RESUMEN
Lung cancer is a malignant tumor with high mortality and drug resistance. Therefore, it is urgent to explore natural and nontoxic drugs to treat lung cancer. In this study, the natural active ingredient AANL extracted from Agrocybe aegirita was used to modify nanoselenium by an oxidation-reduction method. Transmission electron microscope detection and infrared spectroscopy showed that a novel selenium nanocomposite named AANL-SeNPs was successfully prepared. The results of nanoscale characterization showed that AANL-SeNPs had good stability and uniform dispersion in aqueous solution by zeta potential and spectrum analysis. At the cellular level, we found that AANL-SeNPs significantly inhibited the cell viability of lung cancer cells, and the cell inhibition rate of 60 nM AANL-SeNPs was 39 % in H157 cells, 67 % in H147 cells, and 62 % in A549 cells. The IC50 value of AANL-SeNPs was 51.85 nM in A549 cells and 81.57 nM in H157 cells. Moreover, AANL-SeNPs could inhibit the cell proliferation and migration, and enhance the sensitivity of lung cancer cells to osimertinib and has no toxic to normal cells. In vivo, AANL-SeNPs significantly slowed tumor growth in tumor-bearing mice by establishing a subcutaneous transplantation tumor model for lung cancer, and the tumor size was smaller and was reduced about 79 % in 2 mg/kg AANL-SeNPs group compared with PBS group. Mechanistically, a total of 38 differentially expressed proteins were identified by data-independent acquisition mass spectrometry. A significantly upregulated protein, CDC-like kinase 2 (CLK2), was screened and validated for further analysis, which showed that the expression levels of CLK2 were increased in H157 and H1437 cells after AANL-SeNPs treatment. The results obtained in this study suggest that a novel selenium nanocomposite AANL-SeNPs, which inhibits lung cancer by upregulating the expression of CLK2.
Asunto(s)
Antineoplásicos , Proliferación Celular , Neoplasias Pulmonares , Nanocompuestos , Proteínas Tirosina Quinasas , Selenio , Regulación hacia Arriba , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Nanocompuestos/química , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Animales , Selenio/química , Selenio/farmacología , Ratones , Regulación hacia Arriba/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Estructura Molecular , Relación Estructura-Actividad , Supervivencia Celular/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neoplasias Experimentales/metabolismo , Línea Celular Tumoral , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Ubiquitin-specific protease 31 (USP31) is a member of deubiquitinase family that is involved in nuclear factor-κB activation and sarcomagenesis. However, little is known about posttranslational modification in the regulation of its activity and cervical cancer cell growth. In our study, we found that the Lys1264 residue of USP31 can be modified with an acetyl group by high-resolution mass spectrometry in HeLa cell line, and site-specific mutagenesis can significantly increase USP31 ubiquitin hydrolase activity and decrease the expression of p65. When being transfected with a plasmid expressing mutated USP31, the number of cancer cells was significantly decreased. We also observed that mutated USP31 could promote apoptosis but not cell cycle by flow cytometer analysis. Overexpression of mutated USP31 could reverse the effect in USP31 knockdown cell line. To further investigate its activity in tumorigenesis, deacetylase sirtuin 1 (Sirt1) was shown to interact with USP31 by co-immunoprecipitation and blocking the function of Sirt1 by knockdown or the inhibitor nicotinamide could increase the acetylation of USP31. When Lys1264 of USP31 mutated, Sirt1 could not remove its acetylation and alter the expression level of p65. Finally, inhibition or knockdown of Sirt1 suppressed USP31 activity in HeLa cell line, leading to cisplatin-induced apoptosis resistance. Therefore, acetylation at Lys1264 suppresses USP31 activity and plays a protective role in cancer cell growth. Our study contributes to understanding the mechanism of USP31 activity regulation and its role in tumorigenesis.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Neoplasias del Cuello Uterino/enzimología , Acetilación , Femenino , Células HeLa , Humanos , Lisina , Proteínas de Neoplasias/genética , Proteasas Ubiquitina-Específicas/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
Pseudomonas sp. P11 was isolated from the industrial wastewater sediment nearby the Daye Non-ferrous Metals Company, China. This strain possesses the ability to resist various heavy metals and efficiently precipitate arsenic. We here present a summary classification and a set of features of Pseudomonas sp. P11, together with the description of the genomic sequencing and annotation. The genomic sequence is 6,644,817 bp with a G+C content of 62.20% and contains 6143 protein-coding genes, 250 pseudo genes, and 76 tRNAs/rRNAs genes. Operons and gene clusters responsible for multiple heavy metal tolerance or detoxification were identified and accounted for the observed resistance phenotypes. Phylogenetic analysis revealed that the paralogous arsenic resistant genes possess different evolutionary paths. This study provides important insights to illuminate the versatility and adaptation of this strain to the heavy metal-contaminated environment.
