RESUMEN
Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (SclN93A), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that SclN93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of SclN93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (SclN93AFc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Osteogénesis/efectos de los fármacos , Proteínas Recombinantes/farmacología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Células Cultivadas , Femenino , Células HEK293 , Humanos , Proteínas Relacionadas con Receptor de LDL/antagonistas & inhibidores , Proteínas Relacionadas con Receptor de LDL/química , Proteínas Relacionadas con Receptor de LDL/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/química , Proteínas Mutantes/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Osteogénesis/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/genética , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes/químicaRESUMEN
The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.
Asunto(s)
Unión Competitiva/fisiología , Neoplasias Encefálicas/genética , Glioblastoma/genética , Factores de Transcripción SOXB1/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Dominio B30.2-SPRY , Unión Competitiva/genética , Femenino , Técnicas de Silenciamiento del Gen , Glioblastoma/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteómica , Factores de Transcripción SOXB1/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The aims of this study was to investigate the influences of hsa_circ_0056558/miR-1290/CDK6 axis in ankylosing spondylitis (AS). The differentially expressed has_circ_0056558 and miR-1290 in AS tissue were analysed based on RNA-seq data and microarray data, respectively. qRT-PCR was performed for detection of relative expression levels of hsa_circ_0056558, miR-1290, CDK6, osteogenic differentiation markers (Runx2 and Osterix) and other inflammatory factors (TNF-α, IL-1ß, and IL-6). Western blotting analysis was conducted to test the protein levels of CDK6, osteogenic differentiation markers (Runx2 and Osterix), and PI3K/AKT/NF-κB pathway-associated proteins. CCK8 assay and flow cytometry were conducted to determine cell proliferation and cell apoptotic ability, respectively. Targeted relationships were predicted by bioinformatic analysis and verified by dual-luciferase reporter assay. The differentiation of fibroblast cells was analysed by alkaline phosphatase (ALP) activity assay. Our findings revealed that the expression levels of both circ_0056558 and CDK6 in AS tissue were significantly higher than that in normal samples. Besides, hsa_circ_0056558 could suppress cell proliferation and differentiation by facilitating CDK6 expression and suppressing miR-1290 expression in AS. Over-expression of miR-1290 negatively regulated CDK6 expression to enhance cell proliferation. The protein levels of p-AKT, p-NF-κB p65, and p-IκBα were promoted by hsa_circ_0056558 or CDK6 over-expression while suppressed by miR-1290 up-regulation. In conclusion, our study demonstrated that hsa_circ_0056558 and CDK6 suppressed cell proliferation and differentiation while enhanced cell apoptosis by competitive binding to miR-1290 in AS, which might be possibly achieved by PI3K/AKT/NF-κB pathway, providing us novel therapeutic strategy for AS.
Asunto(s)
Quinasa 6 Dependiente de la Ciclina/genética , MicroARNs/metabolismo , ARN Circular/metabolismo , Espondilitis Anquilosante/genética , Apoptosis/genética , Apoptosis/inmunología , Unión Competitiva/genética , Unión Competitiva/inmunología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Biología Computacional , Fibroblastos , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/agonistas , MicroARNs/antagonistas & inhibidores , ARN Circular/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Espondilitis Anquilosante/inmunología , Espondilitis Anquilosante/patologíaRESUMEN
BACKGROUND: Long intergenic non-protein coding RNA00665 (LINC00665) plays a crucial tumorigenic role in many cancers, such as gastric cancer and lung adenocarcinoma. However, its role and mechanism of action in the progression of breast cancer (BC) are unknown. METHODS: LINC00665 expression levels were determined using quantitative polymerase chain reaction analysis with BC tissues and cell lines. BC cell proliferation was tested by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, whereas BC cell migration and invasion capabilities were analyzed by performing transwell migration assays. Percentages of apoptotic cells were measured by flow cytometry. Interactions between LINC00665 and miR-3169-5p were examined by performing luciferase reporter assays, and the expression levels of proteins, such as ß-catenin, were examined by western blot analysis. RESULTS: LINC00665 was expressed at high levels in BC