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1.
PLoS One ; 18(2): e0279431, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36827343

RESUMO

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that eventually develops into cirrhosis and even liver cancer. In recent years, the incidence rate has been increasing, and the early diagnosis and treatment of PBC are crucial. In the early diagnosis method of PBC, anti-mitochondrial antibodies (AMAs) are an important diagnostic basis, especially the M2 subtype (AMA-M2) with almost 100% specificity. We selected the BCOADC-E2 protein, a mitochondrial autoantigen that reacts specifically with AMA-M2 antibodies, and carried out DNA recombination and protein mutation experiments by cloning in vitro the homologous target gene sequence BCKD that expresses the antigenic epitope of BCOADC-E2 protein, to provide experience for later exploring the effect of mutations of amino acids around the lysine in the active center of BCOADC-E2 protein on its specific binding to AMA-M2, and to lay the foundation for determining the key amino acids of BCOADC-E2 for the diagnosis and treatment of PBC. In addition, we apply this scientific research content to graduate course teaching. Experimental technology of microbial molecular ecology is a course with the cross-integration of multidisciplinary knowledge and experimental skills offered at our college since 2018. This article derives from the part of this course on the construction of recombinant plasmids. The students first constructed the recombinant plasmid pGEX-BCKD using the vector plasmid pGEX-4T1 and the target gene fragment BCKD provided by the laboratory and used this as a template to construct the pGEX-BCKD-E4A point mutation plasmid by the overlap extension PCR (SOE PCR) technique to achieve the effect of mutating the fifth amino acid glutamate in front of lysine, the active centre of the BCOADC-E2 lipid acyl binding domain, to alanine for subsequent studies. Through the research experiment, combining theoretical knowledge and experimental operation, we aim to deepen the student's understanding of DNA recombination technology, let them feel the practical application prospect of experimental technology, stimulate students' interest in professional knowledge learning, and cultivate students' scientific thinking and innovation consciousness. We examined the quality of the teaching through the process and summative evaluation of the students. In this study, the students successfully completed the construction of pGEX-BCKD-E4A point mutant plasmid, and the average test score increased from 40.4% before teaching to 91.1%. The teaching effect was remarkable. This kind of research experimental teaching mode has good application prospects, and other education and teachers can refer to and reference it.


Assuntos
Cetona Oxirredutases , Cirrose Hepática Biliar , Humanos , Complexos Multienzimáticos/metabolismo , Complexo Piruvato Desidrogenase , Cetona Oxirredutases/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida) , Projetos de Pesquisa , Lisina , DNA , Autoanticorpos
2.
Folia Histochem Cytobiol ; 60(4): 335-343, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36583336

RESUMO

INTRODUCTION: Aberrant fucosylation is closely related to malignant transformation, cancer detection, and evaluation of treatment efficacy. The fucosylation process requires GDP-L-fucose, fucosyltransferases, and fucosidases. In gastric cancer (GC), fucosylation alterations were associated with tumor formation, metastasis inhibition, and multi-drug resistance. It is not clear whether tissue-specific transplantation antigen P35B (TSTA3) and alpha-L-fucosidase 2 (FUCA2) have any effect on the development of GC. MATERIALS AND METHODS: We used immunohistochemistry to assess the expression of TSTA3 and FUCA2 in 71 gastric adenocarcinoma samples and their relationship with clinicopathological parameters. RESULTS: TSTA3 expression was associated with lower histological grade I and II (P = 0.0120) and intestinal type Lauren classification (P = 0.0120). TSTA3 immunopositivity could predict Lauren's classification. Analysis of mRNA expression in GC validation cohorts corroborates the significant TSTA3 association with histological grade observed in our study. However, no associations were found between TSTA3 staining and overall survival. FUCA2 expression was markedly increased in GC tissues compared with non-tumoral tissues (P < 0.0001) and was associated with surgical staging III and IV (P = 0.0417) and advanced histological grade tumor states (P = 0.0125). CONCLUSIONS: Alterations of FUCA2 and TSAT3 immunoexpression could lay the basis for future studies using cell glycosylation as a biomarker for the planning of therapeutic strategy in primary gastric cancer.


