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1.
Methods Mol Biol ; 2554: 123-139, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36178624

RESUMO

Saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy is an established technique for detecting and characterizing the binding of small molecules, such as metabolites, to biological macromolecules like proteins and nucleic acids. STD NMR allows detection of binding in complex mixtures of potential ligands, which is often used for library screening in the pharmaceutical industry but may also be beneficial for binding studies with metabolite mixtures. The nature of the ligand is normally restricted to small molecules in terms of NMR spectroscopy, and the size of the macromolecule on the other side should be larger than 10-15 kDa. This technique is especially applicable to detecting binders of intermediate to low affinity with the dissociation constant (KD) above 1 µM. In this chapter, we focus on recent developments and the applications of STD NMR to studying interactions of natural products and metabolites, in particular. The reader is also referred to excellent reviews of the field and the literature cited therein. This chapter also provides a detailed experimental protocol for performing the STD NMR measurement based on the example of the subunit A of the Na+-transporting NADH/ubiquinone oxidoreductase (Na+-NQR) from V. cholerae interacting with its natural quinone substrate and inhibitors.


Assuntos
Produtos Biológicos , Ácidos Nucleicos , Vibrio cholerae , Misturas Complexas , Ligantes , Espectroscopia de Ressonância Magnética/métodos , NAD/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Proteínas/química , Ubiquinona/metabolismo , Vibrio cholerae/metabolismo
2.
Gene ; 850: 146926, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36191825

RESUMO

Arsenic transforming bacterial strains belong to genus Pseudomonas sp.AK9 (KY569424), were isolated from the middle Gangetic plains of Bihar, India. The Pseudomonas sp. AK9 strains were able to transform toxic arsenite to a less toxic arsenate. In the present work, the presence of different arsenic resistance genes (aoxB, arsB, acr3 and aoxAB) were observed in isolated strain. Furthermore, the aoxB gene was amplified from genomic DNA of AK9, cloned in E.coli/DH5αcells, and sequenced. The BLASTn results and phylogenetic study of the aoxB gene showed 95.32 % and 90.07 % identity with the large subunit of aoxB gene of previous reported Thiomonas arsenivorans strain DSM16361 and Thiomonas arsenivorans strain b6, respectively. Further overhang primers were designed for amplifications of full length aoxB gene (∼1200 bp), and cloned in to the expression vector and host E.coli/BL21 cells. The GST-aoxB gene was expressed in BL21 cells, and a profound expression product of âˆ¼ 72 kDa was observed in SDS PAGE. The detection of a large subunit (aoxB) of arsenate oxidase protein in western blotting assay affirmed the expression of aoxB gene in recombinant E.coli/BL21 clone. Further, the recombinant E.coli/BL21cells showed increased growth than the normal E.coli/BL21 cells against As (III). Thus, this study showed the presence of aoxB gene in Pseudomonas sp. AK9 genome which regulates the resistant ability to arsenic toxicity.


Assuntos
Arsênio , Arsenitos , Oxirredutases , Arseniatos/metabolismo , Arsênio/toxicidade , Arsenitos/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Filogenia , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
3.
Protein Expr Purif ; 202: 106187, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36216219

RESUMO

Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may help obtain biochemical and biophysical parameters of a known enzyme and even uncover unknown characteristics indicative of novel enzymatic functions. Such information is often necessary to prepare adequate screening assays and drug-discovery experiments in general. Toxoplasma gondii is an obligate protozoan parasite that is a member of the phylum Apicomplexa, can develop several neuro-degenerative symptoms and, in specific cases, certain death for human hosts. Its relict non-photosynthetic plastid, the apicoplast, harbours a unique de novo long-chain fatty acid synthesis pathway of a prokaryotic character, FASII. The FASII pathway shows plasticity and, is essential for many intracellular and membranal components, along with fatty acid uptake via salvaging from the host, therefore, its disruption causes parasite death. TgFabG, a FASII enzyme responsible for a single reduction step in the pathway, was recombinantly expressed, purified and biochemically and biophysically characterised in this study. The bioengineering hurdle of expressing the recombinant gene of a eukaryotic, signal peptide-containing protein in a prokaryotic system was overcome for the apicomplexan enzyme TgFabG, by truncating the N-terminal signal peptide. TgFabG was ultimately recombinantly produced in a plasmid expression vector from its 1131 base pair gene, purified as 260 and 272 amino acid proteins using a hexahistidine (6 × Histag) affinity chromatography and its biochemical (enzyme activity and kinetics) and biophysical characteristics were analysed in vitro.


