Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RARa, RAR(3, and RARly. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RARa antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyconal activation.Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.The natural retinol (vitamin A) derivative retinoic acid (RA) is known to have profound effects on cell growth and differentiation (1) and to be essential for normal embryonic development (2). While RA and some synthetic analogs (retinoids) are useful in the control of some tumors (3) as well as of nonmalignant hyperproliferative conditions of the skin (4), they are, at high concentrations, teratogenic (5).The pleiotropic effects of retinoids are mediated by two known families of nuclear receptors, both belonging to the steroid-thyroid hormone receptor superfamily of ligandinducible transcriptional regulators (6, 7). The RA receptor (RAR) gene family comprises three subtypes-RARa (8, 9), RAR,[8][9][10][11][12], and RAR'y (13, 14)-with each gene encoding a variable number of isoforms arising by differential splicing of two primary . All receptors of the RAR family bind RA with comparable affinity (18). The retinoid receptors of the second family (RXR) do not bind the major form of RA (all-trans-RA) (19). They bind instead the 9-cis stereoisomer of RA (20, 21).Transcription of some RAR genes themselves is RA sensitive (22-25). Also, the expression of some of the cellular retinol-or RA-binding proteins (CRBP and CRABP), putatively involved in the storage, transport, and/or metabolism of retinol and RA, is differentially regulated by RA in a receptor-specific manner (26-28). The RA-related molecules represent, therefore, an autoregulated system. RAR types and isoforms, as well as RXRa and RXRB, are differentially expressed both spatially and temporally (15-18, 29-32). They might therefore regulate different target genes during embryonic and adult life, as well as in specific cell types at different stages of differentiation. RARa is the most ubiquitously expressed, while RAR8 and RARy display a more restricted pattern of distribution, with RARy being predominantly expressed in the skin (31).It seems reasonable to assume that the multiple effects of RA could be dissociated by specific ligands for each of the known receptors, and/or by receptor-specific antagonists, so as to obtain the desired beneficial effects while limiting the unwanted side effects. Retinoids with a good degree of selectivity have been described (33), and we have o...

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC2.5.1.31 ) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E,E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphos-phate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coliproteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675 ; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and inCaenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli,Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

Metabolic defects in phytanic acid catabolism have been shown to be connected with a number of human diseases which can lead to lethal defects of the nervous system and other organs. These effects are probably a result of the very high accumulation of phytanic acid in tissues throughout the body, due to defects in phytanic acid oxidation, the peroxisome being a major site for this process. The nuclear hormone receptors peroxisome proliferator-activated receptor and retinoid X receptor (RXR) have been shown to function as transcription factors in the control of the peroxisomal enzyme expression. Known activators of peroxisome proliferator-activated receptor include polyunsaturated fatty acids and, for RXR, the 9 4 s isomer of retinoic acid. Here we report that phytanic acid is also a natural ligand for RXRa, being able to activate a RXR-responsive promoter. We present evidence that phytanic acid binds to RXRa, promotes formation of an RXRa/RXR response element complex (as detected by gel retardation), and induces a RXRa conformational change similar to that induced by 9-cis-retinoic acid (as detected by protease sensitivity). These results suggest an involvement of RXRa in the control of fatty acid metabolism and could imply that RXRs have a role in the disease effects resulting from defective phytanic acid catabolism.

New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10 ؊7 ). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.Antibiotic resistance is a major health concern, and the existing antibiotics target only a handful of molecules. Therefore, there is an urgent need for antibiotics with novel mechanisms of action. Peptide deformylase (PDF; EC 3.5.1.27) is essential in a variety of pathogenic bacteria but is not required for cytoplasmic protein synthesis in eukaryotes and is therefore an interesting potential target for antibacterial agents. Protein synthesis in eubacteria, under normal conditions, is initiated by formyl-methionyl-tRNA (19). Consequently, all nascent polypeptides are synthesized with N-formyl-methionine at the N terminus. The formyl group is removed by PDF during elongation of the polypeptide chain (1, 7). As methionine aminopeptidase (EC 3.4.11.18) cannot hydrolyze N-blocked polypeptides, deformylation is also a prerequisite for protein maturation (10,22,27). Both PDF and MAP, are essential for growth in Escherichia coli (10,19,21). pdf gene mutants can only be obtained in E. coli strains lacking the gene for formyltransferase, the enzyme that N-formylates the methionyl-tRNA f Met (EC.2.1.2.9) (20). In a recent publication, we described the identification, optimization, and biological characterization of novel PDF inhibitors (3). These compounds were potent inhibitors of the isolated enzyme but only moderately active as antibacterials. In the accompanying paper, we describe transcription-translation assays that allowed us to demonstrate that the inhibitors were active as inhibitors of PDF in cell homogenates as well as in intact cells (4a). The experimental evidence presented here demonstrates that (i) antibacteri...

The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs).Recently the synthetic retinoid Ro 41-5253 was identified as a selective RARa antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RARa antagonists do not influence RARcWRXRa heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RARca with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RARorRXRc heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RARaL and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RARe conformation seems to allow RARW/RXRcr binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RARa. Complexation of RARI3, RARy, and RXRe, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.