Asunto(s)
Genoma Bacteriano , Sedimentos Geológicos/microbiología , Metales Pesados/metabolismo , Pseudomonas/aislamiento & purificación , Aguas Residuales/microbiología , Arsénico/análisis , Arsénico/metabolismo , Composición de Base , China , Metales Pesados/análisis , Filogenia , Pseudomonas/clasificación , Pseudomonas/genética , Pseudomonas/metabolismoRESUMEN
BACKGROUND: The acid glycoprotein 1 (AGP1) is downregulated in lung cancer. However, the performance of AGP1 in distinguishing benign from malignant lung lesions is still unknown. METHODS: The expression of AGP1 in benign diseases and lung cancer samples was detected by Western blot. The receiver operating characteristic curves, bivariate correlation, and multivariate analysis was analyzed by SPSS software. RESULTS: AGP1 expression levels were significantly downregulated in lung cancer and correlated with carcinoembryonic antigen (CEA), CA199, and CA724 tumor biomarkers. The diagnostic performance of AGP1 for distinguishing malignant from benign pulmonary lesions was better than the other four clinical biomarkers including CEA, squamous cell carcinoma-associated antigen, neuron-specific enolase, and cytokeratin 19 fragment 21-1, with an area under the curve value of 0.713 at 88.8% sensitivity. Furthermore, the multivariate analysis indicated that the variates of thrombin time and potassium significantly affected the AGP1 levels in lung cancer. CONCLUSIONS: Our study indicates that AGP1 expression is decreased in lung cancer compared to benign samples, which helps distinguish benign and malignant pulmonary lesions.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Antígeno Carcinoembrionario , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Pulmón/química , Pulmón/metabolismo , Pulmón/patología , Antígenos de Neoplasias , Biomarcadores de Tumor/metabolismo , Glicoproteínas , Queratina-19RESUMEN
Congenital disorders of glycosylation (CDG) are severe metabolic disorders caused by an imbalance in the glycosylation pathway. Phosphomannomutase2 (PMM2-CDG), the most prevalent CDG, is mainly due to the disorder of PMM2. Pathogenic variants in cysteine have been found in various diseases, and cysteine residues have a potential as therapeutic targets. PMM2 harbor six cysteines; the variants Cys9Tyr (C9Y) and Cys241Ser (C241S) of PMM2 have been identified to associate with CDG, but the underlying molecular mechanisms remain uncharacterized. Here, we purified PMM2 wild type (WT), C9Y, and C241S to investigate their structural characteristics and biophysical properties by spectroscopic experiments under physiological temperature and environmental stress. Notably, the variants led to drastic changes in the protein properties and were prone to aggregate at physiological temperature. Meanwhile, PMM2 was sensitive to oxidative stress, and the cysteine pathogenic variants led to obvious aggregate formation and a higher cellular apoptosis ratio under oxidative stress. Molecular dynamic simulations indicated that the pathogenic variants changed the core domain of homomeric PMM2 and subunit binding free energy. Moreover, we tested the potential drug targeting PMM2-celastrol in cell level and explained the result by molecular docking simulation. In this study, we delineated the pathological mechanism of the cysteine substitution in PMM2, which addressed the vital role of cysteine in PMM2 and provided novel insights into prevention and treatment strategies for PMM2-CDG.