Assuntos
Apicoplastos , Toxoplasma , Humanos , Apicoplastos/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Proteína de Transporte de Acila/metabolismo , Oxirredutases/metabolismo , Ácidos Graxos/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
4.
Biomolecules ; 12(11)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36358916

RESUMO

LHON is a common blinding inherited optic neuropathy caused by mutations in mitochondrial genes. In this study, by using skin fibroblasts derived from LHON patients with the most common m.G11778A mutation and healthy objects, we performed proteomic analysis to document changes in molecular proteins, signaling pathways and cellular activities. Furthermore, the results were confirmed by functional studies. A total of 860 differential expression proteins were identified, containing 624 upregulated and 236 downregulated proteins. Bioinformatics analysis revealed increased glycolysis in LHON fibroblasts. A glycolysis stress test showed that ECAR (extra-cellular acidification rate) values increased, indicating an enhanced level of glycolysis in LHON fibroblasts. Downregulated proteins were mainly enriched in oxidoreductase activity. Cellular experiments verified high levels of ROS in LHON fibroblasts, indicating the presence of oxidative damage. KEGG analysis also showed the metabolic disturbance of fatty acid in LHON cells. This study provided a proteomic profile of skin fibroblasts derived from LHON patients bearing m.G11778A. Increased levels of glycolysis, decreased oxidoreductase activity and fatty acid metabolism could represent the in-depth mechanisms of mitochondrial dysfunction mediated by the mutation. The results provided further evidence that LHON fibroblast could be an alternative model for investigating the devastating disease.


Assuntos
Atrofia Óptica Hereditária de Leber , Humanos , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/metabolismo , DNA Mitocondrial/genética , Proteômica , Oxirredutases/metabolismo , Mutação , Fibroblastos/metabolismo , Glicólise , Ácidos Graxos/metabolismo
5.
Int J Mol Sci ; 23(21)2022 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-36362382

RESUMO

Pyranose oxidase (POx, glucose 2-oxidase; EC 1.1.3.10, pyranose:oxygen 2-oxidoreductase) is an FAD-dependent oxidoreductase and a member of the auxiliary activity (AA) enzymes (subfamily AA3_4) in the CAZy database. Despite the general interest in fungal POxs, only a few bacterial POxs have been studied so far. Here, we report the biochemical characterization of a POx from Streptomyces canus (ScPOx), the sequence of which is positioned in a separate, hitherto unexplored clade of the POx phylogenetic tree. Kinetic analyses revealed that ScPOx uses monosaccharide sugars (such as d-glucose, d-xylose, d-galactose) as its electron-donor substrates, albeit with low catalytic efficiencies. Interestingly, various C- and O-glycosides (such as puerarin) were oxidized by ScPOx as well. Some of these glycosides are characteristic substrates for the recently described FAD-dependent C-glycoside 3-oxidase from Microbacterium trichothecenolyticum. Here, we show that FAD-dependent C-glycoside 3-oxidases and pyranose oxidases are enzymes belonging to the same sequence space.


Assuntos
Flavina-Adenina Dinucleotídeo , Oxirredutases , Filogenia , Oxirredutases/genética , Oxirredutases/metabolismo , Monossacarídeos , Cinética , Bactérias/metabolismo , Glicosídeos
6.
J Chromatogr A ; 1685: 463576, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36323109

RESUMO

The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-ß-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.