Assuntos
Adenocarcinoma , Cetona Oxirredutases , Neoplasias Gástricas , Humanos , alfa-L-Fucosidase/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologia , Adenocarcinoma/patologia , Biomarcadores , Biomarcadores Tumorais , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismo
3.
Med Eng Phys ; 110: 103883, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36075788

RESUMO

Ovarian cancer (OC) is one of the most lethal malignancies in the female reproductive system. To find genes related to cancer progression targeting specific biological factors for targeted therapy, bioinformatics technology has been widely used. To screen the prognostic gene markers of OC by bioinformatics and explore their potential molecular biological mechanisms. Two data sets related to OC, GSE54388, and GSE119056, were rooted in the open comprehensive gene expression database (GEO). To correct the background of the data, standardize and screen differentially expressed genes (DEGs) using the R software limma package. The selected DEGs were enriched by Gene Ontology (GO) and through DAVID online database. Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway analysis and protein-protein interaction network (PPI-network) map were constructed by STRING online database and Cytoscape software. Combined with the TCGA database, univariate and multivariate COX regression were used to screen prognostic genes. QRT-PCR was used to verify DEGs in clinical tissue samples. Eventually, the function of RBMS3 on the viability, migration, invasion, and apoptosis of OC cells was tested through functional experiments in vitro. 352 common DEGs were screened from GSE54388 and GSE119056 data sets. Survival analysis showed that MEIS2, TSTA3, CNTN1, RBMS3, and TRA2A were considered to be connected with the prognosis of OC. We discover that the expression level of RBMS3 was positively connected with the overall survival (OS) rate of sufferers with OC. The level of RBMS3 in OC tissues was markedly lower than that in neighboring structures and the outcomes of the GEPIA database were consistent with those of the qRT-PCR experiment. Through gene transfection technology it was found that overexpression of RBMS3 in OC cells substantially suppressed the vitality, migration, and invasion of OC cells and raised the rates of apoptosis in the OC cells. In this experiment, we distinguish 5 genes that may participate in the prognosis of OC and showed the key genes and pathways related to OC. It is speculated that RBMS3, a tumor suppressor gene, can be applied as a potential biological marker for the treatment of OC, gene expression summary, and prognosis.


Assuntos
Cetona Oxirredutases , Neoplasias Ovarianas , Humanos , Feminino , Perfilação da Expressão Gênica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Biologia Computacional , Transdução de Sinais , Bases de Dados Factuais , Transativadores/genética , Transativadores/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Carboidratos Epimerases/metabolismo , Cetona Oxirredutases/metabolismo
4.
Bioengineered ; 13(3): 5551-5563, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35184647

RESUMO

Eriodictyol is a natural flavonoid with many pharmacological effects, such as anti-oxidation, anti-inflammation, anti-tumor, and neuroprotection. Besides, it has been reported that flavonoids play an important role in protein glycosylation. The fucosylation structure is closely associated with processes of various tumor metastases. TSTA3 is involved in the de novo synthesis and can convert cellular GDP-D-mannose into GDP-L-fucose. It was predicted on the STITCH database that eriodictyol interacted with TSTA3. In addition, literature has confirmed that TSTA3 is upregulated in CRC and can regulate the proliferation and migration of breast cancer cells. Herein, the precise effects of eriodictyol on the clone-forming, proliferative, migratory and invasive abilities of CRC cells as well as EMT process were assessed. Moreover, the correlation among eriodictyol, TSTA3, and fucosylation in these malignant behaviors of CRC cells was evaluated, in order to elucidate the underlying mechanism. The current work discovered that eriodictyol inhibited the viability, clone-formation, proliferation, migration, invasion, and EMT of CRC cells, and that these inhibitory effects of eriodictyol on the malignant behavior of CRC cells were reversed by TSTA3 overexpression. Additionally, eriodictyol suppresses fucosylation by downregulating the TSTA3 expression. Results confirmed that fucosylation inhibitor (2-F-Fuc) inhibited clone formation, proliferation, migration, invasion, as well as EMT of CRC cells and eriodictyol treatment further reinforced the suppressing effects of 2-F-Fuc on the malignant behavior of CRC cells. We conclude that eriodictyol suppresses the clone-forming, proliferative, migrative and invasive abilities of CRC cells as well as represses the EMT process by downregulating TSTA3 expression to restrain fucosylation.


