RESUMEN
Recently, a number of reports on the importance of USP35 in cancer have been published. However, very little is known about the exact mechanism by which USP35 activity is regulated. Here, we show the possible regulation of USP35 activity and the structural specificity affecting its function by analyzing various fragments of USP35. Interestingly, the catalytic domain of USP35 alone does not exhibit deubiquitinating activity; in contrast, the C-terminal domain and insertion region in the catalytic domain is required for full USP35 activity. Additionally, through its C-terminal domain, USP35 forms a homodimer that prevents USP35 degradation. CHIP bound to HSP90 interacts with and ubiquitinates USP35. However, when fully functional USP35 undergoes auto-deubiquitination, which attenuates CHIP-mediated ubiquitination. Finally, USP35 dimer is required for deubiquitination of the substrate Aurora B and regulation of faithful mitotic progression. The properties of USP35 identified in this study are a unique homodimer structure, regulation of deubiquitinating activity through this, and utilization of a novel E3 ligase involved in USP35 auto-deubiquitination, which adds another complexity to the regulation of deubiquitinating enzymes.
Asunto(s)
Neoplasias , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: Heat shock protein 27 (HSP27) is overexpressed during pulmonary fibrosis (PF) and exacerbates PF; however, the upregulation of HSP27 during PF and the therapeutic strategy of HSP27 inhibition is not well elucidated. METHODS: We have developed a mouse model simulating clinical stereotactic body radiotherapy (SBRT) with focal irradiation and validated the induction of RIPF. HSP25 (murine form of HSP27) transgenic (TG) and LLC1-derived orthotropic lung tumor models were also used. Lung tissues of patients with RIPF and idiopathic pulmonary fibrosis, and lung tissues from various fibrotic mouse models, as well as appropriated cell line systems were used. Public available gene expression datasets were used for therapeutic response rate analysis. A synthetic small molecule HSP27 inhibitor, J2 was also used. RESULTS: HSP27 expression with its phosphorylated form (pHSP27) increased during PF. Decreased mRNA expression of SMAD-specific E3 ubiquitin-protein ligase 2 (Smurf2), which is involved in ubiquitin degradation of HSP27, was responsible for the increased expression of pHSP27. In addition, increased expression of miRNA15b was identified with decreased expression of Smurf2 mRNA in PF models. Inverse correlation between pHSP27 and Smurf2 was observed in the lung tissues of PF animals, an irradiated orthotropic lung cancer models, and PF tissues from patients. Moreover, a HSP27 inhibitor cross-linked with HSP27 protein to ameliorate PF, which was more effective when targeting the epithelial to mesenchymal transition (EMT) stage of PF. CONCLUSIONS: Our findings identify upregulation mechanisms of HSP27 during PF and provide a therapeutic strategy for HSP27 inhibition for overcoming PF.
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MicroARNs , Fibrosis Pulmonar , Ratones , Animales , Fibrosis Pulmonar/genética , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/farmacología , Transición Epitelial-Mesenquimal , Ubiquitina-Proteína Ligasas/genética , MicroARNs/metabolismo , ARN MensajeroRESUMEN
BACKGROUND: Although quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma. METHODS: The annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol. RESULTS: An acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction. CONCLUSION: This is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.
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Cardiopatías/sangre , Cardiopatías/diagnóstico , MicroARNs/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Biomarcadores/sangre , Biomarcadores/metabolismo , Cardiopatías/genética , Humanos , MicroARNs/sangre , MicroARNs/aislamiento & purificaciónRESUMEN
Ubiquitination and deubiquitination are protein post-translational modification processes that have been recognized as crucial mediators of many complex cellular networks, including maintaining ubiquitin homeostasis, controlling protein stability, and regulating several signaling pathways. Therefore, some of the enzymes involved in ubiquitination and deubiquitination, particularly E3 ligases and deubiquitinases, have attracted attention for drug discovery. Here, we review recent findings on USP15, one of the deubiquitinases, which regulates diverse signaling pathways by deubiquitinating vital target proteins. Even though several basic previous studies have uncovered the versatile roles of USP15 in different signaling networks, those have not yet been systematically and specifically reviewed, which can provide important information about possible disease markers and clinical applications. This review will provide a comprehensive overview of our current understanding of the regulatory mechanisms of USP15 on different signaling pathways for which dynamic reverse ubiquitination is a key regulator.
