RESUMO
Aggregation of the amyloid beta (Aß) peptide and hyperphosphorylation of tau protein, which are markers of Alzheimer's disease (AD), have been reported also in diabetes mellitus (DM). One regulator of tau phosphorylation is O-GlcNAcylation, whereas for hyperphosphorylation it could be GSK3beta, which is activated in hyperglycemic conditions. With this in mind, both O-GlcNAcylation and phosphorylation of tau protein were evaluated in the brain of rats with streptozotocin (STZ)-induced hyperglycemia and hyperinsulinemia and treated with the Aß25-35 peptide in the hippocampal region CA1. Weight, glycated hemoglobin, glucose, and insulin were determined. Male Wistar rats were divided in groups (N=20): a) control, b) treated only with the Aß25-35 peptide, c) treated with Aß25-35 and STZ, and d) treated only with STZ. Results showed statistically significant differences in the mean weight, glucose levels, insulin concentration, and HbA1c percentage, between C- and D-treated groups and not STZ-treated A and B (P<0.05). Interestingly, our results showed diminution of O-GlcNAcylation and increase in P-tau-Ser-396 in the hippocampal area of the Aß25-35- and STZ-treated groups; moreover, enhanced expression of GSK3beta was observed in this last group. Our results suggest that hyperinsulinemia-Aß25-35-hyperglycemia is relevant for the down regulation of O-GlcNAcylation and up-regulation of the glycogen synthase kinase-3 beta (GSK3beta), favoring Aß25-35-induced neurotoxicity in the brain of rats.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Região CA1 Hipocampal/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas tau/metabolismo , Acilação/efeitos dos fármacos , Animais , Glicemia , Região CA1 Hipocampal/metabolismo , Insulina/sangue , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
BACKGROUND: Tuberculosis (TB) remains a serious human health problem that affects millions of people in the world. Understanding the biology of Mycobacterium tuberculosis (Mtb) is essential for tackling this devastating disease. Mtb possesses a very complex cell envelope containing a variety of lipid components that participate in the establishment of the infection. We have previously demonstrated that di-O-acylated trehalose (DAT), a non-covalently linked cell wall glycolipid, inhibits the proliferation of T lymphocytes and the production of cytokines. RESULTS: In this work we show that DAT and the closely related tri-O-acylated trehalose (TAT) inhibits nitric oxide (NO) production and the inducible nitric oxide synthase (iNOS) expression in macrophages (MØ). CONCLUSIONS: These findings show that DAT and TAT are cell-wall located virulence factors that downregulate an important effector of the immune response against mycobacteria.
Assuntos
Glicolipídeos/farmacologia , Macrófagos/enzimologia , Mycobacterium/química , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/biossíntese , Trealose/farmacologia , Acilação/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Regulação para Baixo/efeitos dos fármacos , Glicolipídeos/isolamento & purificação , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Trealose/isolamento & purificaçãoRESUMO
O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.
Assuntos
Animais , Masculino , Músculo Liso Vascular/fisiologia , Cadeias Leves de Miosina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Vasoconstrição/fisiologia , Aorta Torácica , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acilação/efeitos dos fármacos , Acilação/fisiologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Azepinas/farmacologia , Western Blotting , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Oxazóis/farmacologia , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Fenilefrina/agonistas , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos Wistar , Ribonucleotídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.
Assuntos
Músculo Liso Vascular/fisiologia , Cadeias Leves de Miosina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Vasoconstrição/fisiologia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acilação/efeitos dos fármacos , Acilação/fisiologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Aorta Torácica , Azepinas/farmacologia , Western Blotting , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Masculino , Toxinas Marinhas , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Oxazóis/farmacologia , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Fenilefrina/agonistas , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos Wistar , Ribonucleotídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.