Asunto(s)
Trastornos Congénitos de Glicosilación , Cisteína , Fosfotransferasas (Fosfomutasas) , Humanos , Trastornos Congénitos de Glicosilación/metabolismo , Trastornos Congénitos de Glicosilación/patología , Cisteína/genética , Cisteína/metabolismo , Glicosilación , Simulación del Acoplamiento Molecular , Fosfotransferasas (Fosfomutasas)/genéticaRESUMEN
The pathogenesis mechanism of lung cancer is very complex, with high incidence and mortality. Serpin family A member 3 (SERPINA3) expression levels were reduced in the sera of patients with lung cancer and may be a candidate diagnostic and prognostic survival biomarker in lung cancer, as previously reported. However, the detailed biological functions of SERPINA3 in the pathogenesis of lung cancer remain unknown. In the present study, it was aimed to explore the effects of SERPINA3 on the occurrence of lung cancer. SERPINA3 expression was assessed using bioinformatics database analysis and experimental detection. Then, the biological effects of SERPINA3 were investigated in a cell culture system and a xenograft model of human lung cancer. The potential regulatory mechanism of SERPINA3 in lung cancer was explored by dataindependent acquisition mass spectrometry (DIAMS) detection and further validated by western blotting (WB). The results indicated that SERPINA3 expression levels were significantly downregulated in lung cancer tissues and cell lines. At the cellular level, it was revealed that overexpressed SERPINA3 inhibited cell growth, proliferation, migration and invasion and promoted the apoptosis of lung cancer cells. Moreover, overexpressed SERPINA3 enhanced the sensitivity of lung cancer cells to osimertinib. In vivo, a xenograft model of human lung cancer was established with BALB/c nude mice. After the injection of A549 cells, the tumor growth of the tumorbearing mice in the SERPINA3overexpressing group increased more slowly, and the tumor volume was smaller than that in the emptyvector group. Mechanistically, a total of 65 differentially expressed proteins were identified. It was found that the speckletype POZ protein (SPOP) was significantly upregulated in SERPINA3overexpressing H157 cells using DIAMS detection and analysis. WB validation showed that SPOP expression increased, and NFkappaB (NFκB) p65 was inhibited in cell lines and tumor tissues of mice when SERPINA3 was overexpressed. The present findings suggest that SERPINA3 is involved in the development of lung cancer and has an antineoplastic role in lung cancer.
Asunto(s)
Neoplasias Pulmonares , Serpinas , Humanos , Animales , Ratones , FN-kappa B/metabolismo , Ratones Desnudos , Línea Celular Tumoral , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proliferación Celular , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Serpinas/genética , Serpinas/metabolismoRESUMEN
An MEKC method combining field-amplified sample injection (FASI) has been developed for the analysis of albumin (Alb) and transferrin (TRF) in human urines. Various parameters affecting FASI preconcentration were optimized systematically. Under the optimum conditions, using electrokinetic sample injection for 90 s, the sensitivity was improved 77.6- and 45.3-fold for TRF and Alb, respectively, without loss of separation efficiency when compared with hydrodynamical sample injection for 5 s (0.5 psi). The detection limits of TRF and Alb were found to be 0.31 and 0.14 mg/L, respectively. The proposed method has been successfully applied to the determination of TRF and Alb in desalted urines from patients with kidney diseases, and the recoveries of TRF and Alb were 91.1-101.7%.
Asunto(s)
Albúminas/aislamiento & purificación , Albuminuria , Cromatografía Capilar Electrocinética Micelar/métodos , Transferrina/orina , Acetonitrilos/química , Humanos , Enfermedades Renales/metabolismo , Sensibilidad y Especificidad , Cloruro de Sodio/química , Solventes/química , Transferrina/aislamiento & purificación , Ácido Trifluoroacético/química , Agua/químicaRESUMEN
We report the draft genome of Vibrio parahaemolyticus ATCC 17802(T), containing 5067729 bp. The G+C content of the genome is 45.24 %. This strain possesses genes encoding a Type III secretion system 1, a Type III secretion system 2 and a Tdh related hemolysin (TRH). Its taxonomically important phenotypes were also experimentally characterized.
Asunto(s)
Genoma Bacteriano/genética , Vibrio parahaemolyticus/genética , ADN Bacteriano/genética , Datos de Secuencia MolecularRESUMEN
EphB6 is a member in the receptor tyrosine kinase Eph family in that its kinase domain contains several alterations in conserved amino acids and is catalytically inactive. Although EphB6 is expressed both in a variety of embryonic and adult tissues, biological functions of this receptor are largely unknown. In this study, we examined the expression of EphB6 protein in 54 of tissue specimens of tongue squamous cell carcinoma by using a specific polyclonal anti-EphB6 antibody. The relationship between expression of EphB6 and clinical pathologic parameters was analyzed. The expression level of EphB6 in carcinoma cells from 34 out of 54 (63%) specimens was no alterative compared with normal squamous cells in same patient. The level of EphB6 protein staining was increased in carcinoma cells in 20 out of 54 (37%) specimens compared with normal squamous cells in same patient. The high-expression of EphB6 was significantly associated with age (P=0.021), tumor TNM stage (P=0.026) and lymph node metastasis (P=0.046). Patients with high expressed EphB6 protein had a high mortality (P=0.057). No significant relationship between expression of EphB6 and sex, tumor grade, HPV infection, relapse and smoke was found. We showed that patients with high expression of EphB6 had a significantly poor overall survival (OS) compared to patients with negative or weak expression (P=0.042). Our results indicated that EphB6 protein may be used as a new marker for prognosis for tongue squamous cell carcinoma.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Receptores de la Familia Eph/biosíntesis , Neoplasias de la Lengua/patología , Adulto , Anciano , Carcinoma de Células Escamosas/mortalidad , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Pronóstico , Receptores de la Familia Eph/análisis , Análisis de Supervivencia , Neoplasias de la Lengua/mortalidadRESUMEN
The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.
