ABSTRACT
The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.
Subject(s)
Cell Hypoxia/genetics , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Protein Serine-Threonine Kinases/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Binding Proteins , Gene Expression Regulation, Bacterial/genetics , Humans , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/pathogenicity , Oxygen/chemistry , Oxygen/metabolism , Phosphorylation , Protamine Kinase/genetics , Protamine Kinase/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Tuberculosis/genetics , Tuberculosis/microbiologyABSTRACT
Naltrexone, an opioid receptor antagonist, has been approved for clinical use in the treatment of alcohol dependence. In the present study, we examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. A complementary DNA microarray analysis was used to profile gene expression changes in the hippocampus and prefrontal cortex (PFC) of C57BL/6 mice injected with naltrexone following ethanol treatment. Intraperitoneal administration of 200µl (16mg/kg) of naltrexone for 4 weeks caused alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in ethanol-treated mice. Ingenuity Pathway Analysis (IPA) software was used to search for biological pathways and interrelationships between gene networks in the subsets of candidate genes that were altered in the naltrexone-treated PFC and hippocampus. We found gene networks associated with cell morphology, cell death, nervous system development and function, and neurological disease. Confirmation studies using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the expression of transthyretin (TTR) and protein kinase C (PKC)γ were increased in the PFC but not in the hippocampus of naltrexone-treated mice. In conclusion, the present study demonstrates a pharmacogenomic response to naltrexone in the brains of ethanol-consuming mice. These findings provide a basis for conducting pharmacogenetic research on the effect of naltrexone in specific brain areas, which would enhance our understanding of the underlying causes and possible treatments of alcohol use disorders.
Subject(s)
Gene Expression/drug effects , Naltrexone/pharmacology , Narcotic Antagonists/pharmacology , Prealbumin/biosynthesis , Prefrontal Cortex/drug effects , Protein Kinase C/biosynthesis , Alcohol Drinking/drug therapy , Alcohol Drinking/genetics , Animals , Hippocampus/drug effects , Hippocampus/metabolism , Male , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Prealbumin/genetics , Prefrontal Cortex/metabolism , Protein Kinase C/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In the brain, the stress system plays an important role in motivating continued alcohol use and relapse. The neuropeptide substance P and the neurokinin-1 receptor (NK1R) are involved in the stress response and drug reward systems. Recent findings have shown that the binding of ligands to NK1Rs decreases the self-administration of alcohol in mice. We examined the effect of an artificial microRNA (amiRNA) on the functional expression of NK1R in mouse brains. Lentiviruses expressing either an amiRNA targeting the NK1R (amiNK1R) or a negative control amiRNA (amiNC) were injected into mouse brains. Four weeks after amiRNA injection, we found that amiNK1R decreased the voluntary alcohol consumption compared to mice injected with amiNC. We also observed that NK1R expression was reduced in the hippocampus. RNA interference is an effective approach to regulate the expression of specific behavior-related genes. Our results support the potential use of amiRNA as a therapeutic agent for the treatment of alcohol dependence.