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Procesamiento Proteico-Postraduccional/fisiología , Transducción de Señal/fisiología , Proteasas Ubiquitina-Específicas/fisiología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/fisiología , Complejo del Señalosoma COP9/fisiología , Humanos , Inmunidad Innata , Masculino , Ratones , FN-kappa B/metabolismo , Neoplasias de la Próstata/metabolismo , Dominios Proteicos , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Precursores del ARN/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteasas Ubiquitina-Específicas/química , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación , Vía de Señalización Wnt/fisiología , Proteínas de Xenopus/metabolismoRESUMEN
The MDM2-p53 pathway is crucial for maintenance of p53 homeostasis. Some ribosomal proteins (RPs) play critical roles in regulating p53 by interacting with MDM2. However, the role and functional mechanism of each RP in MDM2-p53 pathway still remain unknown. In this study, we found that Ribosomal Protein S2 (RPS2) is a new regulator of MDM2-P53 signaling pathway to regulate p53 protein level. Here, we characterized that RPS2 interacts with MDM2 through the RING finger domain of MDM2. RPS2 is ubiquitinated by MDM2 and the ubiquitinated status of RPS2 regulates the stability of p53, which is activated in response to cellular stresses such as DNA damage, oxidative stress, and especially ribosomal stress. In addition, p53 is not induced in RPS2 knockdown even in the ribosomal stressed condition, indicating that RPS2 is essential for the stabilization of p53. Collectively, our data suggest that RPS2 plays a critical role in the regulation of p53 signaling including the ribosomal stress response.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Ribosómicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células HeLa , Humanos , Dominios Proteicos , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Ribosómicas/genética , Transducción de Señal , Estrés Fisiológico , Proteína p53 Supresora de Tumor/genética , UbiquitinaciónRESUMEN
Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein-protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Pruebas de Enzimas/métodos , Animales , Polarización de Fluorescencia , Humanos , Sondas Moleculares/química , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Ubiquitination and deubiquitination play a critical role in all aspects of cellular processes, and the enzymes involved are tightly regulated by multiple factors including posttranslational modifications like most other proteins. Dysfunction or misregulation of these enzymes could have dramatic physiological consequences, sometimes leading to diseases. Therefore, it is important to have a clear understanding of these regulatory processes. Here, we have reviewed the posttranslational modifications of deubiquitinating enzymes and their consequences on the catalytic activity, stability, abundance, localization, and interaction with the partner proteins.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Procesamiento Proteico-Postraduccional , Catálisis , Humanos , Fosforilación , Transporte de Proteínas , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein-protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3. The ubiquitination and deubiquitination status of PRP31 regulates its interaction with the U5 snRNP component PRP8, which is required for the efficient splicing of chromosome segregation related genes, probably by stabilizing the U4/U6.U5 tri-snRNP complex. Collectively, our data suggest that USP15 plays a key role in the regulation of dynamic protein-protein interactions of the spliceosome.
Asunto(s)
Antígenos de Neoplasias/genética , Mapas de Interacción de Proteínas/genética , Proteínas de Unión al ARN/genética , Proteasas Ubiquitina-Específicas/genética , Enzimas Reparadoras del ADN/genética , Células HeLa , Humanos , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , Factores de Empalme de ARN/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Empalmosomas/genética , Ubiquitinación/genéticaRESUMEN
Mitosis is a complex and dynamic process that is tightly regulated by a large number of mitotic proteins. Dysregulation of these proteins can generate daughter cells that exhibit genomic instability and aneuploidy, and such cells can transform into tumorigenic cells. Thus, it is important for faithful mitotic progression to regulate mitotic proteins at specific locations in the cells at a given time in each phase of mitosis. Ubiquitin-dependent modifications play critical roles in this process by regulating the degradation, translocation, or signal transduction of mitotic proteins. Here, we review how ubiquitination and deubiquitination regulate the progression of mitosis. In addition, we summarize the substrates and roles of some deubiquitinating enzymes (DUBs) crucial for mitosis and describe how they contribute error correction during mitosis and control the transition between the mitotic phases.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Mitosis , Animales , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) is a U4/U6 recycling factor as well as a targeting factor of USP4 and USP15. However, the details of how SART3 recognizes these deubiquitinases and how they get subsequently translocated into the nucleus are not known. Here, we present the crystal structures of the SART3 half-a-tetratricopeptide (HAT) repeat domain alone and in complex with the domain present in ubiquitin-specific protease (DUSP)-ubiquitin-like (UBL) domains of ubiquitin specific protease 4 (USP4). The 12 HAT repeats of SART3 are in two sub-domains (HAT-N and HAT-C) forming a dimer through HAT-C. USP4 binds SART3 at the opposite surface of the HAT-C dimer interface utilizing the ß-structured linker between the DUSP and the UBL domains. The binding affinities of USP4 and USP15 to SART3 are 0.9 µM and 0.2 µM, respectively. The complex structure of SART3 nuclear localization signal (NLS) and importin-α reveals bipartite binding, and removal of SART3 NLS prevents the entry of USP4 (and USP15) into the nucleus and abrogates the subsequent deubiquitinase activity of USP4.