Asunto(s)
Arginina Quinasa/química , Decápodos/enzimología , Pliegue de Proteína , Urea/química , Animales , Cinética , Desnaturalización Proteica , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
OBJECTIVES: The aim of this study was to develop a small interfering RNA (siRNA) against the expression of KIR3DL1 receptor on natural killer (NK) cells, in order to promote the ability of NK cells to destroy human immunodeficiency virus (HIV)-infected cells and thus prevent failure of siRNA therapy targeting human immunodeficiency virus type 1 (HIV-1) virus among HIV-1 infected patients in vitro. METHODS: A siRNA targeting KIR3DL1 was synthesized and then modified with cholesterol, methylene, and sulfate. The inhibitory action of the siRNAs on primary cultured NK cells was detected. The amount of IFN-γ and TNF-α secretions in NK cells was measured. The intended functions of NK cells in vitro were analyzed by CFSE and PI methods. RESULTS: There were no significant differences in inhibiting the expression of KIR3DL1 on NK cells between the modified and unmodified siRNAs, while inhibition by each of them differed significantly from controls. The amount of IFN-γ and TNF-α secretions in the NK cells was abundant due to unsuccessful expression of KIR3DL1 on NK cells, which further promoted function of the NK cells. CONCLUSION: The siRNA against KIR3DL1 could enhance the ability of the NK cells to kill the HIV-1 infected cells in vitro and successfully prevented the failure of siRNA therapy targeting the HIV-1 virus. Therefore, it can act as a potential gene therapeutic agent among HIV-1 infected people.
Asunto(s)
Factores Inmunológicos/farmacología , Células Asesinas Naturales/inmunología , ARN Interferente Pequeño/farmacología , Receptores KIR3DL1/antagonistas & inhibidores , Adulto , Células Cultivadas , Femenino , Terapia Genética/métodos , Infecciones por VIH/terapia , Humanos , Factores Inmunológicos/genética , Interferón gamma/metabolismo , Masculino , Persona de Mediana Edad , ARN Interferente Pequeño/genética , Receptores KIR3DL1/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVES: To estimate the prevalence of sexually transmitted infections (STIs) among female sex workers (FSWs) in the Jiangsu Province, China and measure the association of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections with their potential correlates. DESIGN: A cross-sectional study on a representative sample of FSWs in Yangzhou and Changzhou cities of Jiangsu was conducted. METHODS: 185 sex-work venues in Yangzhou and 174 in Changzhou were selected by stratified random sampling. 2972 FSWs (1108 in Yangzhou and 1864 in Changzhou), aged 15 years or more, who agreed to participate and provided blood sample for HIV and syphilis testing were interviewed in these venues. Cervical specimens from 849 randomly chosen participants were then tested for CT and NG. RESULTS: Proportions of young, school-educated, currently married FSWs who were living alone, migrated from other provinces and engaged in unprotected vaginal intercourse in past 3 months (UVI) were relatively high. Prevalence of HIV, syphilis, CT and NG were 0.20%, 4.88%, 14.61% and 5.42% respectively. Younger age, living alone or with persons other than partners/family members, engaging in UVI and having other STIs seemed to be associated with higher risk of CT or NG infection. Being divorced/widowed and working in middle/low-level venues were identified as additional risk factors for NG. CONCLUSIONS: Based on a representative sample, this initial effort to identify the correlates of CT/NG infections among FSWs of Jiangsu revealed that focused interventions targeting high-risk FSWs are urgently required for controlling STI epidemics in Yangzhou and Changzhou where substantial number of STI cases were identified.
