The human multidrug resistance-associated protein functionally complements the yeast cadmium resistance factor 1.

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCF1) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione S-conjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.

An ATP-dependent As(III)-glutathione transport system in membrane vesicles of Leishmania tarentolae.

Membrane preparations enriched in plasma membrane vesicles prepared from promastigotes of Leishmania tarentolae were shown to accumulate thiolate derivatives of 73As(III). Free arsenite was transported at a low rate, but rapid accumulation was observed after reaction with reduced glutathione (GSH) conditions that favor the formation of As(GS)3. Accumulation required ATP but not electrochemical energy, indicating that As(GS)3 is transported by an ATP-coupled pump. Pentostam, a Sb(V)-containing drug that is one of the first-line therapeutic agents for treatment of leishmaniasis, inhibited uptake after reaction with GSH. Vesicles prepared from a strain in which both copies of the pgpA genes were disrupted accumulated As(GS)3 at wild-type levels, demonstrating that the PgpA protein is not the As(GS)3 pump. These results have important implications for the mechanism of drug resistance in the trypanosomatidae, suggesting that a plasma membrane As(GS)3 pump catalyzes active extrusion of metal thiolates, including the Pentostam-glutathione conjugate.

Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

Persistent inhibition of cell respiration by nitric oxide: Crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S-transferase family

The class I glutathione S-transferases (GSTs) of Anopheles gambiae are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes, aggst1-2, is intronless and has been described. In contrast, the two A. gambiae GST genes (aggst1α and aggst1β) reported within are interrupted by introns. The gene aggst1α contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5′ exon encoding the N termini of the GST protein spliced to one of four distinct 3′ exons encoding the carboxyl termini. All four of the alternative transcripts of aggst1α are expressed in A. gambiae larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the aggst1β gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.

Solid and liquid phase 59Co NMR studies of cobalamins and their derivatives

We describe the application of 59Co NMR to the study of naturally occurring cobalamins. Targets of these investigations included vitamin B12, the B12 coenzyme, methylcobalamin, and dicyanocobyrinic acid heptamethylester. These measurements were carried out on solutions and powders of different origins, and repeated at a variety of magnetic field strengths. Particularly informative were the solid-state central transition NMR spectra, which when combined with numerical line shape analyses provided a clear description of the cobalt coupling parameters. These parameters showed a high sensitivity to the type of ligands attached to the metal and to the crystallization history of the sample. 59Co NMR determinations also were carried out on synthetic cobaloximes possessing alkyl, cyanide, aquo, and nitrogenated axial groups, substituents that paralleled the coordination of the natural compounds. These analogs displayed coupling anisotropies comparable to those of the cobalamins, as well as systematic up-field shifts that can be rationalized in terms of their stronger binding affinity to the cobalt atom. Cobaloximes also displayed a higher regularity in the relative orientations of their quadrupole and shielding coupling tensors, reflecting a higher symmetry in their in-plane coordination. For the cobalamines, poor correlations were observed between the values measured for the quadrupole couplings in the solid and the line widths observed in the corresponding solution 59Co NMR resonances.

Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet

The availability of cysteine is thought to be the rate limiting factor for synthesis of the tripeptide glutathione (GSH), based on studies in rodents. GSH status is compromised in various disease states and by certain medications leading to increased morbidity and poor survival. To determine the possible importance of dietary cyst(e)ine availability for whole blood glutathione synthesis in humans, we developed a convenient mass spectrometric method for measurement of the isotopic enrichment of intact GSH and then applied it in a controlled metabolic study. Seven healthy male subjects received during two separate 10-day periods an l-amino acid based diet supplying an adequate amino acid intake or a sulfur amino acid (SAA) (methionine and cysteine) free mixture (SAA-free). On day 10, l-[1-13C]cysteine was given as a primed, constant i.v. infusion (3μmol⋅kg−1⋅h−1) for 6 h, and incorporation of label into whole blood GSH determined by GC/MS selected ion monitoring. The fractional synthesis rate (mean ± SD; day-1) of whole blood GSH was 0.65 ± 0.13 for the adequate diet and 0.49 ± 0.13 for the SAA-free diet (P < 0.01). Whole blood GSH was 1,142 ± 243 and 1,216 ± 162 μM for the adequate and SAA-free periods (P > 0.05), and the absolute rate of GSH synthesis was 747 ± 216 and 579 ± 135 μmol⋅liter−1⋅day−1, respectively (P < 0.05). Thus, a restricted dietary supply of SAA slows the rate of whole blood GSH synthesis and diminishes turnover, with maintenance of the GSH concentration in healthy subjects.