Assuntos
Carboidratos Epimerases , Neoplasias Colorretais , Cetona Oxirredutases , Carboidratos Epimerases/antagonistas & inibidores , Carboidratos Epimerases/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Transição Epitelial-Mesenquimal , Flavanonas , Glicosilação , Guanosina Difosfato Fucose/metabolismo , Guanosina Difosfato Fucose/farmacologia , Humanos , Cetona Oxirredutases/antagonistas & inibidores , Cetona Oxirredutases/metabolismo
6.
Int J Mol Sci ; 23(2)2022 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-35055169

RESUMO

The aim of this study was to characterize the distribution of the thrombin receptor, protease activated receptor 1 (PAR1), in the neuroretina. Neuroretina samples of wild-type C57BL/6J and PAR1-/- mice were processed for indirect immunofluorescence and Western blot analysis. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to determine mRNA expression of coagulation Factor X (FX), prothrombin (PT), and PAR1 in the isolated neuroretina. Thrombin activity following KCl depolarization was assessed in mouse neuroretinas ex vivo. PAR1 staining was observed in the retinal ganglion cells, inner nuclear layer cells, and photoreceptors in mouse retinal cross sections by indirect immunofluorescence. PAR1 co-localized with rhodopsin in rod outer segments but was not expressed in cone outer segments. Western blot analysis confirmed PAR1 expression in the neuroretina. Factor X, prothrombin, and PAR1 mRNA expression was detected in isolated neuroretinas. Thrombin activity was elevated by nearly four-fold in mouse neuroretinas following KCl depolarization (0.012 vs. 0.044 mu/mL, p = 0.0497). The intrinsic expression of coagulation factors in the isolated neuroretina together with a functional increase in thrombin activity following KCl depolarization may suggest a role for the PAR1/thrombin pathway in retinal function.


Assuntos
Carboidratos Epimerases/metabolismo , Cetona Oxirredutases/metabolismo , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Neurônios Retinianos/metabolismo , Animais , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cloreto de Potássio/farmacologia , Protrombina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Ganglionares da Retina/metabolismo , Segmento Interno das Células Fotorreceptoras da Retina/metabolismo , Rodopsina/metabolismo
7.
Biochem Biophys Res Commun ; 587: 153-159, 2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-34875534

RESUMO

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 µM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Dissulfiram/análogos & derivados , Inibidores Enzimáticos/farmacologia , Glicólise/efeitos dos fármacos , Morfolinas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/genética , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Conjuntos de Dados como Assunto , Dissulfiram/farmacologia , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Humanos , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Fosforilação/efeitos dos fármacos , Plasmócitos/efeitos dos fármacos , Plasmócitos/enzimologia , Plasmócitos/patologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo
8.
Mol Oncol ; 16(9): 1816-1840, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34942055

