RESUMEN
Irg1 is an enzyme that generates itaconate, a metabolite that plays a key role in the regulation of inflammatory responses. Previous studies have implicated Irg1 as an important mediator in preventing excessive inflammation and tissue damage in Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the pattern recognition receptors and signaling pathways by which Mtb triggers Irg1 gene expression by comparing the responses of control and genetically deficient BMDMs. Using this approach, we demonstrated partial roles for TLR-2 (but not TLR-4 or -9), MyD88 and NFκB signaling in Irg1 induction by Mtb bacilli. In addition, drug inhibition studies revealed major requirements for phagocytosis and endosomal acidification in Irg1 expression triggered by Mtb but not LPS or PAM3CSK4. Importantly, the Mtb-induced Irg1 response was highly dependent on the presence of the bacterial ESX-1 secretion system, as well as host STING and Type I IFN receptor (IFNAR) signaling with Type II IFN (IFN-γ) signaling playing only a minimal role. Based on these findings we hypothesize that Mtb induces Irg1 expression in macrophages via the combination of two independent triggers both dependent on bacterial phagocytosis: 1) a major signal stimulated by phagocytized Mtb products released by an ESX-1-dependent mechanism into the cytosol where they activate the STING pathway leading to Type I-IFN production, and 2) a secondary TLR-2, MyD88 and NFκB dependent signal that enhances Irg1 production independently of Type I IFN induction.
Asunto(s)
Hidroliasas , Macrófagos , Proteínas de la Membrana , Mycobacterium tuberculosis , Receptor de Interferón alfa y beta , Receptor Toll-Like 2 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Inducción Enzimática , Hidroliasas/biosíntesis , Hidroliasas/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Proteínas de la Membrana/metabolismo , Ratones , Mycobacterium tuberculosis/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Fagocitosis , Receptor de Interferón alfa y beta/metabolismo , Receptor Toll-Like 2/metabolismo , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to ß-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.
Asunto(s)
Proteínas Bacterianas/química , Ácido Graso Sintasas/química , Hidroliasas/química , Algoritmos , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Ácido Graso Sintasas/biosíntesis , Ácido Graso Sintasas/genética , Ácidos Grasos Insaturados/metabolismo , Hidroliasas/biosíntesis , Hidroliasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Protein restriction (PR) significantly inhibits spontaneous and chemical carcinogenesis. Several factors seem to be involved in this effect, including a decrease in body weight, cellular proliferation and DNA damage and an increase in antioxidant defenses. The current study was designed to determine modifications in some hepatic cytochromes P450 (CYPs) due to a hypoproteic diet and to investigate its implications on chemical mutagenesis. Western blot analysis showed decreases of 73, 40 and 74% in CYP1A, CYP2B and CYP2E1 protein concentrations in hepatic microsomes from animals fed a protein-restricted (6% protein) diet for 6 weeks in comparison with microsomes from rats fed a 24% protein diet during the same period. In the same way, low protein fed animals showed a 3.5-fold decrease in hepatic CYP1A1-associated ethoxyresorufin O-deethylase activity, a 6-fold decrease in CYP1A2-associated methoxyresorufin O-demethylase activity, a 1.7-fold decrease in CYP2B1-associated penthoxyresorufin O-dealkylase activity, a 9-fold decrease in CYP2B2-associated benzyloxyresorufin O-dealkylase and, finally, a 3.4-fold decrease in CYP2E1-associated 4-nitrophenol hydroxylase activity. As a result of decreased CYP hepatic protein concentrations and enzymatic activities, liver S9 from rats fed a hypoproteic diet was less efficient in activating promutagens than S9 prepared from rats fed a 24% protein diet in the Ames test. Mutagenic potency obtained with protein-restricted S9 was reduced 25-fold for 2-aminoanthracene, 1.5-fold for N-nitrosodipropylamine, 12.5-fold for N-nitrosodibutylamine, 2-fold for cyclophosphamide and N-nitrosopyrrolidine and 71-fold for N-nitrosodimethylamine. However, the mutagenic potency of benzo[a]pyrene was the same (4 revertants/ microg) with S9 derived from rats fed either a 6 or 24% protein diet.