tissues and cells. Upregulated LINC00665 expression correlated with tumor size and tumor, node, and metastasis stages, but not with the age of patients. LINC00665 knockdown inhibited BC cell proliferation, migration, and invasion, whereas it promoted apoptosis. Moreover, bioinformatics analysis and the luciferase reporter assay revealed that LINC00665 bound the microRNA (miR) miR-3619-5p. miR-3619-5p expression correlated negatively with LINC00665 expression in BC tissues. miR-3619-5p overexpression inhibited BC cell proliferation, migration, and invasion, but promoted apoptosis. Simultaneous knockdown of LINC00665 and miR-3619-5p led to increased cell proliferation, migration, and invasion, and inhibited apoptosis. Additionally, catenin beta 1, which encodes the ß-catenin protein, was the target gene of miR-3619-5p. ß-catenin expression clearly decreased after LINC00665 knockdown and miR-3619-5p overexpression, but increased after simultaneous knockdown of LINC00665 and miR-3619-5p. CONCLUSION: LINC00665 knockdown inhibited BC cell proliferation and invasion by binding miR-3619-5p and inhibiting ß-catenin expression.
Asunto(s)
Neoplasias de la Mama/genética , Proliferación Celular/genética , MicroARNs/genética , ARN Largo no Codificante/genética , beta Catenina/genética , Apoptosis/genética , Unión Competitiva/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Células MCF-7 , Regulación hacia Arriba/genéticaRESUMEN
Circular RNAs are a class of endogenous noncoding RNAs that play an important role in gene regulation. These RNAs are involved in the development and progression of various cancers, but their roles in gastric cancer have not yet been thoroughly elucidated. This study showed that hsa_circ_0000467 expression was higher in gastric cancer tissues than in corresponding adjacent tissues (P < 0.050) and that hsa_circ_0000467 expression levels were correlated with gastric cancer histological grade (P < 0.050). In addition, hsa_circ_0000467 was remarkably upregulated in gastric cancer cell lines (P < 0.001). Cell function experiments indicated that hsa_circ_0000467 downregulation decreased the proliferation and invasion ability of BGC-823 and SGC-7901 cells and the number of cells entering the G2/M phase. A direct binding interaction was detected between hsa_circ_0000467 and miR-326-3p by dual-luciferase reporter assays. In addition, the results showed that inhibition of miR-326-3p reversed the decreases in the proliferation and invasion of BGC-823 and SGC-7901 cells caused by hsa_circ_0000647 downregulation. Inhibition of miR-326-3p also decreased the number of cells entering the G2/M phase and the expression of cyclin D1. In conclusion, hsa_circ_0000467 plays a regulatory role in the development and progression of gastric cancer by regulating miR-326-3p, and this circRNA may be a potential diagnostic marker and therapeutic target of gastric cancer.
Asunto(s)
Unión Competitiva/genética , Carcinogénesis/genética , Carcinogénesis/patología , MicroARNs/metabolismo , ARN Circular/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Anciano , Sitios de Unión/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Masculino , MicroARNs/genética , Invasividad Neoplásica , ARN Circular/genética , Regulación hacia ArribaRESUMEN
We aimed to assess the roles of small nucleolar RNA host gene 6 (SNHG6) in hepatocellular carcinoma (HCC) progression, and establish the lncRNA-miRNA-mRNA regulation mechanism for HCC therapy. SNHG6 is one of the host genes in small nucleolar RNAs (snoRNAs), which make a difference in the development of human cancers. SERPINH1 is a gene encoding a member of the serpin superfamily of serine proteinase inhibitors with miRNA predicted by TargetScan and DIANA Tools. SNHG6, serpin family H member 1 (SERPINH1) and miR-139-5p expression levels in HCC tissues and cells were determined by quantitative real-time PCR (qRT-PCR). Migration and invasion of HCC cells were measured by transwell assay. Cell cycle analysis was determined by using flow cytometry. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay were performed for cell viability analysis. The expression of SERPINH1 was detected by qRT-PCR and western blot. Dual-luciferase reporter gene assay was conducted to identify the targeted relationship between miR-139-5p and SNHG6, as well as SERPINH1 and miR-139-5p. The positive regulation between SNHG6 and SERPINH1 was demonstrated in this study. In contrast, miR-139-5p was significantly down-regulated in HCC cells, the inhibition of miR-139-5p promotes the proliferation of HCC cells, and accelerated the cell cycle of HCC cells. Our study demonstrated the co-expression of SNHG6 and SERPINH1 in HCC cells for the first time, which revealed that SNHG6 could serve as a novel oncogene for the HCC therapy by its regulation.
