THE DOGS (First Agaricus Ode)

THE DOGS (First Agaricus Ode

Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro

Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (Kd 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60–70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions

Transcriptional regulation of the hepatic cytochrome <em>P450 2a5</em> gene

Abstract Cytochrome P450 (CYP) enzymes are the major metabolizers of xenobiotics, e.g. drugs, and environmental toxins. Thus, changes in CYP expression have an important impact on drug metabolism and susceptibility to chemical toxicity. In the present study, the transcriptional mechanisms of both constitutive and inducible regulation of the Cyp2a5 gene in mouse liver were investigated. Mouse primary hepatocyte cultures were used as the main model system together with cell and molecular biology methods. The key activation regions of the Cyp2a5 5' promoter were determined using reporter gene assays. Two major transcription activation sites of the Cyp2a5 5' promoter, called the proximal and the distal, were found. Transcription factors hepatocyte nuclear factor-4 (HNF-4) and nuclear factor I were shown to bind to the proximal promoter. Aryl hydrocarbon receptor nuclear translocator (ARNT) and upstream stimulatory factor bound to a common palindromic E-box element in the distal promoter region. All three response elements were shown to be essential for constitutive expression of CYP2A5 in murine hepatocytes. ARNT appeared to control Cyp2a5 transcription without a heterodimerization partner suggesting active involvement of the ARNT homodimer in mammalian gene regulation. Aryl hydrocarbon receptor (AHR) ligands were shown to induce Cyp2a5 transcriptionally by an AHR-dependent mechanism, and established Cyp2a5 as a novel AHR-regulated gene. The AHR response element and the E-box, identified in these studies, were located near to each other and close to a separately defined nuclear factor (erythroid-derived 2)-like 2 binding site in the distal region of the Cyp2a5 promoter, suggesting cooperation between these elements. Peroxisome proliferator-activated receptor gamma coactivator-1α was shown to up-regulate Cyp2a5 transcription through coactivation of HNF-4α. This indicates that xenobiotic metabolism can be regulated by modification of co-activation. The present results show that CYP2A5 is regulated by several different cross-regulatory pathways. The regulatory mechanisms involved in the transcription of the Cyp2a5 gene may also control other CYP genes, especially the human ortholog CYP2A6, and may explain some of the individual variations in the metabolism of xenobiotics