RESUMO

Metabolic rewiring is one of the indispensable drivers of epithelial-mesenchymal transition (EMT) involved in breast cancer metastasis. In this study, we explored the metabolic changes during spontaneous EMT in three separately established breast EMT cell models using a proteomic approach supported by metabolomic analysis. We identified common proteomic changes, including the expression of CDH1, CDH2, VIM, LGALS1, SERPINE1, PKP3, ATP2A2, JUP, MTCH2, RPL26L1 and PLOD2. Consistently altered metabolic enzymes included the following: FDFT1, SORD, TSTA3 and UDP-glucose dehydrogenase (UGDH). Of these, UGDH was most prominently altered and has previously been associated with breast cancer patient survival. siRNA-mediated knock-down of UGDH resulted in delayed cell proliferation and dampened invasive potential of mesenchymal cells and downregulated expression of the EMT transcription factor SNAI1. Metabolomic analysis revealed that siRNA-mediated knock-down of UGDH decreased intracellular glycerophosphocholine (GPC), whereas levels of acetylaspartate (NAA) increased. Finally, our data suggested that platelet-derived growth factor receptor beta (PDGFRB) signalling was activated in mesenchymal cells. siRNA-mediated knock-down of PDGFRB downregulated UGDH expression, potentially via NFkB-p65. Our results support an unexplored relationship between UGDH and GPC, both of which have previously been independently associated with breast cancer progression.


Assuntos
Neoplasias da Mama , Cetona Oxirredutases , Neoplasias da Mama/patologia , Carboidratos Epimerases , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Glucose Desidrogenase , Humanos , Proteômica , RNA Interferente Pequeno , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Difosfato de Uridina , Uridina Difosfato Glucose Desidrogenase/metabolismo
9.
Sci Rep ; 11(1): 21852, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34750441

RESUMO

In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV-FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin-antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.


Assuntos
Fatores de Coagulação Sanguínea/biossíntese , Fatores de Coagulação Sanguínea/genética , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Trombina/biossíntese , Antitrombina III/genética , Antitrombina III/metabolismo , Carboidratos Epimerases/biossíntese , Carboxiliases/genética , Linhagem Celular , Fator V/genética , Fator V/metabolismo , Fator Xa/metabolismo , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Cetona Oxirredutases/biossíntese , Modelos Biológicos , Fragmentos de Peptídeos/metabolismo , Proteólise , Protrombina/biossíntese , Protrombina/genética , Protrombina/metabolismo , Trombomodulina/genética , Trombomodulina/metabolismo , Vitamina K Epóxido Redutases/genética
10.
Cell Mol Life Sci ; 78(23): 7451-7468, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34718827

RESUMO

In human metabolism, pyruvate dehydrogenase complex (PDC) is one of the most intricate and large multimeric protein systems representing a central hub for cellular homeostasis. The worldwide used antiepileptic drug valproic acid (VPA) may potentially induce teratogenicity or a mild to severe hepatic toxicity, where the underlying mechanisms are not completely understood. This work aims to clarify the mechanisms that intersect VPA-related iatrogenic effects to PDC-associated dihydrolipoamide dehydrogenase (DLD; E3) activity. DLD is also a key enzyme of α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, α-ketoadipate dehydrogenase, and the glycine decarboxylase complexes. The molecular effects of VPA will be reviewed underlining the data that sustain a potential interaction with DLD. The drug-associated effects on lipoic acid-related complexes activity may induce alterations on the flux of metabolites through tricarboxylic acid cycle, branched-chain amino acid oxidation, glycine metabolism and other cellular acetyl-CoA-connected reactions. The biotransformation of VPA involves its complete ß-oxidation in mitochondria causing an imbalance on energy homeostasis. The drug consequences as histone deacetylase inhibitor and thus gene expression modulator have also been recognized. The mitochondrial localization of PDC is unequivocal, but its presence and function in the nucleus were also demonstrated, generating acetyl-CoA, crucial for histone acetylation. Bridging metabolism and epigenetics, this review gathers the evidence of VPA-induced interference with DLD or PDC functions, mainly in animal and cellular models, and highlights the uncharted in human. The consequences of this interaction may have significant impact either in mitochondrial or in nuclear acetyl-CoA-dependent processes.


