RESUMEN
SIRT1 is the closest mammalian homologue of yeast SIR2, an important ageing regulator that prolongs lifespan in response to caloric restriction. Despite its importance, the mechanisms that regulate SIRT1 activity are unclear. Our study identifies a novel post-translational modification of SIRT1, namely sumoylation at Lys 734. In vitro sumoylation of SIRT1 increased its deacetylase activity. Conversely, mutation of SIRT1 at Lys 734 or desumoylation by SENP1, a nuclear desumoylase, reduced its deacetylase activity. Stress-inducing agents promoted the association of SIRT1 with SENP1 and cells depleted of SENP1 (but not of SENP1 and SIRT1) were more resistant to stress-induced apoptosis than control cells. We suggest that stress-inducing agents counteract the anti-apoptotic activity of SIRT1 by recruiting SENP1 to SIRT1, which results in the desumoylation and inactivation of SIRT1 and the consequent acetylation and activation of apoptotic proteins.
Asunto(s)
Acetilesterasa/efectos de los fármacos , Daño del ADN , Procesamiento Proteico-Postraduccional , Sirtuinas/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Acetilación , Animales , Apoptosis , Línea Celular , Cisteína Endopeptidasas , Endopeptidasas/metabolismo , Humanos , Proteínas/metabolismo , Sirtuina 1 , Sirtuinas/genética , Sirtuinas/farmacología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
A horizontal clinostat which mimics the microgravity in space was used to study its effects on carrot cells. After using boric acid buffer (pH 8.8) as extraction medium, PAGE pattern of esterase isozymes of carrot callus cells displayed 8 bands of which the activities of only 2 were affected by microgravity. They were named grEST1 and grEST2, where grEST stands for gravity-related esterase. The rates of increase in activity of them in carrot cells when cultured on a rotating horizontal clinostat were lower than that cultured in normal gravitational environments, and the difference increased with the culture time. The activities of grEST1 and grEST2 in carrot callus cells subjected to horizontal rotation were found to return to their original levels after being placed under normal gravity (1 x g) for 7 days. We suggest that the effect of simulated microgravity conditions on grEST1 and grEST2 activities in carrot callus cells is through affecting their synthesis. In addition, the activities of grEST1 and grEST2 were not inhibited by eserine, acetylcholine iodide, diisopropyl fluorophosphate and p-chloromercuribenzoate, which indicates that they are acetylesterases.
Asunto(s)
Daucus carota/citología , Daucus carota/enzimología , Esterasas/aislamiento & purificación , Simulación de Ingravidez , Acetilcolina/farmacología , Acetilesterasa/efectos de los fármacos , Acetilesterasa/aislamiento & purificación , Acetilesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Daucus carota/efectos de los fármacos , Esterasas/efectos de los fármacos , Esterasas/metabolismo , Gravitación , Isoflurofato/farmacología , Fisostigmina/farmacología , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Rotación , Ácido p-Cloromercuribenzoico/farmacologíaRESUMEN
The influenza C glycoprotein HEF was analyzed for acetylesterase activity after SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes. Using a histological esterase assay, the glycoprotein was detected as a colored band indicating that it is enzymatically active. The enzyme activity was not affected by low pH, but was abolished after denaturation by SDS as well as after breaking the disulfide bonds by reducing agents. Glycoprotein inactivated by SDS regained its enzyme activity if the ionic detergent was displaced by either bovine serum albumin or a nonionic detergent. The stability of the enzyme combined with the color assay provides a convenient tool to study the acetylesterase activity of the influenza C virus glycoprotein.
Asunto(s)
Acetilesterasa/análisis , Gammainfluenzavirus/enzimología , Proteínas del Envoltorio Viral/química , Acetilesterasa/efectos de los fármacos , Acetilesterasa/metabolismo , Colodión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Concentración de Iones de Hidrógeno , Desnaturalización Proteica , Albúmina Sérica Bovina/farmacologíaRESUMEN
Besides the well established role of low density lipoproteins (LDL), the phospholipid PAF-acether (paf) seems to be involved in atherogenesis. The effect of LDL (10 micrograms/ml for 24 h, n = 3) on paf binding characteristics of monocyte/macrophage-like U 937 cells was investigated using the radioligand [3H]paf, unlabeled paf and the paf receptor antagonist WEB 2086. The specific [3H]paf binding significantly increased at 1.4 nM (P less than 0.02) and 2.8 nM (P less than 0.01) added [3H]paf with an increased number of paf binding sites in the Scatchard plot analysis of the data. Specific paf binding was functionally active since paf mediated a cellular [Ca2+]i rise. The protein kinase C (PKC) activator PMA (1 nM, 37 degrees C) expressed specific [3H]paf binding already after a 15-min incubation period, indicating a PKC activation as the decisive step of paf receptor expression. LDL also stimulated the paf degrading cellular acetylhydrolase significantly by increasing both Km (9.4 +/- 1.9 vs. 2.0 +/- 0.5 microM, P less than 0.02) and vmax (0.5 +/- 0.2 vs. 0.2 +/- 0.0 nmol/min per mg cell protein, P less than 0.02). The data demonstrate that LDL increases the number of paf receptors on monocyte/macrophage-like U 937 cells and interferes with the dynamics and/or synthesis of the cellular acetyl hydrolase. These effects could be of importance in the pathogenesis of atherosclerosis.