Asunto(s)
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Modelos Moleculares , Conformación Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Empalmosomas/metabolismo , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Cristalografía por Rayos X , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Señales de Localización Nuclear/química , Señales de Localización Nuclear/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas de Unión al ARN/genética , Relación Estructura-Actividad , Secuencias Repetidas en Tándem , Ubiquitina Tiolesterasa/genética , Proteasas Ubiquitina-Específicas/metabolismo , alfa Carioferinas/química , alfa Carioferinas/metabolismoRESUMEN
The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for alpha-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Ciclo Celular/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalmosomas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Células HeLa , Humanos , Empalme de Proteína/fisiología , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ubiquitina/metabolismo , Proteasas Ubiquitina-EspecíficasRESUMEN
The transforming growth factor-ß (TGF-ß) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-ß signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-ß-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , MicroARNs/genética , Proteína Smad2/genética , Proteína Smad4/genética , Factor de Crecimiento Transformador beta/metabolismo , Ciclo Celular , Línea Celular Tumoral , Humanos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Transducción de SeñalRESUMEN
MicroRNAs (miRNAs) are short noncoding RNA molecules that control the expression of mRNAs associated with various biological processes. Therefore, deregulated miRNAs play important roles in the pathogenesis of diseases. Numerous studies are aimed at discovering biomarkers of diseases or determining miRNA functions by monitoring circulating miRNAs in various biological sources such as plasma and urine. However, the analysis of miRNA in such fluids presents problems related to accuracy and reproducibility because of their low levels in biological fluids. Therefore, better extraction kits and more sensitive detection systems have been developed for improved and reproducible analysis of circulating miRNAs. However, new extraction methods are also needed to improve the yield of miRNAs for their reliable analysis from biological fluids. The combination of yeast transfer RNA (tRNA) and glycogen as carrier molecules and incubation durations were optimized to maximize extraction efficiency. The extraction recovery using a combination of yeast tRNA and glycogen was approximately threefold more than that by using glycogen or yeast tRNA alone. In addition, reproducible and accurate analysis of miRNAs can be carried out after extraction using a combination of yeast tRNA and glycogen without an impact on plasma components. Graphical abstract Steps of miRNA extraction in plasma.
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MicroARNs/sangre , MicroARNs/aislamiento & purificación , Fraccionamiento Químico , Electroforesis Capilar , Glucógeno/química , Humanos , MicroARNs/genética , Reacción en Cadena de la Polimerasa , ARN de Hongos/química , ARN de Transferencia/química , Reproducibilidad de los Resultados , Levaduras/químicaRESUMEN
MicroRNAs (miRNAs) are short RNA molecules that control the expression of mRNAs associated with various biological processes. Therefore, deregulated miRNAs play an important role in the pathogenesis of diseases. Numerous studies aimed at developing novel miRNA-based drugs or determining miRNA functions have been conducted by inhibiting miRNAs using anti-miRNA oligonucleotides (AMOs), which inhibit the function by hybridizing with miRNA. To increase the binding affinity and specificity to target miRNA, AMOs with various chemical modifications have been developed. Evaluating the potency of these various types of AMOs is an essential step in their development. In this study, we developed a capillary electrophoresis with laser-induced fluorescence (CE-LIF) method to evaluate the potency of AMOs by measuring changes in miRNA levels with fluorescence-labeled ssDNA probes using AMO-miR-23a, which inhibits miR-23a related to lung cancer. In order to eliminate interference by excess AMOs during hybridization of the ssDNA probe with the miR-23a, the concentration of the ssDNA probe was optimized. This newly developed method was used to compare the potency of two different modified AMOs. The data were supported by the results of a luciferase assay. This study demonstrated that CE-LIF analysis could be used to accurately evaluate AMO potency in biological samples.
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Antagomirs/administración & dosificación , Antagomirs/genética , Regulación hacia Abajo/genética , Electroforesis Capilar/métodos , Regulación de la Expresión Génica/genética , MicroARNs/biosíntesis , Espectrometría de Fluorescencia/métodos , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , MicroARNs/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Valosin-containing protein (VCP), also known as p97, is an AAA(+) ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXLOTU1) complexed to the N-terminal domain of VCP (NVCP) at 1.8-Å resolution reveals that UBXLOTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of NVCP using the (39)GYPP(42) loop of UBXLOTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a KD of 0.71 µM. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.
