RESUMEN
Growth factor receptor-bound 2 (Grb2) is an important link in the receptor tyrosine kinase signaling cascades. It is involved in crucial processes, both physiological (mainly embryogenesis) and pathological (different types of cancer). Several binding partners of all three domains (SH3-SH2-SH3) of this adaptor protein are well described, such as ErbB family members for the SH2 domain and Sos for the SH3 domains. How the different domains interact with each other, both structurally and functionally, is still unclear. These interactions could be essential for regulation processes, and therefore are of great interest. Although a lot of structural data on Grb2 exist, they describe either individual domains, ligand-bound conformations, or frozen pictures of the protein captured by crystallography. Here we report the assignment of backbone and of [Formula: see text] chemical shifts of full-length, apo-Grb2 in solution. In addition to the assigned conformation corresponding to three well-folded domains, a set of peaks compatible with the presence of an unfolded conformation of the N-terminal SH3 domain is observed. This assignment paves the way for future studies of inter-domain interactions and dynamics that have to be taken into account when studying the regulation of Grb2 interactions and signaling pathways.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Proteína Adaptadora GRB2/análisis , Espectroscopía de Protones por Resonancia Magnética , Secuencia de Aminoácidos , Proteína Adaptadora GRB2/química , Humanos , Ligandos , Isótopos de NitrógenoRESUMEN
Protein-protein interactions (PPIs) form the basis of cellular processes, regulating cell behavior and fate. PPIs can be extremely transient in nature, which hinders their detection. In addition, traditional biochemical methods provided limited information on the spatial distribution and temporal dynamics of PPIs that is crucial for their regulation in the crowded cellular environment. Given the pivotal role of membrane micro- and nanodomains in the regulation of PPIs at the plasma membrane, the development of methods to visualize PPIs with a high spatial resolution is imperative. Here, we present a super-resolution fluorescence microscopy technique that can detect and map short-lived transient protein-protein interactions on a nanometer scale in the cellular environment. This imaging method is based on single-molecule fluorescence microscopy and exploits the effect of the difference in the mobility between cytosolic and membrane-bound proteins in the recorded fluorescence signals. After the development of the proof of concept using a model system based on membrane-bound modular protein domains and fluorescently labeled peptides, we applied this imaging approach to investigate the interactions of cytosolic proteins involved in the epidermal growth factor signaling pathway (namely, Grb2, c-Raf, and PLCγ1). The detected clusters of Grb2 and c-Raf were correlated with the distribution of the receptor at the plasma membrane. Additionally, the interactions of wild type PLCγ1 were compared with those detected with truncated mutants, which provided important information regarding the role played by specific domains in the interaction with the membrane. The results presented here demonstrate the potential of this technique to unravel the role of membrane heterogeneity in the spatiotemporal regulation of cell signaling.
Asunto(s)
Familia de Proteínas EGF/análisis , Proteína Adaptadora GRB2/análisis , Nanotecnología , Neoplasias del Cuello Uterino/diagnóstico por imagen , Familia de Proteínas EGF/genética , Femenino , Células HeLa , Humanos , Microscopía Fluorescente , Tamaño de la Partícula , Unión Proteica , Células Tumorales CultivadasRESUMEN
The adapter protein growth factor receptor-bound 2 (GRB2) is essential for various basic cellular functions by mediating the regulation of receptor tyrosine kinase (RTK) signaling, however, little is known about GRB2 expression in esophageal squamous cell carcinoma (ESCC). We sought to characterize GRB2 expression and its relationship with clinicopathological parameters and prognostic significance in ESCC patients. Here, it was presented that GRB2 was overexpressed in cytoplasm in 58.1% (100/172) of ESCC cases by immunohistochemistry. Survival analysis demonstrated overexpression of GRB2 protein was significantly related to poor prognosis of ESCC patients (P = 0.021). Furthermore, overexpression of GRB2 was significantly associated with the lymph node metastases. In addition, subgroup analysis according to lymph node metastasis revealed a shorter disease-free survival (DFS) in the ESCC patients with GRB2 overexpression than the patients with GRB2 low-expression (Means for DFS months: 33.8 versus 52.1). Finally, the significant difference between overexpression of GRB2 and poor survival rates exhibited in univariate analysis (P = 0.022) and multivariate Cox analysis (close to significance, P = 0.065), demonstrated that GRB2 was an independent factor in prognosis of ESCC patients. In conclusion, GRB2 expression status could be as a positive biomarker of ESCC progression and lymph node metastasis.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Proteína Adaptadora GRB2/biosíntesis , Adulto , Anciano , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Supervivencia sin Enfermedad , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidad , Carcinoma de Células Escamosas de Esófago , Femenino , Proteína Adaptadora GRB2/análisis , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Regulación hacia ArribaRESUMEN
