The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1-and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1-and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5 regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.Important gene hierarchies, in part coding for components of signal transduction pathways, regulate growth and differentiation during development. One such pathway is the Sonic hedgehog-Patched-Gli pathway (1). SHH 1 signaling is critical to the genetic specification of fate of many tissues during early organogenesis including the central nervous system (2, 3), lung (4), prostate (5), bone (6 -8), and muscle (9). SHH signaling is mediated by the GLI family of transcription factors (10). One of these genes, GLI1, has been shown to be a transcriptional activator operating through a C-terminal VP-16-like acidic helical domain (11). GLI1 transforms cells in culture, and its expression is associated with significant human cancers including basal cell carcinoma (12), medulloblastoma (13), and sarcomas (14). Few downstream targets of GLI1 are known, which precludes a clear understanding of its action in carcinogenesis. Genetic evidence suggests that PTCH and Wnt genes are downstream targets of GLI1 (15), and biochemical evidence has established HNF-3␤ (Hepatocyte Nuclear Factor-3␤) as a target of GLI1 during development (16).Microarray technology has provided a methodology to study the expression of thousands of genes simultaneously and has been used in many important settings (17). Among these is the dissection of signal transduction pathways. To identify unique downstream targets of GLI1, we have utilized a cell transformation phenotype as a selection system for the stable integration and expression of either GLI1 or Ha-ras in RK3...

Cytochromes P450 form a large and important class of heme monooxygenases with a broad spectrum of substrates and corresponding functions, from steroid hormone biosynthesis to the metabolism of xenobiotics. Despite decades of study, the molecular mechanisms responsible for the complex non-Michaelis behavior observed with many members of this super-family during metabolism, often termed ‘cooperativity,’ remain to be fully elucidated. Although there is evidence that oligomerization may play an important role in defining the observed cooperativity, some monomeric cytochromes P450, particularly those involved in xenobiotic metabolism, also display this behavior due to their ability to simultaneously bind several substrate molecules. As a result, formation of distinct enzyme-substrate complexes with different stoichiometry and functional properties can give rise to homotropic and heterotropic cooperative behavior. This review aims to summarize the current understanding of cooperativity in cytochromes P450, with a focus on the nature of cooperative effects in monomeric enzymes.

Summary Degradation of the cholesterol side-chain in M. tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from M. smegmatis mc2 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4-cholesten-3-one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4-cholesten-3-one to the C-26 alcohol and subsequently to the acid. The X-ray structures of both substrate-free CYP125A3 and CYP142A2 and of cholest-4-en-3-one-bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion of cyp125A3 or cyp142A2 does not impair growth of M. smegmatis mc2 155 on cholesterol. However, deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatis Δcyp125Δcyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome.

Cytochrome P450 3A4 (CYP3A4) displays non-MichaelisMenten kinetics for many of the substrates it metabolizes, including testosterone (TST) and ␣-naphthoflavone (ANF). Heterotropic effects between these two substrates can further complicate the metabolic profile of the enzyme. In this work, monomeric CYP3A4 solubilized in Nanodiscs has been studied for its ability to interact with varying molar ratios of ANF and TST. Comparison of the observed heme spin state, NADPH consumption, and product formation rates with a non-cooperative model calculated from a linear combination of the global analysis of each substrate reveals a detailed landscape of the heterotropic interactions and indicates negligible binding cooperativity between ANF and TST. The observed effect of ANF on the kinetics of TST metabolism is due to the additive action of the second substrate with no specific allosteric effects.Hepatic cytochromes P450 play a fundamental role in the breakdown of xenobiotics from the blood. The most abundant of these in the adult human liver is cytochrome P450 3A4 (CYP3A4) 2 (1), which metabolizes approximately half of the most commonly prescribed drugs (2). Its ability to interact simultaneously with multiple substrate molecules leads to atypical kinetic phenomena, termed homotropic or heterotropic cooperativity (3-6), the former describing interactions of molecules of the same substrate and the latter referring to interactions of different substrates. Evidence of CYP3A4 simultaneously interacting with multiple substrate molecules comes from the crystal structures of the enzyme, which show a large, plastic active site (7), substrate bound at a peripheral binding site (8), changes in kinetic behavior in the presence of so-called effector molecules (9 -12), as well as a global analysis of multiple observable enzyme properties (13)(14)(15)(16).Recent work from our laboratory has shown that the apparent heterotropic cooperativity in Type I spin transition between ANF and TST can be accounted for by the additive effect of the ability of each substrate to induce the spin transition (17). Differences in their relative spectral affinities may give the appearance of a stimulatory effect of ANF on TST-induced spin transition; however, this is not indicative of any true cooperative behavior in binding (17). Here, we extend this analysis to include the contributions of NADPH oxidation and product-forming rates from each of the substrates to elucidate a more complete understanding of the heterotropic interactions of this important drug-metabolizing enzyme.