RESUMO
During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1-/- mice failed to efficiently prime CD8+ T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos , Endocitose , Proteínas Citotóxicas Formadoras de Poros , Animais , Camundongos , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/genética , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Endocitose/genética , Endocitose/imunologia , Testes Genéticos , Antígenos de Histocompatibilidade Classe I , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , ProteóliseRESUMO
Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/enzimologia , Nicotinamida N-Metiltransferase/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotinamida N-Metiltransferase/antagonistas & inibidoresRESUMO
Nicotinamide N-methyltransferase (NNMT) is a S-adenosyl-l-methionine (SAM)-dependent enzyme that catalyzes N-methylation of nicotinamide (NA) and other pyridines to form N-methyl pyridinium ions. Here we report the first ternary complex X-ray crystal structures of monkey NNMT and mouse NNMT in bound form with the primary endogenous product, 1-methyl nicotinamide (MNA) and demethylated cofactor, S-adenosyl-homocysteine (SAH) determined at 2.30 Å and 1.88 Å respectively. The structural fold of these enzymes is identical to human NNMT. It is known that the primary endogenous product catalyzed by NNMT, MNA is a specific inhibitor of NNMT. Our data clearly indicates that the MNA binds to the active site and it would be trapped in the active site due to the formation of the bridge between the pole (long helix, α3) and long C-terminal loop. This might explain the mechanism of MNA acting as a feedback inhibitor of NNMT.