OBJECTIVE: The aims of this study were to investigate microRNA (miRNA) expression profiles in the periurethral vaginal wall tissues of postmenopausal women with and without stress urinary incontinence (SUI) and to explore the putative target genes associated with SUI via miRNA-messenger RNA (mRNA) pair prediction. METHODS: Periurethral vaginal wall tissues of postmenopausal women with SUI (n = 13) and matched continent postmenopausal women (n = 13) were collected during transvaginal surgical operation. Total RNAs were extracted and miRNAs were profiled by TaqMan Array Human MicroRNA assays in three case-control pairs. TargetScanS, PicTar, and miRanda were used to obtain the putative miRNA-mRNA pairs based on sequence data, and three pairs were predicated. The relative expression levels of miRNAs in predicated miRNA-mRNA pairs were quantified in 10 other case-control pairs by real-time polymerase chain reaction. The expression levels of mRNAs and corresponding proteins were estimated via real-time polymerase chain reaction and Western blot analysis. RESULTS: Twelve miRNAs were identified to be differentially expressed between two groups: the significantly up-regulated let-7a, miR-101#, miR-125b-2#, miR-190b, and miR-892b, and the down-regulated miR-124, miR-330-3p, miR-485-3p, miR-517b, miR-523, miR-589, and miR-93#. Moreover, three miRNA-mRNA pairs of interest were established via computational algorithms: miR-124 and growth factor receptor-bound protein 2; miR-330-3p and bicaudal D homolog 2; and miR-93# and signal transducer and activator of transcription 3. The expression levels of the three miRNAs were quantified, and a reduction in SUI was revealed. On the other hand, increased expression levels of predicated mRNAs and their protein products were detected. CONCLUSIONS: This study reports the differential expression of 12 miRNAs in SUI and predicates three miRNA-mRNA pairs. Interestingly, all three predicated target genes are associated with neurodegenerative conditions, indicating the potential significance of neurodegenerative mechanisms in the etiology of SUI.
Asunto(s)
Expresión Génica , MicroARNs/análisis , Posmenopausia , Uretra/química , Incontinencia Urinaria de Esfuerzo/genética , Vagina/química , Anciano , Algoritmos , China , Femenino , Proteína Adaptadora GRB2/análisis , Proteína Adaptadora GRB2/genética , Humanos , MicroARNs/genética , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Enfermedades Neurodegenerativas/genética , ARN Mensajero/análisis , Factor de Transcripción STAT3/análisis , Factor de Transcripción STAT3/genética , Análisis de Matrices TisularesRESUMEN
Aquaporin-4 (AQP4) is a water channel protein mainly located in the astroglial plasma membrane, the precise function of which in the brain edema that accompanies hepatic encephalopathy (HE) is unclear. Since ammonia is the main pathogenic agent in HE, its effect on AQP4 expression and distribution in confluent primary astroglial cultures was examined via their exposure to ammonium chloride (1, 3 and 5 mM) for 5 and 10 days. Ammonia induced the general inhibition of AQP4 mRNA synthesis except in the 1 mM/5 day treatment. However, the AQP4 protein content measured was dependent on the method of analysis; an apparent increase was recorded in treated cells in in-cell Western assays, while an apparent reduction was seen with the classic Western blot method, perhaps due to differences in AQP4 aggregation. Ammonia might therefore induce the formation of insoluble AQP4 aggregates in the astroglial plasma membrane. The finding of AQP4 in the pellet of classic Western blot samples, plus data obtained via confocal microscopy, atomic force microscopy (using immunolabeled cells with gold nanoparticles) and scanning electron microscopy, all corroborate this hypothesis. The effect of ammonia on AQP4 seems not to be due to any osmotic effect; identical osmotic stress induced by glutamine and salt had no significant effect on the AQP4 content. AQP4 functional analysis (subjecting astrocytes to a hypo-osmotic medium and using flow cytometry to measure cell size) demonstrated a smaller water influx in ammonia-treated astrocytes suggesting that AQP4 aggregates are representative of an inactive status; however, more confirmatory studies are required to fully understand the functional status of AQP4 aggregates. The present results suggest that ammonia affects AQP4 expression and distribution, and that astrocytes change their expression of AQP4 mRNA as well as the aggregation status of the ensuing protein depending on the ammonia concentration and duration of exposure.
Asunto(s)
Amoníaco/farmacología , Acuaporina 4/metabolismo , Astrocitos/metabolismo , Membrana Celular/química , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Acuaporina 4/química , Acuaporina 4/genética , Astrocitos/ultraestructura , Edema Encefálico/etiología , Edema Encefálico/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Medios de Cultivo Condicionados/química , Citometría de Flujo , Proteína Adaptadora GRB2/análisis , Glutamina/farmacología , Encefalopatía Hepática/complicaciones , Encefalopatía Hepática/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Cloruro de Sodio/farmacología , Solubilidad , Taurina/análisis , alfa-Sinucleína/análisisRESUMEN
ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor-bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.