Asunto(s)
Unión Competitiva/genética , Carcinoma Hepatocelular/metabolismo , Progresión de la Enfermedad , Proteínas del Choque Térmico HSP47/metabolismo , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Regulación hacia Arriba/genética , Animales , Carcinoma Hepatocelular/patología , Línea Celular , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Proteínas del Choque Térmico HSP47/genética , Células Hep G2 , Hepatocitos/metabolismo , Xenoinjertos , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , ARN Largo no Codificante/genética , TransfecciónRESUMEN
Integrin ß3 (ITGB3), which is the target gene of the miRNA let-7 that can be antagonized by long noncoding RNA (lncRNA) H19, is well known to have a critical role in endometrium receptivity. However, the regulation of ITGB3 in cell-cell or cell-extracellular matrix adhesion and invasion for the maintenance of early pregnancy remains unknown. This study aimed to explore the role of the H19/let-7/ITGB3 axis in regulating trophoblastic spheroid adhesion and in vitro invasion ability using the HTR-8/SVneo cell line and to investigate the expression levels of lncRNA H19 and ITGB3 in human products of conception. The in vitro knockdown of H19 resulted in decreased expression of ITGB3 at the mRNA and protein levels and reduced the adhesion and invasion ability. In the embryonic chorion tissue of spontaneous abortion (SA), the expressions of H19 and ITGB3 at both the mRNA and protein levels decreased. The results of quantitative RT-PCR, Western blot analysis, dual-luciferase report gene and functional miRNA let-7 rescue experiments, adhesion assay and in vitro transwell invasion assay confirmed that H19 regulated trophoblastic spheroid adhesion with endometrial stromal cells through the H19/let-7/ITGB3 axis, thereby providing an improved understanding of the molecular mechanism of SA.
Asunto(s)
Adhesión Celular/genética , MicroARNs/metabolismo , ARN Largo no Codificante/fisiología , Esferoides Celulares/fisiología , Trofoblastos/fisiología , Aborto Espontáneo/genética , Aborto Espontáneo/metabolismo , Aborto Espontáneo/patología , Adulto , Unión Competitiva/genética , Estudios de Casos y Controles , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , MicroARNs/genética , Embarazo , Trofoblastos/patología , Adulto JovenRESUMEN
Crystal structures can identify ligand-receptor interactions and assist the development of novel therapeutics, but experimental challenges sometimes necessitate the use of homologous proteins. Tropisetron is an orthosteric ligand at both 5-HT3 and α7 nACh receptors and its binding orientation has been determined in the structural homologue AChBP (pdbid: 2WNC). Co-crystallisation with a structurally-related ligand, granisetron, reveals an almost identical orientation (pdbid; 2YME). However, there is a >1000-fold difference in the affinity of tropisetron at 5-HT3 versus α7 nACh receptors, and α7 nACh receptors do not bind granisetron. These striking pharmacological differences prompt questions about which receptor the crystal structures most closely represent and whether the ligand orientations are correct. Here we probe the binding orientation of tropisetron and granisetron at 5-HT3 receptors by in silico modelling and docking, radioligand binding on cysteine-substituted 5-HT3 receptor mutants transiently expressed in HEK 293 cells, and synthetic modification of the ligands. For 15 of the 23 cysteine substitutions, the effects on tropisetron and granisetron were different. Structure-activity relationships on synthesised derivatives of both ligands were also consistent with different orientations, revealing that contrary to the crystallographic evidence from AChBP, the two ligands adopt different orientations in the 5-HT3 receptor binding site. Our results show that even quite structurally similar molecules can adopt different orientations in the same binding site, and that caution may be needed when using homologous proteins to predict ligand binding.