Assuntos
Di-Hidrolipoamida Desidrogenase/metabolismo , Inibidores de Histona Desacetilases/efeitos adversos , Doença Iatrogênica , Complexo Piruvato Desidrogenase/metabolismo , Ácido Valproico/efeitos adversos , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Acetilcoenzima A/biossíntese , Acetilação , Animais , Glicina Desidrogenase (Descarboxilante)/metabolismo , Humanos , Complexo Cetoglutarato Desidrogenase/metabolismo , Cetona Oxirredutases/metabolismo , Fígado/patologia , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Teratógenos/metabolismo
11.
Bol Med Hosp Infant Mex ; 78(5): 474-478, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34571524

RESUMO

Background: Charcot-Marie-Tooth disease type 2Q (CMT2Q) is a rare disorder (< 1/1,000,000 individuals worldwide) linked to chromosome 10p14 in the DHTKD1 gene. This phenotype is characterized by an adolescent or adulthood-onset, slowly progressive distal muscle weakness and symmetrical atrophy associated with reduced or absent deep tendon reflexes. Currently, only two familiar cases from China have been reported: one familiar case of eight individuals affected by isolated DHTKD1 gene mutation and one familiar case of two individuals affected by DHTKD1 gene mutation and GJB1 gene mutation. Case report: We present the case of a 10-year-old male patient with obesity, frequent falls, swollen legs and thighs, and pain in the lower and upper limbs. We performed the clinical evaluation and a clinical targeted exome test, which reported mutations on DHTKD1 y NTRK2 genes. Conclusions: Due to scientific and technological advances, genetic dysfunctions that can cause different diseases have been identified with greater sensitivity. Globally, this is the eleventh case reported of DHTKD1 gene mutation linked to CMT2Q. Moreover, this is the first case related to NTRK2 gene mutation (linked to obesity, hyperphagia, and delayed development). The patient showed an atypical CMT2Q phenotype additional to obesity. Therefore, we propose to study metabolic disorders linked to hereditary peripheral neuropathies.


Introducción: La enfermedad de Charcot-Marie-Tooth tipo 2Q (CMT2Q) es una alteración poco frecuente (< 1/1,000,000 habitantes en todo el mundo) condicionada por mutaciones en el gen DHTKD1, localizado en el cromosoma 10p14. El padecimiento inicia en la adolescencia o la edad adulta de manera lenta y progresiva, con debilidad muscular y atrofia distal simétrica, y afecta predominantemente las extremidades inferiores y los reflejos tendinosos profundos, que se encuentran reducidos o ausentes. Solo se ha reportado un caso familiar de ocho personas afectadas con la mutación aislada en el gen DHTKD1 y un caso familiar de dos personas afectadas con mutaciones en los genes DHTKD1 y GJB1, ambas familias de China. Caso clínico: Se presenta el caso de un paciente de sexo masculino de 10 años y 11 meses de edad con obesidad, caídas frecuentes, edema de miembros pélvicos y dolor en las extremidades inferiores y superiores. Se realizaron valoración clínica y estudio genético molecular de exoma dirigido, el cual reportó mutaciones en los genes DHTKD1 y NTRK2. Conclusiones: Gracias al avance científico y tecnológico se han podido identificar con mayor precisión las alteraciones genéticas causantes de diferentes enfermedades. Este es el undécimo caso reportado en el mundo de una mutación en el gen DHTKD1 asociada con la enfermedad de CMT2Q. También es el primer caso relacionado con una mutación del gen NTRK2 (asociada con obesidad, hiperfagia y retraso en el desarrollo). El paciente presentó un cuadro clínico atípico de enfermedad de CMT2Q agregado a obesidad. Por ello, se sugiere estudiar a fondo la conexión entre trastornos metabólicos y neuropatías periféricas hereditarias.


