RESUMEN
DJ-1, a causative gene for hereditary recessive Parkinsonism, is evolutionarily conserved across eukaryotes and prokaryotes. Structural analyses of DJ-1 and its homologs suggested the 106th Cys is a nucleophilic cysteine functioning as the catalytic center of hydratase or hydrolase activity. Indeed, DJ-1 and its homologs can convert highly electrophilic α-oxoaldehydes such as methylglyoxal into α-hydroxy acids as hydratase in vitro, and oxidation-dependent ester hydrolase (esterase) activity has also been reported for DJ-1. The mechanism underlying such plural activities, however, has not been fully characterized. To address this knowledge gap, we conducted a series of biochemical assays assessing the enzymatic activity of DJ-1 and its homologs. We found no evidence for esterase activity in any of the Escherichia coli DJ-1 homologs. Furthermore, contrary to previous reports, we found that oxidation inactivated rather than facilitated DJ-1 esterase activity. The E. coli DJ-1 homolog HchA possesses phenylglyoxalase and methylglyoxalase activities but lacks esterase activity. Since evolutionary trace analysis identified the 186th H as a candidate residue involved in functional differentiation between HchA and DJ-1, we focused on H186 of HchA and found that an esterase activity was acquired by H186A mutation. Introduction of reverse mutations into the equivalent position in DJ-1 (A107H) selectively eliminated its esterase activity without compromising α-oxoaldehyde hydratase activity. The obtained results suggest that differences in the amino acid sequences near the active site contributed to acquisition of esterase activity in vitro and provide an important clue to the origin and significance of DJ-1 esterase activity.
Asunto(s)
Escherichia coli , Enfermedad de Parkinson , Proteína Desglicasa DJ-1 , Proteína Desglicasa DJ-1/metabolismo , Proteína Desglicasa DJ-1/genética , Proteína Desglicasa DJ-1/química , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Esterasas/metabolismo , Esterasas/genética , Esterasas/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Evolución Molecular , Oxidación-ReducciónRESUMEN
Neurons containing neural nitric oxide synthase (nNOS) are found in various locations in the hypothalamus and, in particular, in the paraventricular and supraoptic nuclei with axons which project to the median eminence and extend into the neural lobe where the highest concentrations of NOS are found in the rat. Furthermore, nNOS is also located in folliculostellate cells and LH gonadotropes in the anterior pituitary gland. To define the role of NO in the release of hypothalamic peptides and pituitary hormones, we inected an inhibitor of NOS, Ng- monomethyl-L-arginine (NMMA) or a releasor of NO, nitroprusside (NP) into the third ventricle (3V) of conscious castrate rats and determined the effect on the release of various pituitary hormones. In vitro, we incubated medial basal hypothalamic (MBH) fragments and studied inhibitors of NO synthase and also releasors of NO. The results indicate that NOergic neurons play an important role in stimulating the release of corticotrophin-releasing hormone (CRH), luteinizing hormone releasing-hormone (LHRH), prolactin-RH's, particularly oxytocin, growth hormone-RH (GHRH) and somatostatin, but not FSH-releasing factor from the hypothalamus. NO stimulates the release of LHRH, which induces sexual behavior, and causes release of LH from the pituitary gland. The intrahypothalamic pathway by which NO controls LHRH release is as follows: glutamergic neurons synapse with noradrenergic terminals in the MBH which release nonepinephrine (NE) that acts on alpha1 receptors on the NOergic neuron to increase intracellular free Ca++ which combines with calmodulin to activate NOS. The NOS diffuses to the LHRH terminal and activates guanylate cyclase (GC), cyclooxygenase and lipoxygenase causing release of LHRH via release of cyclic GMP, PGE2 and leukotrienes, respectively. Alcohol and cytokines can block LHRH release by blocking the activation of cyclooxygenase and lipoxygenase without interfering with the activation of GC. GABA also blocks the response of the LHRH neurons to NO and recent experiments indicate that granulocyte macrophage colony-stimulating factor (GMCSF) blocks the response of the LHRH neuron to NP by activation of GABA neurons since the blockase can be reversed by the competitive inhibitor of GABAa receptors, bicuculine.