Azole-antifungal binding to a novel cytochrome P450 from Mycobacterium tuberculosis: implications for treatment of tuberculosis.

Although antibiotics against Mycobacterium tuberculosis have decreased the incidence of tuberculosis infections significantly, the emergence of drug-resistant strains of this deadly pathogen renders current treatments ineffective. Therefore, it is imperative to identify biochemical pathways in M. tuberculosis that can serve as targets for new anti-mycobacterial drugs. We recently cloned, expressed, and purified MT CYP51, a soluble protein from M. tuberculosis that is similar in sequence to CYP51 (lanosterol-14alpha-demethylase) isozymes, pharmacological targets for several anti-mycotic compounds. Its striking amino acid sequence similarity to that of mammalian and fungal CYP51s led to the hypothesis that MT CYP51 plays an important role in mycobacterial biology that can be targeted for drug action. In this manuscript, we established through spectral analysis that several azole antifungals bind MT CYP51 with high affinity. The effects of several azole compounds on the growth of M. bovis and M. smegmatis, two mycobacterial species that closely resemble M. tuberculosis were examined. We established a correlation between the affinity of azole compounds to MT CYP51 and their ability to impair the growth of M. bovis and M. smegmatis. These results suggest that the metabolic functions of MT CYP51 may be comparable to those of CYP51 in yeast and fungi and may lead to the development of a new generation of anti-mycobacterial agents.

Rat peroxisome proliferator-activated receptors and brown adipose tissue function during cold acclimatization.

Brown adipose tissue (BAT) hyperplasia is a fundamental physiological response to cold; it involves an acute phase of mitotic cell growth followed by a prolonged differentiation phase. Peroxisome proliferator-activated receptors (PPARs) are key regulators of fatty acid metabolism and adipocyte differentiation and may therefore mediate important metabolic changes during non-shivering thermogenesis. In the present study we have investigated PPAR mRNA expression in relation to peroxisome proliferation in rat BAT during cold acclimatization. By immunoelectron microscopy we show that the number of peroxisomes per cytoplasmic volume and acyl-CoA oxidase immunolabeling density remained constant (thus increasing in parallel with tissue mass and cell number) during the initial proliferative phase and the acute thermogenic response but increased after 14 days of cold exposure, correlating with terminal differentiation of BAT. A pronounced decrease in BAT PPARalpha and PPARgamma mRNA levels was found within hours of exposure to cold, which was reversed after 14 days, suggesting a role for either or both of these subtypes in the proliferation and induction of peroxisomes and peroxisomal beta-oxidation enzymes. In contrast, PPARdelta mRNA levels increased progressively during cold exposure. Transactivation assays in HIB 1B and HEK-293 cells demonstrated an adrenergic stimulation of peroxisome proliferator response element reporter activity via PPAR, establishing a role for these nuclear receptors in hormonal regulation of gene transcription in BAT.

Negative glucorticoid regulation of cyclic adenosine 3', 5'-monophosphate-stimulated corticotropin-releasing hormone-reporter expression in AtT-20 cells.

The negative glucocorticoid regulation of CRH gene expression is a critical control element in the hypothalamic-pituitary-adrenal axis. In this study, the molecular mechanisms mediating the glucocorticoid repression of cAMP-induced CRH-reporter expression in AtT-20 cells have been examined. In these cells, dexamethasone decreases forskolin-induced expression of CRH-reporter activity in a dose-dependent manner. This repression is mediated by the glucocorticoid receptor (GR) and does not require ongoing protein synthesis. Several binding sites for the GR DNA-binding domain were identified within the CRH 5'-flanking and 5'-untranslated regions utilizing in vitro DNase I protection assays. These sites were independently mutated and/or deleted. Functional studies in transfected cells suggest that none of the protected DNA sequences mediate the glucocorticoid regulation and that the regulatory element(s) mediating negative glucocorticoid regulation is contained within the CRH DNA sequences from -248 to +4 bp relative to the major transcription initiation site. To further localize the DNA sequence(s) responsive to glucocorticoids, DNA fragments containing various amounts of human CRH 5'flanking sequences were inserted 5' to the SV40 promoter. An 18-bp DNA fragment containing the CRH cAMP-responsive element is sufficient to confer both positive cAMP regulation and glucocorticoid repression of cAMP-stimulated expression to the SV40 promoter. These results suggest that glucocorticoid repression of forskolin-activated CRH-reporter expression in AtT-20 cells occurs via interference with the cAMP-mediated activation of gene expression, possibly via direct or indirect interactions between the GR and the cAMP-responsive element-binding proteins.

The cAMP-responsive element in the corticotropin-releasing hormone gene mediates transcriptional regulation by depolarization.

Membrane depolarization is a critical element of neuronal signaling. In this study, the biochemical and molecular mechanisms involved in transcriptional regulation of the corticotropin-releasing hormone (CRH) gene by depolarization were investigated. In PC-12 cells, potassium-induced membrane depolarization increased expression of a CRH-reporter construct in a cAMP-dependent manner. This synergistic activation was mediated via calcium influx, predominantly via L-type calcium channels, and calmodulin. RNase protection assays demonstrated increased levels of CRH-reporter transcripts in stably transfected cells after treatment with cAMP and potassium, with the induced transcripts initiating at the major transcription initiation site of the human CRH gene. At the genomic level, the CRH cAMP-responsive element conferred both positive cAMP and synergistic cAMP/depolarization regulation to a heterologous promoter. Additionally, DNase I protection assays demonstrated similar nuclear protein/DNA binding profiles across the cAMP-responsive element after treatment of PC-12 cells with potassium or potassium/cAMP. These results support a model in which the protein(s) binding to the cAMP-responsive element integrates signals initiated by multiple pathways (cAMP and calcium) and transmits that integrated signal to the basal transcription machinery, resulting in increased levels of gene expression.