Assuntos
Detergentes , Próstata , Masculino , Humanos , Próstata/metabolismo , Próstata/patologia , Antígenos de Neoplasias/metabolismo , Níquel , Reatores Biológicos , Oxirredutases/metabolismo
7.
Metab Eng ; 74: 178-190, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36336174

RESUMO

3-Hydroxypropionate (3-HP) is a versatile compound for chemical synthesis and a potential building block for biodegradable polymers. Cupriavidus necator H16, a facultative chemolithoautotroph, is an attractive production chassis and has been extensively studied as a model organism for biopolymer production. Here, we engineered C. necator H16 for 3-HP biosynthesis from its central metabolism. Wild type C. necator H16 can use 3-HP as a carbon source, a highly undesirable trait for a 3-HP production chassis. However, deletion of its three (methyl-)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) resulted in a strain that cannot grow on 3-HP as the sole carbon source, and this strain was selected as our production host. A stepwise approach was used to construct pathways for 3-HP production via ß-alanine. Two additional gene deletion targets were identified during the pathway construction process. Deletion of the 3-hydroxypropionate dehydrogenase, encoded by hpdH, prevented the re-consumption of the 3-HP produced by our engineered strains, while deletion of gdhA1, annotated as a glutamate dehydrogenase, prevented the utilization of aspartate as a carbon source, one of the key pathway intermediates. The final strain carrying these deletions was able to produce up to 8 mM 3-HP heterotrophically. Furthermore, an engineered strain was able to produce 0.5 mM 3-HP under autotrophic conditions, using CO2 as sole carbon source. These results form the basis for establishing C. necator H16 as an efficient platform for the production of 3-HP and 3-HP-containing polymers.


Assuntos
Cupriavidus necator , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Engenharia Metabólica , Oxirredutases/metabolismo , Carbono/metabolismo , Polímeros/metabolismo
8.
Molecules ; 27(22)2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36431769

RESUMO

MDM2 and MDM4 are cancer drug targets validated in multiple models for p53-based cancer therapies. The RING domains of MDM2 and non-p53-binder MDM2 splice isoforms form RING domain heterodimer polyubiquitin E3 ligases with MDM4, which regulate p53 stability in vivo and promote tumorigenesis independent of p53. Despite the importance of the MDM2 RING domain in p53 regulation and cancer development, small molecule inhibitors targeting the E3 ligase activity of MDM2-MDM4 are poorly explored. Here, we describe the synthesis and characterization of quinolinol derivatives for the identification of analogs that are capable of targeting the MDM2-MDM4 heterodimer E3 ligase and inducing apoptosis in cells. The structure-activity-relationship (SAR) study identified structural moieties critical for the inhibitory effects toward MDM2-MDM4 E3 ligase, the targeted degradation of MDM4 and FTH1 in cells, and anti-proliferation activity. Lead optimization led to the development of compound MMRi71 with improved activity. In addition to accumulating p53 proteins in wt-p53 bearing cancer cells as expected of any MDM2 inhibitors, MMRi71 effectively kills p53-null leukemia cells, an activity that conventional MDM2-p53 disrupting inhibitors lack. This study provides a prototype structure for developing MDM4/FTH1 dual-targeting inhibitors as potential cancer therapeutics.