Asunto(s)
Infecciones por Chlamydia/epidemiología , Gonorrea/epidemiología , Trabajadores Sexuales , Adulto , China/epidemiología , Estudios Transversales , Femenino , Infecciones por VIH/epidemiología , Humanos , Prevalencia , Factores de Riesgo , Sífilis/epidemiología , Sexo Inseguro , Adulto JovenRESUMEN
Whether Cyclophilin A (CyPA) functions as a foldase or a chaperone when assisting protein folding has long been argued. In this study, we engineered four variants of recombinant human Cyclophilin A (rhCyPA), all of which were inactive to tetrapeptide substrate Suc-AAPF-pNA. However, these variants were able to suppress aggregation during arginine kinase (AK) refolding as efficient as wild-type rhCyPA, especially, variant Q63A had even more efficiency to suppress aggregation and improve reactivation yields of AK. These results indicate that rhCyPA have peptidyl-prolyl cis-trans isomerase (PPIase) independent chaperone-like activity during AK folding. In addition, results suggest that surface hydrophobicity of rhCyPA can suppress AK aggregation and binding to rhCyPA hydrophobic active pocket is a prerequisite for chaperoning AK folding.
Asunto(s)
Arginina Quinasa/química , Ciclofilina A/química , Chaperonas Moleculares/química , Arginina Quinasa/metabolismo , Sitios de Unión , Ciclofilina A/genética , Ciclofilina A/metabolismo , Escherichia coli/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagénesis Sitio-Dirigida , Oligopéptidos/química , Unión Proteica , Ingeniería de Proteínas , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Propiedades de SuperficieRESUMEN
Arginine kinase (AK), a crucial enzyme for the energy metabolism of invertebrates, catalyzes the reversible phosphorylation of arginine by Mg(2+)ATP to form phosphoarginine and Mg(2+)ADP. Arginine 330 (R330), not involved in the catalysis of phosphoryl transfer, is a residue highly conserved in the phosphagen kinase family. In order to investigate the role of R330 in AK, it was replaced by lysine (R330K). Non-reduced SDS-PAGE analysis suggested that wild type AK (Wt-AK) and R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K), whereas O-R330K was more susceptible to generate than O-AK. Subsequently, an intramolecular disulfide bond in O-R330K was demonstrated to be formed between Cys201 and Cys271 by site-directed mutagenesis. Biochemical analysis revealed that conformational changes of R330K were concomitant with the sharp decline of catalytic activity. These results were further confirmed by structure modeling of AK and R330K. Therefore, it can be concluded that R330 residue plays an important role in the structural stability and activity of AK.
Asunto(s)
Arginina Quinasa/genética , Arginina/genética , Crustáceos/enzimología , Mutación/genética , Sustitución de Aminoácidos/genética , Animales , Arginina Quinasa/química , Cromatografía en Gel , Dicroismo Circular , Cisteína/genética , Disulfuros/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Oxidación-Reducción , Estructura Secundaria de Proteína , Espectrometría de FluorescenciaRESUMEN
Arginine kinase (AK), a crucial enzyme in energy metabolism, buffers cellular ATP levels by catalyzing the reversible phosphoryl transfer between ATP and arginine. To better understand the role of Cys271 in conformational changes of AK from greasyback shrimp (Metapenaeus ensis), we replaced the residue with serine and alanine. A detailed comparison of the catalytic activity and conformation was made between wild-type AK and the mutants by means of activity analysis, ultraviolet (UV) difference, fluorescence spectrum and size exclusion chromatography (SEC). The results indicated that the catalytic activity of the two mutants was gone. The substrates, arginine-ADP-Mg(2+) could induce conformational changes, and additional NO(3)(-) could induce further changes in both the native enzyme and the variants. We speculated that Cys271 might be located in the hinge region between the two domains of AK and cause enzyme conformational changes upon addition of substrate.
