Human microRNA (miR29b) expression controls the amount of branched chain alpha-ketoacid dehydrogenase complex in a cell.

Branched chain amino acids (BCAAs) play critical roles in cell and tissue functions in addition to being important components of protein structure. The multifunctional roles speak to the need for maintaining tight control of their concentration within cells. As the BCAA cannot be made de novo in mammals, their cellular concentration is a function of dietary intake, endogenous protein turnover and catabolism of the three amino acids. The branched chain alpha-ketoacid dehydrogenase (BCKD) complex commits the BCAA to degradation and thus is vital in controlling their concentration within a cell. In mammals, BCKD activity state depends on the presence of a covalently bound phosphate on one protein component of the complex. Phosphate is added to the protein through the action of the complex-specific kinase and results in BCKD inactivation. Here, we demonstrate that another reaction plays a role in determining the total amount of BCKD present in a cell. The microRNA (miR29b) molecule tested is targeted to the mRNA for the dihydrolipoamide branched chain acyltransferase component of BCKD and prevents translation when bound. This is the first demonstration of the use of a microRNA to exert control on a metabolic pathway of amino acid catabolism in mammals and offers an explanation for the observed differences in the amount of the BCKD complex present in different tissues and under varying nutritional states.

Protective role of arginase in a mouse model of colitis.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Cutting edge: urease release by Helicobacter pylori stimulates macrophage inducible nitric oxide synthase.

Inducible NO synthase (iNOS) expression and production of NO are both up-regulated with Helicobacter pylori infection in vivo and in vitro. We determined whether major pathogenicity proteins released by H. pylori activate iNOS by coculturing macrophages with wild-type or mutant strains deficient in VacA, CagA, picB product, or urease (ureA(-)). When filters were used to separate H. pylori from macrophages, there was a selective and significant decrease in stimulated iNOS mRNA, protein, and NO(2)(-) production with the ureA(-) strain compared with wild-type and other mutants. Similarly, macrophage NO(2)(-) generation was increased by H. pylori protein water extracts of all strains except ureA(-). Recombinant urease stimulated significant increases in macrophage iNOS expression and NO(2)(-) production. Taken together, these findings indicate a new role for the essential H. pylori survival factor, urease, implicating it in NO-dependent mucosal damage and carcinogenesis.