Assuntos
Leucemia , Neoplasias , Humanos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/química , Ubiquitina-Proteína Ligases/metabolismo , Apoptose , Leucemia/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Ferritinas , Oxirredutases/metabolismo
9.
Molecules ; 27(21)2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36364214

RESUMO

The tumor-suppressor gene, WW domain-containing oxidoreductase (WWOX), has been found to be lost in various types of cancers. ROS result as a tightly regulated signaling process for the induction of cell senescence. The aim of this study was to investigate the role of WWOX in the regulation of ROS and cell senescence, which is intriguing in terms of the possible mechanism of WWOX contributing to bladder cancer. In this study, we used the AY-27 rat bladder tumor cell line and F344 orthotopic bladder tumor models to reveal the pro-senescence effects of WWOX and the corresponding underlying mechanism in bladder cancer. WWOX-overexpressing lentivirus (LV-WWOX) remarkably stimulated cellular senescence, including increased senescence-associated secretory phenotype (SASP) formation, enlarged cellular morphology, and induced SA-ß-Gal-positive staining. A further mechanism study revealed that the pro-senescence effect of LV-WWOX was dependent on increased intercellular reactive oxygen species (ROS) generation, which subsequently triggered p21/p27. Moreover, LV-WWOX significantly inhibited the tumor size by 30.49% in the F344/AY-27 rat orthotopic model (p < 0.05) by activating cellular senescence. The expression of p21 was significantly enhanced in the orthotopic bladder tumors under WWOX treatment. The orthotopic bladder tumors in the groups of rats verified the effect in vivo. Our study suggests that WWOX, an ROS-dependent senescence-induced gene, could be further studied for its therapeutic implications in bladder cancer.


Assuntos
Neoplasias da Bexiga Urinária , Ratos , Animais , Neoplasias da Bexiga Urinária/genética , Espécies Reativas de Oxigênio/metabolismo , Oxirredutases/metabolismo , Ratos Endogâmicos F344 , Senescência Celular/genética , Linhagem Celular Tumoral , Oxidorredutase com Domínios WW/genética , Proteínas Supressoras de Tumor/genética
10.
Chem Commun (Camb) ; 58(95): 13222-13225, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36353944

RESUMO

An engineered (S)-selective imine reductase from Streptomyces sp. GF3546 (SIR46) showed twice the activity of the wild type and high thermostability at 50 °C. Using a biocatalyst based on SIR46, we investigated the activity toward high-concentration 2-methyl-1-pyrroline up to 500 mM, conversion rates of other imines, and reductive amination activity.


Assuntos
Iminas , Streptomyces , Oxirredutases/metabolismo , Aminação , Streptomyces/metabolismo , Aminas , Estereoisomerismo
11.
BMC Urol ; 22(1): 172, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36344974

RESUMO

BACKGROUND: 5-α reductase inhibitors (5-ARIs) are first-line drugs for managing benign prostatic hyperplasia (BPH). Unfortunately, some patients do not respond to 5-ARI therapy and may even show worsening symptoms. The decreased expression of steroid 5-α reductase type 2(SRD5A2) in BPH tissues may explain the failure of 5-ARI therapy, however, the mechanisms underlying SRD5A2 decreased remained unelucidated. OBJECTIVES: To investigate microRNA-mediated regulation of the expression of SRD5A2 resulting in 5-ARI therapy failure. MATERIALS AND METHODS: The expression of SRD5A2 and microRNAs in BPH tissues and prostate cells were detected by immunohistochemistry, western blotting, and quantitative real-time PCR. Dual-luciferase reporter assay was performed to confirm that microRNA directly combine to SRD5A2 mRNA. The apoptosis of prostatic cells was detected by flow cytometry. RESULTS: SRD5A2 expression was variable; it was negative, weak, and strong in 13.6%, 28.8%, and 57.6% of BPH tissues respectively. The normal human prostatic epithelial cell line RWPE-1 strongly expressed SRD5A2, whereas the immortalized human prostatic epithelial cell line BPH-1 weakly expressed SRD5A2. miR-1199-5p expression was remarkably higher in BPH-1 than in RWPE-1 cells(P<0.001), and miR-1199-5p expression was significantly upregulated in BPH tissues with negative SRD5A2 expression than those with positive SRD5A2 expression. Transfection of miR-1199-5p mimics in RWPE-1 cells led to a marked decrease in SRD5A2 expression, whereas miR-1199-5p inhibitor increased SRD5A2 expression in BPH-1 cells. Dual-luciferase reporter assay showed that miR-1199-5p could bind the 3'untranslated region of SRD5A2 mRNA. miR-1199-5p also decreased the RWPE-1 sensibility to finasteride, an inhibitor of SRD5A2. CONCLUSION: Our results show that SRD5A2 expression varies in BPH tissues and miR-1199-5p might be one of the several factors contributing to differential SRD5A2 expression in BPH patients.


