RESUMEN
Trigger factor (TF) is an important catalyst of nascent peptide folding and possesses both peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. TF has a modular structure, containing three domains with distinct structural and functional properties. The guanidine hydrochloride (GuHCl) induced unfolding of TF was investigated by monitoring Trp fluorescence, far-UV CD, second-derivative UV absorption, enzymatic and chaperone activities, chemical crosslinking and binding of the hydrophobic dye, 1-anilinonaphthalene-8-sulfonate (ANS); and was compared to the urea induced unfolding. The native state of TF was found to bind ANS in 1:1 stoichiometry with a K(d) of 84 microM. A native-like state, N', is stable around 0.5 M GuHCl, and shows increased ANS binding, while retaining PPIase activity and most secondary and tertiary structure, but loses chaperone and dimerization activities, consistent with slight conformational rearrangement. A compact denatured state, I, is populated around 1.0 M GuHCl, is inactive and does not show significant binding to ANS. The data suggest that TF unfolds in a stepwise manner, consistent with its modular structure. The ability of TF to undergo structural rearrangement to maintain enzymatic activity while reducing chaperone and dimerization abilities may be related to the physiological function of TF.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/efectos de los fármacos , Guanidina/farmacología , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/efectos de los fármacos , Naftalenosulfonatos de Anilina , Dicroismo Circular , Reactivos de Enlaces Cruzados , Dimerización , Electroforesis en Gel de Poliacrilamida , Chaperonas Moleculares , Muramidasa/química , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína/efectos de los fármacos , Espectrometría de Fluorescencia , Succinimidas , TermodinámicaRESUMEN
Cyclooxygenase-2 (COX-2) plays an important role in the carcinogenesis and progression of gastric cancer. It has been demonstrated that COX-2 overexpression depends on different cellular pathways, involving both transcriptional and post-transcriptional regulation. MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators. Here, we characterize miR-101 expression and its role in the regulation of COX-2 expression, which in turn, will provide us with additional insights into the potential therapeutic benefits of exogenous miR-101 for treatment of gastric cancer. Our results showed that miR-101 levels in gastric cancer tissues were significantly lower than those in the matched normal tissue (P < 0.01). Furthermore, lower levels of miR-101 were associated with increased tumor invasion and lymph node metastasis (P < 0.05). We also found an inverse correlation between miR-101 and COX-2 expression in both gastric cancer specimens and cell lines. Significant decreases in COX-2 mRNA and COX-2 levels were observed in the pre-miR-101-infected gastric cancer cells. One possible mechanism of interaction is that miR-101 inhibited COX-2 expression by directly binding to the 3'-UTR of COX-2 mRNA. Overexpression of miR-101 in gastric cancer cell lines also inhibited cell proliferation and induced apoptosis in vitro, as well as inhibiting tumor growth in vivo. These results collectively indicate that miR-101 may function as a tumor suppressor in gastric cancer, with COX-2 as a direct target.
Asunto(s)
Apoptosis , Ciclooxigenasa 2/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Regiones no Traducidas 3' , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patología , Animales , Secuencia de Bases , Western Blotting , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Carcinoma de Células en Anillo de Sello/genética , Carcinoma de Células en Anillo de Sello/metabolismo , Carcinoma de Células en Anillo de Sello/patología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Ciclooxigenasa 2/genética , Femenino , Humanos , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Neoplasias Gástricas/genéticaRESUMEN
Reduced denatured lysozyme tends to aggregate at neutral pH and competition between productive folding and aggregation substantially reduces the efficiency of refolding. Trigger factor, a folding catalyst and chaperone can, depending on the concentration of trigger factor and the solution conditions, cause either a substantial increase (chaperone activity) or a substantial decrease (antichaperone activity) in the recovery of native lysozyme as compared with spontaneous refolding. When trigger factor is working as a chaperone, the reactivation rates of lysozyme are decelerated and aggregation decreases with increasing trigger factor concentrations. Under conditions where antichaperone activity of trigger factor dominates, the reactivation rates of lysozyme are accelerated and aggregation is increased. Trigger factor and lysozyme were both released from the aggregates on re-solubilization with urea indicating that trigger factor participates directly in aggregate formation and is incorporated into the aggregates. The apparently dual effect of trigger factor toward refolding of lysozyme is a consequence of the peptide binding ability and may be important in regulation of protein biosynthesis.