Asunto(s)
Unión Competitiva/efectos de los fármacos , Modelos Moleculares , Receptores de Serotonina 5-HT3/química , Receptores de Serotonina 5-HT3/genética , Sitios de Unión/efectos de los fármacos , Unión Competitiva/genética , Cristalografía por Rayos X , Granisetrón/química , Granisetrón/farmacología , Células HEK293 , Humanos , Indoles/química , Indoles/farmacología , Ligandos , Estructura Molecular , Mutación/genética , Antagonistas de la Serotonina/farmacología , Relación Estructura-Actividad , Tritio/farmacología , TropisetrónRESUMEN
The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development. Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase. The hormone-dependent formation of the COI1-JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.
Asunto(s)
Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transducción de Señal , Transactivadores/antagonistas & inhibidores , Transactivadores/química , Secuencias de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Unión Competitiva/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN , Modelos Moleculares , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/genética , Conformación Proteica , Proteínas Represoras/genética , Transactivadores/genética , Transactivadores/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. METHODS: Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. RESULTS: Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. CONCLUSIONS: Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. GENERAL SIGNIFICANCE: Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins.
Asunto(s)
Acetilgalactosamina/metabolismo , Galactosa/metabolismo , Lectinas Tipo C/metabolismo , Manosa/metabolismo , Acetilgalactosamina/química , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Unión Competitiva/genética , Calorimetría/métodos , Cromatografía de Afinidad , Dicroismo Circular , Cristalografía por Rayos X , Cucumaria/genética , Cucumaria/metabolismo , Galactosa/química , Lectinas Tipo C/química , Lectinas Tipo C/genética , Manosa/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica/genética , Ingeniería de Proteínas/métodos , Estructura Terciaria de ProteínaRESUMEN
Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [³H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious 'mixing' experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among five pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [³H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer. The dopamine transporter (DAT) can exist as an oligomer but it is unknown whether the protomers function independently. The present results indicate that protomers that are superpotent or deficient in cocaine analog binding can confer enhanced or reduced potency to the oligomer, respectively. In this respect, positive or negative cooperativity is revealed in the DAT oligomer.
Asunto(s)
Cocaína/análogos & derivados , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/farmacocinética , Subunidades de Proteína/metabolismo , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Biotinilación , Cocaína/farmacocinética , Simulación por Computador , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Mutación/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Subunidades de Proteína/genética , Análisis de Regresión , Relación Estructura-Actividad , Transfección , Tritio/farmacocinéticaRESUMEN
Post-transcriptional gene regulation (PTGR) of mRNA turnover, localization and translation is mediated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). These regulators exert their effects by binding to specific sequences within their target mRNAs. Increasing evidence suggests that competition for binding is a fundamental principle of PTGR. Not only can miRNAs be sequestered and neutralized by the targets with which they interact through a process termed 'sponging', but competition between binding sites on different RNAs may also lead to regulatory crosstalk between transcripts. Here, we quantitatively model competition effects under physiological conditions and review the role of endogenous sponges for PTGR in light of the key features that emerge.