Assuntos
MicroRNAs , Hiperplasia Prostática , Masculino , Humanos , Hiperplasia Prostática/tratamento farmacológico , Colestenona 5 alfa-Redutase/genética , Colestenona 5 alfa-Redutase/metabolismo , Regulação para Cima , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases/uso terapêutico , RNA Mensageiro , MicroRNAs/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética
12.
Nat Commun ; 13(1): 7282, 2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36435948

RESUMO

Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN+) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN+-based reducing power. This is based on an orthogonal, NMN+-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN+. With a throughput of >106 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme's global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme's access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.


Assuntos
Mononucleotídeo de Nicotinamida , Oxirredutases , Oxirredutases/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Escherichia coli/metabolismo , Modelos Moleculares , Conformação Molecular
13.
Dalton Trans ; 51(46): 17587-17601, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36345601

RESUMO

In this work, we have designed and generated a Fe(III)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a model peptide and on the surface of thioredoxin (TRX) from E. coli. We investigated metal interactions with a family of peptides containing the motif EExxED or altered versions obtained by removing negatively charged residues: EExxEx, xExxED, and xExxEx. The interaction of the metal ion with the peptides was studied by circular dichroism, and our results indicated that the motif EExxED retained its functional properties and also that this motif is able to bind Ga(III) and Al(III). The interaction of the grafted TRX with iron(III) was investigated by NMR, showing that the motif was functional in the context of the protein structure, and also the binding of two equivalents of Fe(III) per TRX molecule was stable in a non-chelating neutral buffer. Protein conformation, stability, and enzymatic activity were studied by applying experimental and computational approaches. Interestingly, the thiol oxidoreductase activity was modulated by interaction with Ga(III), a Fe(III) mimetic ion. Furthermore, the design of functional proteins with both functions, oxidoreductase activity and metal-ion binding ability, should consider the reorganisation of the electrostatic network. Similarly, studying the crosstalk and electrostatic balance among different metal-binding sites may be critical.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/química , Ferro/química , Proteínas de Escherichia coli/química , Sítios de Ligação , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Compostos de Sulfidrila/química , Oxirredutases/metabolismo
14.
Cancer Biol Ther ; 23(1): 1-16, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36316642

RESUMO

This study was designed to explore the prognostic significance and functionality of STEAP2 (six-transmembrane epithelial antigen of prostate 2) in osteosarcomas and determine whether EFEMP2 (Epidermal growth factor-containing fibulin-like extracellular matrix protein 2) targets STEAP2 to facilitate osteosarcoma cell infiltration and migration. STEAP2 expression in peritumoral tissues, osteosarcoma, benign fibrous dysplasia, osteosarcoma cells, normal osteoblastic hFOB cells, and various invasive subclones was evaluated using IHC, ICC, and qRT-PCR. We also evaluated the association between STEAP2 expression and disease outcome using Kaplan-Meier analyses and then investigated STEAP2 regulation and its functional effects using both in vitro and in vivo assays. The results revealed that the upregulation of STEAP2 in osteosarcoma tissues positively correlated with both the malignant osteosarcoma phenotype and poor patient outcomes. In addition, STEAP2 expression induced epithelial-mesenchymal transition (EMT) via the PI3K/AKT/mTOR axis and facilitated osteosarcoma cell infiltration and migration. Changes in EFEMP2 expression resulted in correlating changes in STEAP2 expression, with EFEMP2-overexpressing osteosarcoma cells exhibiting a less invasive phenotype and reduced EMT following STEAP2 inhibition. It is also worth noting that although EFEMP2 overexpression activated the PI3K/AKT/mTOR pathway promoting EMT, it did not affect osteosarcoma cells in which STEAP2 or Akt was knocked down. Thus, we can conclude that STEAP2 acts as an oncogene in osteosarcoma progression, while EFEMP2 enables PI3K/AKT/mTOR axis initiation and EMT by partly targeting STEAP2, thereby facilitating osteosarcoma cell infiltration and migration.