Asunto(s)
Arginina Quinasa/química , Cisteína/química , Penaeidae/enzimología , Conformación Proteica , Alanina/química , Animales , Catálisis , Cromatografía en Gel , Clonación Molecular , Cartilla de ADN/genética , Fluorescencia , Mutagénesis Sitio-Dirigida , Relación Estructura-ActividadRESUMEN
The purpose of this study is to clarify that the amino acid residues (Asp62 and Arg193) are responsible for the activity and stability of arginine kinase (AK). The amino acid residues Asp62 (D62) and Arg193 (R193) are strictly conserved in monomeric AKs and form an ion pair in the transition state analogue complex. In this research, we replaced D62 with glutamate (E) or glycine (G) and R193 with lysine (K) or glycine (G). The mutants of D62E and R193K retained almost 90% of the wild-type activity, whereas D62G and R193G had a pronounced loss in activity. A detailed comparison was made between the physic-chemical properties and conformational changes of wild-type AK and the mutants by means of ultraviolet (UV) difference and fluorescence spectra. The results indicated that the conformation of all of the mutants had been changed and the stability in a urea solution was also reduced. We speculated that the hydrogen bond and electrostatic interactions formed between residues 62 and 193 play a key role in stabilizing the structure and mediating the synergism in substrate binding of arginine kinase from greasyback shrimp (Metapenaeus ensis).
Asunto(s)
Arginina Quinasa/química , Arginina Quinasa/metabolismo , Animales , Arginina Quinasa/genética , Arginina Quinasa/aislamiento & purificación , Estabilidad de Enzimas , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Penaeidae/enzimología , Unión Proteica , Conformación Proteica , Desplegamiento ProteicoRESUMEN
Cyclophilin A (CyPA) is a cytosolic receptor of immunosuppressive drug cyclosporin A (CsA) which possesses peptidyl-prodyl cis/trans isomerase (PPIase) activity. The recombinant human CyPA (rhCyPA) gene has been expressed in E. coli M15. Purification was performed using salting-out, as well as Sephacryl S-100 and DEAE-Sepharose CL-6B column chromatography. The molecular weight is about 18 kDa, confirmed by SDS-PAGE and mass spectrum. The results of Native-PAGE and immunoblotting showed the existence of three bands, which agreed well with the gel filtration results. The molecular mass of the three bands determined via CTAB gel electrophoresis and SDS-PAGE (rhCyPA cross-linked with glutaraldehyde) was 18 kDa, 36 kDa and 54 kDa respectively. Further more, the native rhCyPA and the cross-linked rhCyPA had the similar chromatographic behavior in gel filtration. All of the evidences above suggest that rhCyPA exists in forms of monomer, dimer and trimer. Moreover, we observed that even at low protein concentrations CyPA largely occurs as a dimer in solution, and enzyme kinetic parameters showed that activity of dimer was much higher than monomer or trimer, which probably have some biological significances.
Asunto(s)
Ciclofilina A/metabolismo , Western Blotting , Ciclofilina A/genética , Electroforesis , Electroforesis en Gel de Poliacrilamida , Humanos , Multimerización de Proteína/genética , Multimerización de Proteína/fisiologíaRESUMEN
Arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrate. The encoding AK gene from Shrimp Metapenaeus ensis (M. ensis) was cloned in prokaryotic expression plasmid pET-28a, and it was then expressed in Escherichia coil in dissoluble form. The recombinant protein was purified by following three chromatography steps in turn: CM-Cellulose cation-exchange, Sephacryl S-100HR gel filtrate and DEAE-Sepharose anion-exchange. The purified AK's apparent K(m) was 2.33+/-0.1 and 1.59+/-0.2 mM for ATP and l-arginine, respectively, while its optimum pH and temperature was 8.5 and 30 degrees C in the process of forward reaction, respectively. Phylogenetic analysis of cDNA-derived amino acid sequences for the AKs indicated a close affinity of M. ensis and another shrimp (Litopenaeus vannamei).
Asunto(s)
Arginina Quinasa/metabolismo , Penaeidae/enzimología , Filogenia , Temperatura , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Arginina Quinasa/genética , Arginina Quinasa/aislamiento & purificación , Clonación Molecular , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 micromol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 micromol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (< or = 100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.
Asunto(s)
Arginina Quinasa/química , Decápodos/enzimología , Detergentes/metabolismo , Guanidina/farmacología , Pliegue de Proteína , Animales , Arginina Quinasa/metabolismo , Dicroismo Circular , Desnaturalización Proteica/efectos de los fármacos , SolubilidadRESUMEN
The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase by dithiothreitol has been investigated using the kinetic theory of the substrate reaction during modification of enzyme activity. The results show that the modified arginine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant, while arginine shows no effect. The results of ultraviolet (UV) difference and intrinsic fluorescence spectra indicate that the substrate arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of arginine kinase are discussed. It is suggested that the cysteine may be located in the hinge region of the two domains of arginine kinase. The reactive cysteine of arginine kinase may play an important role not in the binding to the transition-state analog but in the conformational changes caused by the transition-state analog.