Assuntos
Doença de Charcot-Marie-Tooth , Cetona Oxirredutases , Adolescente , Adulto , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/genética , Criança , China , Humanos , Complexo Cetoglutarato Desidrogenase , Cetona Oxirredutases/genética , Masculino , Glicoproteínas de Membrana , Mutação , Fenótipo , Receptor trkB
12.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360775

RESUMO

Coupling glycolysis and mitochondrial tricarboxylic acid cycle, pyruvate dehydrogenase (PDH) complex (PDHC) is highly responsive to cellular demands through multiple mechanisms, including PDH phosphorylation. PDHC also produces acetyl-CoA for protein acetylation involved in circadian regulation of metabolism. Thiamine (vitamin B1) diphosphate (ThDP) is known to activate PDH as both coenzyme and inhibitor of the PDH inactivating kinases. Molecular mechanisms integrating the function of thiamine-dependent PDHC into general redox metabolism, underlie physiological fitness of a cell or an organism. Here, we characterize the daytime- and thiamine-dependent changes in the rat brain PDHC function, expression and phosphorylation, assessing their impact on protein acetylation and metabolic regulation. Morning administration of thiamine significantly downregulates both the PDH phosphorylation at Ser293 and SIRT3 protein level, the effects not observed upon the evening administration. This action of thiamine nullifies the daytime-dependent changes in the brain PDHC activity and mitochondrial acetylation, inducing diurnal difference in the cytosolic acetylation and acetylation of total brain proteins. Screening the daytime dependence of central metabolic enzymes and proteins of thiol/disulfide metabolism reveals that thiamine also cancels daily changes in the malate dehydrogenase activity, opposite to those of the PDHC activity. Correlation analysis indicates that thiamine abrogates the strong positive correlation between the total acetylation of the brain proteins and PDHC function. Simultaneously, thiamine heightens interplay between the expression of PDHC components and total acetylation or SIRT2 protein level. These thiamine effects on the brain acetylation system change metabolic impact of acetylation. The changes are exemplified by the thiamine enhancement of the SIRT2 correlations with metabolic enzymes and proteins of thiol-disulfide metabolism. Thus, we show the daytime- and thiamine-dependent changes in the function and phosphorylation of brain PDHC, contributing to regulation of the brain acetylation system and redox metabolism. The daytime-dependent action of thiamine on PDHC and SIRT3 may be of therapeutic significance in correcting perturbed diurnal regulation.


Assuntos
Encéfalo/metabolismo , Cetona Oxirredutases/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sirtuínas/metabolismo , Tiamina/farmacologia , Acetilação/efeitos dos fármacos , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de Tempo
13.
J Biol Chem ; 297(2): 100961, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34265301

RESUMO

The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S-S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His-Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe-His. Here, we propose that this difference is important for coupling carboxylation with C-S bond cleavage. We substituted the Phe-His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.


Assuntos
Dipeptídeos , Cetona Oxirredutases , Mesna , Carboxiliases/metabolismo , Domínio Catalítico , Oxirredutases/metabolismo , Especificidade por Substrato , Xanthobacter/metabolismo
14.
Hematology ; 26(1): 453-459, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34165034

RESUMO

PURPOSE: Multiple myeloma (MM) is a malignant disease with abnormal proliferation of clonal plasma cells. Hypoxia is an important factor in the pathogenesis and development of MM. However, the underlying mechanisms are not fully understood. MATERIAL & METHODS: To determine hub genes related to hypoxia in MM, this study took integrated bioinformatics analysis with two expression datasets (GSE80140 and GSE80545) downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were filtrated under the condition of both p-value < 0.05 and [log2FoldChange (log2FC)] > 1. Then, gene ontology (GO) and Kyoto encyclopedia of genes and genomes enrichment (KEGG) analysis, and protein-protein interaction (PPI) network construction were utilized to further explore these DEGs. PrognoScan evaluated all the candidate hub genes for survival analysis. RESULTS: In total, three hub genes, including FH, TSTA3, and POLR3G, were screened out to be related to hypoxia in MM. Patients with the lower expression level of FH, TSTA3, and POLR3G have statistically significantly longer disease- specific survival (Cox p < 0.05). CONCLUSION: We identified FH, TSTA3, and POLR3G as hub genes which can affect MM patients'outcome and new biomarkers for diagnosis and prognosis of MM. Further functional and mechanistic studies are need to develop in order to make them as potential target for clinical treatment.