Assuntos
Neoplasias Ósseas , Transição Epitelial-Mesenquimal , Proteínas da Matriz Extracelular , Osteossarcoma , Oxirredutases , Humanos , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Osteossarcoma/patologia , Oxirredutases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas da Matriz Extracelular/metabolismo
15.
PLoS One ; 17(10): e0275487, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36191023

RESUMO

Thermostable enzymes have the potential for use in a wide variety of biotechnological applications. Cryo-electron microscopy (cryo-EM) enables the imaging of biomolecules in their native aqueous environment. Here, we present high resolution cryo-EM structures of two thermostable enzymes that exhibit multimeric cage-like structures arranged into two different point-group symmetries. First, we determined the structure of the Sulfur Oxygenase Reductase (SOR) enzyme that catalyzes both the oxygenation and disproportionation of elemental sulfur in Archea and is composed of 24 homomeric units each of MW ≃ 35 kDa arranged in octahedral symmetry. The structure of SOR from Acidianus ambivalens (7X9W) was determined at 2.78 Å resolution. The active site of each subunit inside the central nanocompartment is composed of Fe3+ coordinated to two water molecules and the three amino acids (H86, H90 and E114). Second, we determined the structure of Lumazine Synthase (LS) from Aquifex aeolicus (7X7M) at 2.33 Å resolution. LS forms a cage-like structure consisting of 60 identical subunits each of MW ≃ 15 kDa arranged in a strict icosahedral symmetry. The LS subunits are interconnected by ion-pair network. Due to their thermostability and relatively easy purification scheme, both SOR and LS can serve as a model for the catalytic and structural characterization of biocatalysts as well as a benchmark for cryo-EM sample preparation, optimization of the acquisition parameters and 3D reconstruction.


Assuntos
Elétrons , Oxigenases , Aminoácidos , Microscopia Crioeletrônica , Complexos Multienzimáticos , Oxirredutases/metabolismo , Enxofre/metabolismo , Água
16.
Sci Rep ; 12(1): 17081, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224252

RESUMO

In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.


Assuntos
Oxirredutases , Porfiria Variegada , Ácido Aminolevulínico/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Heme , Humanos , Oxirredutases/genética , Oxirredutases/metabolismo , Porfiria Variegada/genética , Protoporfirinogênio Oxidase/genética , Protoporfirinogênio Oxidase/metabolismo , Protoporfirinas
17.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 53(5): 805-814, 2022 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-36224682