Asunto(s)
Unión Competitiva/fisiología , Epigénesis Genética/fisiología , MicroARNs/metabolismo , Modelos Biológicos , Interferencia de ARN/fisiología , ARN Mensajero/metabolismo , Transcriptoma/genética , Unión Competitiva/genéticaRESUMEN
En el marco de la creciente feminización de la profesión médica en México, el artículo indaga sobre las características de este proceso para el caso de la ginecobstetricia. Considerando la feminización como un proceso de cambio, que se analiza cuantitativa y cualitativamente, el artículo se detiene en especial en las experiencias de las mujeres ginecobstetras, experiencias que se dan en el seno de una especialidad que, desde sus orígenes, funcionó como un dispositivo de control del cuerpo de las mujeres. Basado en una investigación etnográfica, el artículo combina fuentes estadísticas, de archivo y de observación de campo. El material que surge de las entrevistas muestra las experiencias y tensiones que viven las ginecobstetras en este contexto.
In the framework of an increasing feminization of the medical profession in Mexico, this article explores the characteristics of this process in the obstetrics and gynecology specialty. Understanding feminization as a process of change to be analyzed both quantitatively and qualitatively, the article focuses special attention on the experiences of female obstetrician-gynecologists within a medical specialty that has since its origins functioned as a mechanism of control over women's bodies. Based on ethnographic research, the article combines statistical and archival sources and field observation. The interviews reveal the experiences and tensions women obstetrician-gynecologists encounter in this context.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Arginina/química , Pseudomonas putida/enzimología , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/aislamiento & purificación , Unión Competitiva/genética , Catálisis , Activación Enzimática/genética , Mononucleótido de Flavina/metabolismo , Cinética , Ligandos , Ácidos Mandélicos/metabolismo , Mutagénesis Sitio-Dirigida , Fenilacetatos/metabolismo , Unión Proteica/genética , Pseudomonas putida/genética , Especificidad por Sustrato/genética , Sulfitos/metabolismoRESUMEN
Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for cellobiohydrolases remains controversial, and recent reports have alternately suggested either association (on-rate) or dissociation (off-rate) as the overall bottleneck. Obviously, this uncertainty hampers both fundamental mechanistic understanding and rational design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens complex formation, and the population of substrate-bound W38A was only about half of the wild type. Nevertheless, the maximal, steady-state rate was twice as high for the variant enzyme. It is argued that these opposite effects on binding and activity can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process).
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/genética , Proteínas Fúngicas/genética , Mutación , Trichoderma/genética , Alanina/química , Alanina/genética , Alanina/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Unión Competitiva/genética , Biocatálisis , Dominio Catalítico/genética , Celobiosa/metabolismo , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/química , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Cinética , Modelos Moleculares , Unión Proteica/genética , Especificidad por Sustrato , Termodinámica , Trichoderma/enzimología , Trichoderma/metabolismo , Triptófano/química , Triptófano/genética , Triptófano/metabolismoRESUMEN
Insect chemosensory proteins (CSPs) are proposed to capture and transport hydrophobic chemicals across the sensillum lymph to olfactory receptors (ORs), but this has not been clarified in moths. In this study, we built on our previously reported segment sequence work and cloned the full length CSP19 gene (SinfCSP19) from the antennae of Sesamia inferens by using rapid amplification of cDNA ends. Quantitative real time-PCR (qPCR) assays indicated that the gene was expressed in a unique profile, i.e. predominant in antennae and significantly higher in male than in female. To explore the function, recombinant SinfCSP19 was expressed in Escherichia coli cells and purified by Ni-ion affinity chromatography. Binding affinities of the recombinant SinfCSP19 with 39 plant volatiles, 3 sex pheromone components and 10 pheromone analogs were measured using fluorescent competitive binding assays. The results showed that 6 plant volatiles displayed high binding affinities to SinfCSP19 (Ki = 2.12-8.75 µM), and more interesting, the 3 sex pheromone components and analogs showed even higher binding to SinfCSP19 (Ki = 0.49-1.78 µM). Those results suggest that SinfCSP19 plays a role in reception of female sex pheromones of S. inferens and host plant volatiles.
