RESUMEN
O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3',5,5'-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , N-Acetilglucosaminiltransferasas/metabolismo , Acetilglucosamina/metabolismo , Quinasa de la Caseína II/metabolismo , Enzimas Inmovilizadas/metabolismo , Humanos , Especificidad por SustratoRESUMEN
Dysregulated MAPK/ERK signaling is implicated in one-third of human tumors and represents an attractive target for the development of anticancer drugs. Similarly, elevated protein O-GlcNAcylation and O-GlcNAc transferase (OGT) are detected in various cancers and serve as attractive novel cancer-specific therapeutic targets. However, the potential connection between them remains unexplored. Here, a positive correlation was found between the activated MAPK/ERK signaling and hyper-O-GlcNAcylation in various cancer types and inhibition of the MAPK/ERK signaling by 10 µM U0126 significantly decreased the expression of OGT and O-GlcNAcylation in H1299, BPH-1 and DU145 cells; then, the pathway analysis of the potential regulators of OGT obtained from the UCSC Genome Browser was done, and ten downstream targets of ERK pathway were uncovered; the following results showed that ELK1, one of the ten targets of ERK pathway, mediated ERK signaling-induced OGT upregulation; finally, the MTT assay and the soft agar assay showed that the inhibition of MAPK/ERK signaling reduced the promotion effect of hyper-O-GlcNAcylation on cancer cell proliferation and anchorage-independent growth. Taken together, our data originally provided evidence for the regulatory mechanism of hyper-O-GlcNAcylation in tumors, which will be helpful for the development of anticancer drugs targeting to hyper-O-GlcNAcylation. This study also provided a new mechanism by which MAPK/ERK signaling-enhanced cancer malignancy. Altogether, the recently discovered oncogenic factor O-GlcNAc was linked to the classical MAPK/ERK signaling which is essential for the maintenance of malignant phenotype of cancers.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Neoplasias/enzimología , Procesamiento Proteico-Postraduccional , Antineoplásicos/farmacología , Butadienos/farmacología , Línea Celular Tumoral , Proliferación Celular , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Glicosilación , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Nitrilos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Interferencia de ARN , Transfección , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
p120-catenin (p120) contains a large central armadillo repeat domain, via which it binds to Ecadherin to stabilize the latter, thereby regulating celltocell adhesion. A previous study by our group demonstrated that Olinked Nacetylglucosamine (OGlcNAc) is involved in the regulation of the interaction between p120 and Ecadherin. As OGlcNAc transferase (OGT) is able to directly bind to the majority of its target proteins, the present study hypothesized that OGT may additionally regulate the formation of the Ecadherin/catenin complex independent of its catalytic activity. To verify this hypothesis, a catalytically inactive OGT mutant was expressed in H1299 cells, and its effects on the formation of the Ecadherin/catenin complex were assessed. A cytoskeletonbinding protein extraction assay confirmed that OGT inhibited the formation of the Ecadherin/catenin complex independent of its catalytic activity. In addition, coimmunoprecipitation and pulldown assays were used to evaluate the interaction between OGT and p120. Immunoblotting indicated that OGT was able to directly bind to p120. To determine the domain of p120 involved in binding to OGT, a series of deletion mutants of p120 were constructed and subjected to protein binding assays by pulldown assays. Immunoblotting showed that OGT bound to the regulatory and armadillo domains of p120, which might interfere with the interaction between p120 and Ecadherin. Finally, OGT, p120 and Ecadherin cytoplasmic domains (ECD) were recombinantly expressed in BL21 (DE3) recombinant E. coli cells, and a glutathione Stransferase (GST) pulldown assay was performed to assess the interactions among the purified recombinant proteins. Immunoblotting indicated that maltosebinding protein (MBP)OGT inhibited the binding of Hisp120 to GSTECD in a dosedependent manner. All of these results suggested that OGT inhibited the formation of the Ecadherin/catenin complex through reducing the interaction between p120 and Ecadherin. The present study provided a novel underlying mechanism of the regulation of the interaction between p120 and Ecadherin, and thus Ecadherinmediated cellcell adhesion, which has essential roles in cancer development and progression.
Asunto(s)
Biocatálisis , Cadherinas/metabolismo , Cateninas/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Cateninas/química , Línea Celular Tumoral , Células HEK293 , Humanos , Ratones Endogámicos BALB C , Unión Proteica , Estructura Terciaria de Proteína , Eliminación de Secuencia , Catenina deltaRESUMEN
O-GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O-GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O-GlcNAcylated protein production in E. coli. The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl ß-d-thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O-GlcNAcylation levels by altering the inducer concentration. More importantly, the O-GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O-GlcNAcylated proteins in E. coli.
Asunto(s)
Acetilglucosamina/metabolismo , Escherichia coli/genética , N-Acetilglucosaminiltransferasas/metabolismo , Plásmidos , Proteínas Recombinantes/biosíntesis , Arabinosa/metabolismo , Vectores Genéticos , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Temozolomide (TMZ) is an effective agent for clinical glioma treatment, but the innate and acquired resistance of glioma always limits its application. Although some advances have been achieved to elucidate the molecular mechanism underlying TMZ resistance, the role of Nrf2 (a principle regulator of cellular defense against drugs and oxidative stress) has not been well established in the acquisition of this phenotype. Our data showed that TMZ treatment induces the activation of Nrf2 and p38 MAPK signaling in glioma cells, while p38 inhibition abolished the effect of TMZ on Nrf2. Further study revealed that Nrf2 silencing was able to enhance the response of glioma cells to TMZ. Additionally, Nrf2 overexpression overrides the effect of p38 MAPK activation on Temozolomide resistance. In conclusions, we identified a p38 MAPK/Nrf2 signaling as a key molecular network contributing to TMZ resistance of glioma, and provided evidence that suppressing this signaling may be a promising strategy to improve TMZ's therapeutic efficiency.