RESUMO

Objective: To explore the effects of hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) on the migration and invasion of HTR-8/SVneo cells, a human trophoblast cell line, and its potential mechanism of action. Methods: Immunofluorescence staining was done to evaluate the expression levels of HADHA in samples of normal villi and recurrent spontaneous abortion (RSA) villi at 6-8 weeks. Lentiviral infection system was used to construct stable HTR-8/SVneo cell lines with HADHA overexpression and knockdown. Western blot, qRT-PCR, Wound-healing assay, and Transwell assay were used to determine the effect of HADHA on the migration and invasion of HTR-8/SVneo cells and the expression of relevant genes. Transcriptome sequencing and bioinformatics analysis were done to screen for the potential target genes and signaling pathways regulated by HADHA. The specific molecular mechanism of how HADHA regulates the migration and invasion of HTR-8/SVneo cells was examined by adding the inhibitor of protein kinase B (PKB/AKT). Results: HADHA was highly expressed in extravillous trophoblasts (EVT) of RSA villus samples as compared with samples from the normal control group. In HTR-8/SVneo cells overexpressing HADHA, the expression levels of migration and invasion-related genes, including HLA-G, MMP2, MMP9, and NCAD, were decreased (P<0.01,P<0.05), and the migration and invasion abilities of HTR-8/SVneo cells were weakened (P<0.05). HADHA knockdown increased the expression levels of HLA-G, MMP2, MMP9, and NCAD (P<0.01, P<0.05), and promoted the migration and invasion of HTR-8/SVneo cells (P<0.05). In addition, HADHA overexpression decreased the phosphorylation levels of PI3K and AKT (P<0.05) and inhibited the PI3K/AKT signaling pathway. HADHA knockdown activated the PI3K/AKT signaling pathway. When MK-2206, an AKT inhibitor, was added to stable HTR-8/SVneo cell lines with HADHA knockdown, the migration and invasion of the cells were significantly reduced. Conclusion: HADHA inhibits the migration and invasion of HTR-8/SVneo cells by inhibiting the PI3K/AKT signaling pathway.


Assuntos
Pré-Eclâmpsia , Proteínas Proto-Oncogênicas c-akt , Movimento Celular/fisiologia , Coenzima A/metabolismo , Coenzima A/farmacologia , Feminino , Antígenos HLA-G/metabolismo , Antígenos HLA-G/farmacologia , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Oxirredutases/metabolismo , Oxirredutases/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Trofoblastos/metabolismo
18.
Plant Signal Behav ; 17(1): 2129290, 2022 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-36196516

RESUMO

Extracellular vesicles (EVs) are nano-sized membrane vesicles released by various cell types. Mammalian EVs have been studied in-depth, but the role of plant EVs has rarely been explored. For the first time, EVs from Drynariae Rhizoma roots were isolated and identified using transmission electron microscopy and a flow nano analyzer. Proteomics and bioinformatics were applied to determine the protein composition and complete the functional analysis of the EVs. Seventy-seven proteins were identified from Drynariae Rhizoma root-derived EVs, with enzymes accounting for 47% of the proteins. All of the enzymes were involved in important biological processes in plants. Most of them, including NAD(P)H-quinone oxidoreductase, were enriched in the oxidative phosphorylation pathway in plants and humans, and Alzheimer's disease, Huntington's disease, and Parkinson's disease, which are associated with oxidative stress in humans. These findings suggested that EVs from Drynariae Rhizoma roots could alleviate such neurological diseases and that enzymes, especially NAD(P)H-quinone oxidoreductase, might play an important role in the process.


Assuntos
Vesículas Extracelulares , Doenças Neurodegenerativas , Polypodiaceae , Biologia Computacional , Vesículas Extracelulares/metabolismo , Humanos , NAD/metabolismo , Doenças Neurodegenerativas/metabolismo , Oxirredutases/metabolismo , Raízes de Plantas/química , Polypodiaceae/química , Proteômica , Quinonas/metabolismo
19.
Int J Mol Sci ; 23(19)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36232289