Asunto(s)
Antenas de Artrópodos/metabolismo , Unión Competitiva/genética , Proteínas de Insectos/metabolismo , Mariposas Nocturnas/metabolismo , Unión Proteica/genética , Atractivos Sexuales/metabolismo , Compuestos Orgánicos Volátiles/metabolismo , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , Femenino , Proteínas de Insectos/genética , Masculino , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Filogenia , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Alineación de Secuencia , Atractivos Sexuales/genéticaRESUMEN
Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3±7.2%) or plasma membrane (67.3±7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of Gq-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 ± 1.3% reduction in FRET ratios following ligand binding, indicating activation of Gq-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.
Asunto(s)
Hormona Liberadora de Gonadotropina/deficiencia , Mutación Missense , Receptores de Neuroquinina-3/genética , Transducción de Señal/genética , Animales , Sitios de Unión/genética , Unión Competitiva/genética , Western Blotting , Células COS , Membrana Celular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Neuroquinina B/genética , Neuroquinina B/metabolismo , Fosforilación , Multimerización de Proteína , Receptores de Neuroquinina-3/química , Receptores de Neuroquinina-3/metabolismoRESUMEN
This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-ß was examined to determine whether levels of the TGF-ß binding AHSG influenced the effect of TGF-ß on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-ß influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis.
Asunto(s)
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeza y Cuello/genética , alfa-2-Glicoproteína-HS/fisiología , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Carcinogénesis/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Carcinoma de Células Escamosas de Cabeza y Cuello , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/metabolismo , alfa-2-Glicoproteína-HS/antagonistas & inhibidoresRESUMEN
It has been shown recently that PrP (prion protein) and the calcium channel auxiliary α2δ subunits interact in neurons and expression systems [Senatore, Colleoni, Verderio, Restelli, Morini, Condliffe, Bertani, Mantovani, Canovi, Micotti, Forloni, Dolphin, Matteoli, Gobbi and Chiesa (2012) Neuron 74, 300-313]. In the present study we examined whether there was an effect of PrP on calcium currents. We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/ß and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density. This reduction was absent when a PrP construct was used lacking its GPI (glycosylphosphatidylinositol) anchor. We have reported previously that α2δ subunits are able to form GPI-anchored proteins [Davies, Kadurin, Alvarez-Laviada, Douglas, Nieto-Rostro, Bauer, Pratt and Dolphin (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1654-1659] and show further evidence in the present paper. We have characterized recently a C-terminally truncated α2δ-1 construct, α2δ-1ΔC, and found that, despite loss of its membrane anchor, it still shows a partial ability to increase calcium currents [Kadurin, Alvarez-Laviada, Ng, Walker-Gray, D'Arco, Fadel, Pratt and Dolphin (2012) J. Biol. Chem. 1287, 33554-33566]. We now find that PrP does not inhibit CaV2.1/ß currents formed with α2δ-1ΔC, rather than α2δ-1. It is possible that PrP and α2δ-1 compete for GPI-anchor intermediates or trafficking pathways, or that interaction between PrP and α2δ-1 requires association in cholesterol-rich membrane microdomains. Our additional finding that CaV2.1/ß1b/α2δ-1 currents were inhibited by GPI-GFP, but not cytosolic GFP, indicates that competition for limited GPI-anchor intermediates or trafficking pathways may be involved in PrP suppression of α2δ subunit function.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Canales de Calcio/metabolismo , Regulación de la Expresión Génica , Glicosilfosfatidilinositoles/metabolismo , Priones/biosíntesis , Animales , Unión Competitiva/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica/fisiología , Transporte de Proteínas/genética , Ratas , Transducción de Señal/genética , XenopusRESUMEN
Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide. As the majority of patients present with invasive, metastatic disease, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA) for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-ß co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-ß signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.
Asunto(s)
Progresión de la Enfermedad , Proteína HMGA2/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Animales , Proteínas Argonautas/metabolismo , Unión Competitiva/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Proteoglicanos/biosíntesis , Proteoglicanos/deficiencia , Proteoglicanos/genética , Isoformas de ARN/genética , Isoformas de ARN/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/genética , Transcripción Genética/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic ß cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.