-Lactato Desidrogenase , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/genética , RNA Circular , Regulação para CimaRESUMO
Protein design has received much attention in the last decades. With an additional disulfide bond to enhance the protein stability, human A15C neuroglobin (Ngb) is an ideal protein scaffold for heme enzyme design. In this study, we rationally converted A15C Ngb into a multifunctional peroxidase by replacing the heme axial His64 with an Asp residue, where Asp64 and the native Lys67 at the heme distal site were proposed to act as an acid-base catalytic couple for H2O2 activation. Kinetic studies showed that the catalytic efficiency of A15C/H64D Ngb was much higher (â¼50-80-fold) than that of native dehaloperoxidase, which even exceeds (â¼3-fold) that of the most efficient native horseradish peroxidase. Moreover, the dye-decolorizing peroxidase activity was also comparable to that of some native enzymes. Electron paramagnetic resonance, molecular docking, and isothermal titration calorimetry studies provided valuable information for the substrate-protein interactions. Therefore, this study presents the rational design of an efficient multifunctional peroxidase based on Ngb with potential applications such as in bioremediation for environmental sustainability.
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Neuroglobina/química , Peroxidase/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
Synthetic dyes such as malachite green (MG) have a wide range of applications. Meanwhile, they bring great challenges for environmental security and cause potential damages to human health. Compared with traditional approaches, enzymatic catalysis is an emerging technique for wastewater treatment. As alternatives to natural enzymes, artificial enzymes have received much attention for potential applications. In previous studies, we have rationally designed artificial enzymes based on myoglobin (Mb), such as by introducing a distal histidine (F43H mutation) and creating a channel to the heme pocket (H64A mutation). We herein show that the artificial enzyme of F43H/H64A Mb can be successfully applied for efficient biodegradation of MG under weak acid conditions. The degradation efficiency is much higher than those of natural enzymes, such as dye-decolorizing peroxidase and laccase (13-18-fold). The interaction of MG and F43H/H64A Mb was investigated by using both experimental and molecular docking studies, and the biodegradation products of MG were also revealed by UPLC-ESI-MS analysis. Based on these results, we proposed a plausible biodegradation mechanism of MG. With the high-yield of overexpression in E. coli cells, this study suggests that the artificial enzyme has potential applications in the biodegradation of MG in fisheries and textile industries.
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The treatment of environmental pollutants such as synthetic dyes and lignin has received much attention, especially for biotechnological treatments using both native and artificial metalloenzymes. In this study, we designed and engineered an efficient peroxidase using the O2 carrier myoglobin (Mb) as a protein scaffold by four mutations (F43Y/T67R/P88W/F138W), which combines the key structural features of natural peroxidases such as the presence of a conserved His-Arg pair and Tyr/Trp residues close to the heme active center. Kinetic studies revealed that the quadruple mutant exhibits considerably enhanced peroxidase activity, with the catalytic efficiency (kcat/Km) comparable to that of the most efficient natural enzyme, horseradish peroxidase (HRP). Moreover, the designed enzyme can effectively decolorize a variety of synthetic organic dyes and catalyze the bioconversion of lignin, such as Kraft lignin and a model compound, guaiacylglycerol-ß-guaiacyl ether (GGE). As analyzed by HPLC and ESI-MS, we identified several bioconversion products of GGE, as produced via bond cleavage followed by dimerization or trimerization, which illustrates the mechanism for lignin bioconversion. This study indicates that the designed enzyme could be exploited for the decolorization of textile wastewater contaminated with various dyes, as well as for the bioconversion of lignin to produce more value-added products.
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Corantes/química , Lignina/metabolismo , Mioglobina/química , Peroxidase/metabolismo , Engenharia de Proteínas , Animais , Cromatografia Líquida de Alta Pressão , Cor , Guaifenesina/análogos & derivados , Heme/química , Peróxido de Hidrogênio/metabolismo , Cinética , Oxirredução , Polimerização , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , CachaloteRESUMO
Excessive accumulation of cholesterol in ß cells initiates endoplasmic reticulum (ER) stress and associated apoptosis. We have reported that excessive uptake of cholesterol by MIN6 cells decreases the expression of secretagogin (SCGN) and then attenuates insulin secretion. Here, we aimed to determine whether cholesterol-induced SCGN decrease is involved in the modulation of ER stress and apoptosis in pancreatic ß cells. In this study, MIN6 cells were treated with oxidized low-density lipoprotein (ox-LDL) for 24 h, and then intracellular lipid droplets and cell apoptosis were quantified, and SCGN and ER stress markers were identified by western blot analysis. Furthermore, small interfer RNA (siRNA)-mediated SCGN knockdown and recombinant plasmid-mediated SCGN restoration experiments were performed to confirm the role of SCGN in ER stress and associated cell apoptosis. Finally, the interaction of SCGN with ATF4 was computationally predicted and then validated by a co-immunoprecipitation assay. We found that ox-LDL treatment increased the levels of ER stress markers, such as phosphorylated protein kinase-like endoplasmic reticulum kinase, phosphorylated eukaryotic initiation factor 2 alpha, activating transcription factor 4 (ATF4), and transcription factor CCAAT-enhancer-binding protein homologous protein, and promoted MIN6 cell apoptosis; in addition, the expression of SCGN was downregulated. siRNA-mediated SCGN knockdown exacerbated ß-cell ER stress by increasing ATF4 expression. Pretreatment of MIN6 cells with the recombinant SCGN partly antagonized ox-LDL-induced ER stress and apoptosis. Furthermore, a co-immunoprecipitation assay revealed an interaction between SCGN and ATF4 in MIN6 cells. Taken together, these results demonstrated that pancreatic ß-cell apoptosis induced by ox-LDL treatment can be attributed, in part, to an SCGN/ATF4-dependent ER stress response.