RESUMO

A central feature of the skeletal muscle is its ability to regenerate through the activation, by environmental signals, of satellite cells. Once activated, these cells proliferate as myoblasts, and defects in this process profoundly affect the subsequent process of regeneration. High levels of reactive oxygen species such as hydrogen peroxide (H2O2) with the consequent formation of oxidized macromolecules increase myoblasts' cell death and strongly contribute to the loss of myoblast function. Recently, particular interest has turned towards the beneficial effects on muscle of the naturally occurring polyamine spermidine (Spd). In this work, we tested the hypothesis that Spd, upon oxidative challenge, would restore the compromised myoblasts' viability and redox status. The effects of Spd in combination with aminoguanidine (Spd-AG), an inhibitor of bovine serum amine oxidase, on murine C2C12 myoblasts treated with a mild dose of H2O2 were evaluated by analyzing: (i) myoblast viability and recovery from wound scratch; (ii) redox status and (iii) polyamine (PAs) metabolism. The treatment of C2C12 myoblasts with Spd-AG increased cell number and accelerated scratch wound closure, while H2O2 exposure caused redox status imbalance and cell death. The combined treatment with Spd-AG showed an antioxidant effect on C2C12 myoblasts, partially restoring cellular total antioxidant capacity, reducing the oxidized glutathione (GSH/GSSG) ratio and increasing cell viability through a reduction in cell death. Moreover, Spd-AG administration counteracted the induction of polyamine catabolic genes and PA content decreased due to H2O2 challenges. In conclusion, our data suggest that Spd treatment has a protective role in skeletal muscle cells by restoring redox balance and promoting recovery from wound scratches, thus making myoblasts able to better cope with an oxidative insult.


Assuntos
Peróxido de Hidrogênio , Espermidina , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Proliferação de Células , Dissulfeto de Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Camundongos , Mioblastos/metabolismo , Oxirredução , Oxirredutases/metabolismo , Poliaminas/metabolismo , Poliaminas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Espermidina/metabolismo , Espermidina/farmacologia
20.
Int J Mol Sci ; 23(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36232367

RESUMO

Angelica glauca Edgew, which is an endangered medicinal and aromatic herb, is a rich source of numerous industrially important bioactive metabolites, including terpenoids, phenolics, and phthalides. Nevertheless, genomic interventions for the sustainable utilization and restoration of its genetic resources are greatly offset due to the scarcity of the genomic resources and key regulators of the underlying specialized metabolism. To unravel the global atlas of the specialized metabolism, the first spatial transcriptome sequencing of the leaf, stem, and root generated 109 million high-quality paired-end reads, assembled de novo into 81,162 unigenes, which exhibit a 61.53% significant homology with the six public protein databases. The organ-specific clustering grouped 1136 differentially expressed unigenes into four subclusters differentially enriched in the leaf, stem, and root tissues. The prediction of the transcriptional-interactome network by integrating enriched gene ontology (GO) and the KEGG metabolic pathways identified the key regulatory unigenes that correspond to terpenoid, flavonoid, and carotenoid biosynthesis in the leaf tissue, followed by the stem and root tissues. Furthermore, the stem and root-specific significant enrichments of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), and caffeic acid 3-O-methyltransferase (COMT) indicate that phenylalanine mediated the ferulic acid biosynthesis in the stem and root. However, the root-specific expressions of NADPH-dependent alkenal/one oxidoreductase (NADPH-AOR), S-adenosyl-L-methionine-dependent methyltransferases (SDMs), polyketide cyclase (PKC), and CYP72A15 suggest the "root" as the primary site of phthalide biosynthesis. Additionally, the GC-MS and UPLC analyses corresponded to the organ-specific gene expressions, with higher contents of limonene and phthalide compounds in the roots, while there was a higher accumulation of ferulic acid in the stem, followed by in the root and leaf tissues. The first comprehensive genomic resource with an array of candidate genes of the key metabolic pathways can be potentially utilized for the targeted upscaling of aromatic and pharmaceutically important bioactive metabolites. This will also expedite genomic-assisted conservation and breeding strategies for the revival of the endangered A. glauca.


Assuntos
Angelica , Policetídeos , Angelica/genética , Carotenoides/metabolismo , Cinamatos/metabolismo , Ácidos Cumáricos , Flavonoides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genômica , Limoneno , Metiltransferases/metabolismo , Oxigenases de Função Mista/genética , Anotação de Sequência Molecular , NADP/metabolismo , Oxirredutases/metabolismo , Fenilalanina/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Melhoramento Vegetal , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Policetídeos/metabolismo , S-Adenosilmetionina/metabolismo , Transcriptoma
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