Asunto(s)
Proteínas ADAM/metabolismo , Clatrina/metabolismo , Endocitosis , Proteína Adaptadora GRB2/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/análisis , Proteínas ADAM/química , Proteína ADAM12 , Neoplasias de la Mama/química , Neoplasias de la Mama/enzimología , Carcinoma/química , Endosomas/metabolismo , Femenino , Proteína Adaptadora GRB2/análisis , Células HEK293 , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Dominios Proteicos Ricos en Prolina , Dominios y Motivos de Interacción de ProteínasRESUMEN
Adaptor proteins for the various growth factor receptors play a crucial role in signal transduction through tyrosine phosphorylation. Several candidates for adaptor proteins with potential effects on the epidermal growth factor (EGF) receptor-mediated signaling pathway have been identified by recent phosphoproteomic studies. Here, we focus on a novel protein, GAREM (Grb2-associated and regulator of Erk/MAPK) as a downstream molecule of the EGF receptor. GAREM is phosphorylated at tyrosine 105 and 453 after EGF stimulation. Grb2 was identified as its binding partner, and the proline-rich motifs of GAREM are recognized by the N- and C-terminal SH3 domains of Grb2. In addition, the tyrosine phosphorylations of GAREM are necessary for its binding to Grb2. Because the amino acid sequence surrounding tyrosine 453 is similar to the immunoreceptor tyrosine-based inhibitory motif, Shp2, a positive regulator of Erk, binds to GAREM in this phosphorylation-dependent manner. Consequently, Erk activation in response to EGF stimulation is regulated by the expression of GAREM in COS-7 and HeLa cells, which occurs independent of the presence of other binding proteins, such as Gab1 and SOS, to the activated EGF receptor. Furthermore, the expression of GAREM has an effect on the transformation activity of cultured cells. Together, these findings suggest that GAREM plays a key role in the ligand-mediated signaling pathway of the EGF receptor and the tumorigenesis of cells.
Asunto(s)
Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Proteína Adaptadora GRB2/análisis , Proteína Adaptadora GRB2/genética , Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosforilación , Prolina/química , Prolina/metabolismo , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
Stable isotope labeling by amino acids in cell culture (SILAC) has become a popular labeling strategy for peptide quantitation in proteomics experiments. If the SILAC technology could be extended to intact proteins, it would enable direct quantitation of their relative expression levels and of the degree of modification between different samples. Here we show through modeling and experiments that SILAC is suitable for intact protein quantitation and top-down characterization. When SILAC-labeling lysine and/or arginine, peaks of light and heavy SILAC-doublets do not interfere with peaks of different charge states at least between 10 and 200 kDa. Unlike chemical methods, SILAC ensures complete incorporation-all amino acids are labeled. The isotopic enrichment of commercially available SILAC amino acids of nominally 95% to 98% shifts the mass difference between light and heavy state but does not lead to appreciably broadened peaks. We expressed labeled and unlabeled Grb2, a 28 kDa signaling protein, and showed that the two forms can be quantified with an average standard deviation of 6%. We performed on-line top-down sequencing of both forms in a hybrid linear ion trap orbitrap instrument. The quantized mass offset between fragments provided information about the number of labeled residues in the fragments, thereby simplifying protein identification and characterization.
Asunto(s)
Marcaje Isotópico/métodos , Proteínas/análisis , Proteínas/química , Secuencia de Aminoácidos , Escherichia coli/metabolismo , Proteína Adaptadora GRB2/análisis , Proteína Adaptadora GRB2/metabolismo , Datos de Secuencia Molecular , Mapeo Peptídico , ProteómicaRESUMEN
We have reported that oridonin, a diterpenoid isolated from the plant Rabdosia rubescens, had apoptosis-inducing activities in many cell lines (e.g., human melanoma A375-S2, human cervical cancer HeLa, human breast adenocarcinoma MCF-7, and murine fibrosarcoma L929). In this study, we further investigated signaling events involved in oridonin-induced apoptosis in human epidermoid carcinoma A431 cells. It was found that the total tyrosine kinase activity was inhibited and the protein expressions of epidermal growth factor receptor (EGFR) and phosphorylated EGFR were decreased in oridonin-induced A431 cell apoptosis. Expression of EGFR downstream effector proteins, Grb2, Ras, Raf-1, and extracellular signal-regulated kinase (ERK), was also downregulated by oridonin. Moreover, the oridonin-induced apoptosis was augmented by the Ras inhibitor manumycin A, Raf-1 inhibitor GW5074, or ERK inhibitor PD98059, suggesting that inactivation of Ras, Raf, or ERK participates in oridonin-induced apoptosis. Taken together, oridonin-induced apoptosis in A431 cells might be through blocking EGFR and its downstream Ras/Raf/ERK signal pathway.