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Adenosina Trifosfatasas/química , Proteínas de Ciclo Celular/química , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Degradación Asociada con el Retículo Endoplásmico/genética , Células HEK293 , Humanos , Immunoblotting , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/genética , Proteína que Contiene ValosinaRESUMEN
Neurotensin (NT) is distributed throughout the brain and gastrointestinal tract. Although the relationship between NT and matrix metalloproteinase-9 (MMP-9) activity in gastric cancer has not been reported, the elevation of MMP-9 and NT is reported in the breast, lung, prostate, and gastric cancer. The aim of our study is to investigate the relationship between NT and MMP-9 activity and the underlying signaling mechanism in gastric cancer cell lines. Commercial ELISA kits were used for estimation of NT and MMP-9 expression, and fluorescence resonance energy transfer (FRET) assay was used for measurement of MMP-9 activity. Cell migration and invasion were determined by wound healing and transwell assay. The expression of signaling proteins was measured by Western blotting. Our study reveals a positive correlation between increased plasma NT and MMP-9 activity in both of patient's serum and gastric cancer cell lines. A dose-dependent elevation of MMP-9 activity was observed by NT treatment in gastric cancer cells (MKN-1 and MKN-45) compared to untreated gastric cancer and normal epithelial cell (HFE-145). Moreover, NT-mediated migration and invasion were observed in gastric cancer cells unlike in normal cell. The signaling mechanism of NT in gastric cancer cells was confirmed in protein kinase C (PKC), extracellular-signal regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K) pathway. In addition, pretreatment of gastric cancer cells with NTR1 inhibitor SR48692 was shown to significantly inhibit the NT-mediated MMP-9 activity, cell invasion, and migration. Our finding illustrated NTR1 could be a possible therapeutic target for gastric cancer.
Asunto(s)
Metaloproteinasa 9 de la Matriz/biosíntesis , Neurotensina/genética , Neoplasias Gástricas/genética , Activación Transcripcional , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Persona de Mediana Edad , Invasividad Neoplásica/genética , Neurotensina/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas/genética , Pirazoles/administración & dosificación , Quinolinas/administración & dosificación , Neoplasias Gástricas/patologíaRESUMEN
Protein degradation by the ubiquitin-proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS-dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)-fusion of the ubiquitin substrate ribophorin 1 (GFP-RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP-RPN1 and RFP in cell lysates were performed in an untreated capillary (75 µm × 50 cm) and 100 mM Tris-CHES buffer (pH 9.0) containing 10 mM SDS. GFP-RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP-RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP-RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS-dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.
Asunto(s)
Electroforesis Capilar/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Espectrometría de Fluorescencia/métodos , Ubiquitina/metabolismo , Proteínas Fluorescentes Verdes/química , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Proteínas/análisis , Proteínas/química , Proteínas/metabolismo , Ubiquitina/análisis , Ubiquitina/químicaRESUMEN
Introduction: The relationships between the feeding rhythm, sleep and cognition in Alzheimer's disease (AD) are incompletely understood, but meal time could provide an easy-to-implement method of curtailing disease-associated disruptions in sleep and cognition. Furthermore, known sex differences in AD incidence could relate to sex differences in circadian rhythm/sleep/cognition interactions. Methods: The 5xFAD transgenic mouse model of AD and non-transgenic wild-type controls were studied. Both female and male mice were used. Food access was restricted each day to either the 12-h light phase (light-fed groups) or the 12-h dark phase (dark-fed groups). Sleep (electroencephalographic/electromyographic) recording and cognitive behavior measures were collected. Results: The 5xFAD genotype reduces NREM and REM as well as the number of sleep spindles. In wild-type mice, light-fed groups had disrupted vigilance state amounts, characteristics, and rhythms relative to dark-fed groups. These feeding time differences were reduced in 5xFAD mice. Sex modulates these effects. 5xFAD mice display poorer spatial memory that, in female mice, is curtailed by dark phase feeding. Similarly, female 5xFAD mice have decreased anxiety-associated behavior. These emotional and cognitive measures are correlated with REM amount. Discussion: Our study demonstrates that the timing of feeding can alter many aspects of wake, NREM and REM. Unexpectedly, 5xFAD mice are less sensitive to these feeding time effects. 5xFAD mice demonstrate deficits in cognition which are correlated with REM, suggesting that this circadian-timed aspect of sleep may link feeding time and cognition. Sex plays an important role in regulating the impact of feeding time on sleep and cognition in both wild-type and 5xFAD mice, with females showing a greater cognitive response to feeding time than males.
RESUMEN
Micro-RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE-LIF) using fluorescence-labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA-499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA-499 was found when hybridization was conducted at 40°C in 50 mM Tris-acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA-499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA-499 was completed within 1 h using CE-LIF. These results showed the potential of CE for fast, specific, and sensitive high-throughput analysis of low-abundance miRNAs in cell extracts, biofluids, and tissues.