Assuntos
Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/genética , Carboidratos Epimerases/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Genômica , Humanos , Cetona Oxirredutases/genética , Mieloma Múltiplo/diagnóstico , Prognóstico , RNA Polimerase III/genética , Transcriptoma
15.
Protein J ; 40(4): 489-503, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34100161

RESUMO

The accumulation of carbon dioxide in the atmosphere as a result of human activities has caused a number of adverse circumstances in the world. For this reason, the proposed solutions lie within the aim of reducing carbon dioxide emissions have been quite valuable. However, as the human activity continues to increase on this planet, the possibility of reducing carbon dioxide emissions decreases with the use of conventional methods. The emergence of compounds than can be used in different fields by converting the released carbon dioxide into different chemicals will construct a fundamental solution to the problem. Although electro-catalysis or photolithography methods have emerged for this purpose, they have not been able to achieve successful results. Alternatively, another proposed solution are enzyme based systems. Among the enzyme-based systems, pyruvate decarboxylase, carbonic anhydrase and dehydrogenases have been the most studied enzymes. Pyruvate dehydrogenase and carbonic anhydrase have either been an expensive method or were incapable of producing the desired result due to the reaction cascade they catalyze. However, the studies reporting the production of industrial chemicals from carbon dioxide using dehydrogenases and in particular, the formate dehydrogenase enzyme, have been remarkable. Moreover, reported studies have shown the existence of more active and stable enzymes, especially the dehydrogenase family that can be identified from the biome. In addition to this, their redesign through protein engineering can have an immense contribution to the increased use of enzyme-based methods in CO2 reduction, resulting in an enormous expansion of the industrial capacity.


Assuntos
Dióxido de Carbono/química , Formiato Desidrogenases/química , Cetona Oxirredutases/química , Catálise
16.
Biomed Pharmacother ; 140: 111771, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34058441

RESUMO

Danhong injection (DHI) is a compound Chinese medicine widely used in China for treatment of ischemic cardio-cerebrovascular diseases. However, limited data are available regarding the protective effect of DHI on the ischemic penumbra in ischemic stroke. This study aimed to investigate the effect of intravenous DHI on neuronal injure in the ischemic penumbra after cerebral ischemia/reperfusion (CI/R), focusing especially on the involvement of intracellular energy metabolism coupling. Male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion for 60 min followed by reperfusion with or without intravenous DHI (0.5, 1.0, or 2.0 mL/kg) once daily for 7 days. Post-treatment with DHI ameliorated neurological defects, diminished cerebral infarction, alleviated cerebral edema, improved microcirculatory perfusion after 7days of reperfusion, and inhibited apoptosis and enhanced neuronal survival in the ischemic penumbra. In addition, DHI significantly ameliorated oxidative stress, reduced DNA damage, and inhibited the activation of PARP1/AIF pathway, thereby restoring cytoplasmic glycolytic activity. Furthermore, this drug increased PDH activity by inhibiting the HIF1α/PDK1 signaling pathway, thus eliminating the inhibitory effect of CI/R on mitochondrial metabolism. The results of this study suggest that DHI can alleviate cerebral edema after CI/R and rescue the ischemic penumbra, and these protective effects are due to the regulation of intracellular energy metabolic coupling.


Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Dano ao DNA , Medicamentos de Ervas Chinesas/farmacologia , Edema/tratamento farmacológico , Edema/metabolismo , Edema/patologia , Metabolismo Energético/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Cetona Oxirredutases/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Ratos Sprague-Dawley
18.
Blood ; 137(25): 3518-3532, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33720355

RESUMO

Acute myeloid leukemia (AML) cells have an atypical metabolic phenotype characterized by increased mitochondrial mass, as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. To exploit this altered metabolism, we assessed publicly available databases to identify FAO enzyme overexpression. Very long chain acyl-CoA dehydrogenase (VLCAD; ACADVL) was found to be overexpressed and critical to leukemia cell mitochondrial metabolism. Genetic attenuation or pharmacological inhibition of VLCAD hindered mitochondrial respiration and FAO contribution to the tricarboxylic acid cycle, resulting in decreased viability, proliferation, clonogenic growth, and AML cell engraftment. Suppression of FAO at VLCAD triggered an increase in pyruvate dehydrogenase activity that was insufficient to increase glycolysis but resulted in adenosine triphosphate depletion and AML cell death, with no effect on normal hematopoietic cells. Together, these results demonstrate the importance of VLCAD in AML cell biology and highlight a novel metabolic vulnerability for this devastating disease.


Assuntos
Ácidos Graxos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Ácidos Graxos/genética , Glicólise , Humanos , Cetona Oxirredutases/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
19.
Oncol Rep ; 45(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760213

RESUMO

Tissue­specific transplantation antigen P35B (TSTA3) expression is upregulated in esophageal squamous cell carcinoma and breast cancer, and functions as an oncogene in breast cancer. However, the roles and underlying mechanisms of TSTA3 in lung cancer have not been fully elucidated. The current study aimed to reveal the role of TSTA3 in lung cancer and explore whether TSTA3 may be modulated by microRNA (miR)­125a­5p to activate ß­catenin signaling. Immunohistochemical staining and western blotting were used to analyze TSTA3 expression in lung cancer tissues and cells. Cell functions were assessed via Cell Counting Kit­8, flow cytometry, wound­healing, Transwell and in vivo tumor formation assays. The effect of TSTA3 on the activation of ß­catenin signaling was determined using western blot and immunofluorescence analyses. The association between miR­125a­5p and TSTA3 was determined by western blotting and luciferase gene reporter assay. The present study revealed that, compared with normal tissues and cells, TSTA3 expression was significantly increased in lung cancer tissues and cell lines, and high TSTA3 expression predicted a poor prognosis and more malignant clinical features in patients with lung cancer. TSTA3 upregulation significantly enhanced ß­catenin expression and promoted its nuclear accumulation. In addition, TSTA3 expression was negatively regulated by miR­125a­5p, which was downregulated in lung cancer. Furthermore, TSTA3 overexpression markedly promoted cell proliferation, migration, invasion and tumorigenesis, and suppressed cell apoptosis. TSTA3 downregulation abolished the effects of miR­125a­5p downregulation on promoting lung cancer cell malignant transformation. Overall, the current study demonstrates that TSTA3 is regulated by miR­125a­5p and functions as an oncogene in lung cancer via promoting the activation of ß­catenin signaling.


Assuntos
Carboidratos Epimerases/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Cetona Oxirredutases/genética , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Idoso , Animais , Apoptose/genética , Carboidratos Epimerases/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Cetona Oxirredutases/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Oncogênicas/genética , Pneumonectomia , Prognóstico , Carcinoma de Pequenas Células do Pulmão/mortalidade , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/cirurgia , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo
20.
Cancer Immunol Res ; 9(3): 348-361, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33441309

RESUMO

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.


Assuntos
Adenocarcinoma/imunologia , Neoplasias Associadas a Colite/imunologia , Células Dendríticas/imunologia , Receptor Notch2/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Animais , Transplante de Medula Óssea , Linfócitos T CD8-Positivos/imunologia , Carboidratos Epimerases/genética , Carcinogênese/genética , Carcinogênese/imunologia , Linhagem Celular Tumoral , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/mortalidade , Neoplasias Associadas a Colite/patologia , Apresentação Cruzada , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Hidroliases/genética , Cetona Oxirredutases/genética , Camundongos , Camundongos Knockout , Mutação , Prognóstico , Receptor Notch2/genética , Receptores CCR7/genética , Análise de Sobrevida , Quimeras de Transplante
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