Glutathione synthesis is essential for mouse development but not for cell growth in culture

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of γ-glutamylcysteine synthetase (γGCS-HStm1), an essential enzyme in GSH synthesis. Embryos homozygous for γGCS-HStm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

(4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure–activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N′-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1′ enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1′/S2′ subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

The chemistry of the S-nitrosoglutathione/glutathione system

S-Nitrosothiols have generated considerable interest due to their ability to act as nitric oxide (NO) donors and due to their possible involvement in bioregulatory systems—e.g., NO transfer reactions. Elucidation of the reaction pathways involved in the modification of the thiol group by S-nitrosothiols is important for understanding the role of S-nitroso compounds in vivo. The modification of glutathione (GSH) in the presence of S-nitrosoglutathione (GSNO) was examined as a model reaction. Incubation of GSNO (1 mM) with GSH at various concentrations (1–10 mM) in phosphate buffer (pH 7.4) yielded oxidized glutathione, nitrite, nitrous oxide, and ammonia as end products. The product yields were dependent on the concentrations of GSH and oxygen. Transient signals corresponding to GSH conjugates, which increased by one mass unit when the reaction was carried out with 15N-labeled GSNO, were identified by electrospray ionization mass spectrometry. When morpholine was present in the reaction system, N-nitrosomorpholine was formed. Increasing concentrations of either phosphate or GSH led to lower yields of N-nitrosomorpholine. The inhibitory effect of phosphate may be due to reaction with the nitrosating agent, nitrous anhydride (N2O3), formed by oxidation of NO. This supports the release of NO during the reaction of GSNO with GSH. The products noted above account quantitatively for virtually all of the GSNO nitrogen consumed during the reaction, and it is now possible to construct a complete set of pathways for the complex transformations arising from GSNO + GSH.

Glutathione biosynthesis in Arabidopsis trichome cells

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode γ-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238 ± 25, 80 ± 2, and 144 ± 19 μM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH]cyt is reduced to 47 ± 5 μM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.

Inhibition of β-catenin-mediated transactivation by cadherin derivatives

We studied the effect of N-cadherin, and its free or membrane-anchored cytoplasmic domain, on the level and localization of β-catenin and on its ability to induce lymphocyte enhancer-binding factor 1 (LEF-1)-responsive transactivation. These cadherin derivatives formed complexes with β-catenin and protected it from degradation. N-cadherin directed β-catenin into adherens junctions, and the chimeric protein induced diffuse distribution of β-catenin along the membrane whereas the cytoplasmic domain of N-cadherin colocalized with β-catenin in the nucleus. Cotransfection of β-catenin and LEF-1 into Chinese hamster ovary cells induced transactivation of a LEF-1 reporter, which was blocked by the N-cadherin-derived molecules. Expression of N-cadherin and an interleukin 2 receptor/cadherin chimera in SW480 cells relocated β-catenin from the nucleus to the plasma membrane and reduced transactivation. The cytoplasmic tails of N- or E-cadherin colocalized with β-catenin in the nucleus, and suppressed the constitutive LEF-1-mediated transactivation, by blocking β-catenin–LEF-1 interaction. Moreover, the 72 C-terminal amino acids of N-cadherin stabilized β-catenin and reduced its transactivation potential. These results indicate that β-catenin binding to the cadherin cytoplasmic tail either in the membrane, or in the nucleus, can inhibit β-catenin degradation and efficiently block its transactivation capacity.

Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC50 values of 18–100 nM in a standard TRAP assay.

An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives

We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the β-galactosidase/neoR fusion protein and the Cre-ERT2 (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygroR protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4′hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4′hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.

Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups

Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular “sensor” molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the β-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.