Hydrogen peroxide-mediated neuronal cell death induced by an endogenous neurotoxin, 3-hydroxykynurenine.

3-Hydroxykynurenine (3-HK) is a tryptophan metabolite whose level in the brain is markedly elevated under several pathological conditions, including Huntington disease and human immunodeficiency virus infection. Here we demonstrate that micromolar concentrations (1-100 microM) of 3-HK cause cell death in primary neuronal cultures prepared from rat striatum. The neurotoxicity of 3-HK was blocked by catalase and desferrioxamine but not by superoxide dismutase, indicating that the generation of hydrogen peroxide and hydroxyl radical is involved in the toxicity. Measurement of peroxide levels revealed that 3-HK caused intracellular accumulation of peroxide, which was largely attenuated by application of catalase. The peroxide accumulation and cell death caused by 1-10 microM 3-HK were also blocked by pretreatment with allopurinol or oxypurinol, suggesting that endogenous xanthine oxidase activity is involved in exacerbation of 3-HK neurotoxicity. Furthermore, NADPH diaphorase-containing neurons were spared from toxicity of these concentrations of 3-HK, a finding reminiscent of the pathological characteristics of several neurodegenerative disorders such as Huntington disease. These results suggest that 3-HK at pathologically relevant concentrations renders neuronal cells subject to oxidative stress leading to cell death, and therefore that this endogenous compound should be regarded as an important factor in pathogenesis of neurodegenerative disorders.

A plastid enzyme arrested in the step of precursor translocation in vivo.

The key enzyme of chlorophyll biosynthesis in higher plants, NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR, EC 1.3.1.33), accumulates in its precursor form (pPORA) in barley. pPORA is bound to the chloroplasts and is able to interact with the enzyme's substrate, Pchlide, at both the cytosolic as well as the stromal side of the plastid envelope. The interaction with intraplastidic Pchlide, formed in ATP-containing chloroplasts upon feeding with -aminolevulinic acid, drives vectorial translocation of pPORA across the plastid envelope membranes. In contrast, exogenously applied Pchlide causes the release of the envelope-bound precursor protein to the cytosol. Both processes compete with each other if intra- and extraplastidic Pchlide are applied simultaneously. A cytosolic heat shock cognate protein of Mr 70,000 present in wheat germ and barley leaf protein extracts appears to prevent the release of the pPORA to the cytosol in vivo, however.

SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form.

SoxR protein is known to function both as a sensor and as a transcriptional activator for a superoxide response regulon in Escherichia coli. The activity of SoxR was tested by its ability to enable the transcription of its target gene, soxS, in vitro. The activity of the oxidized form was lost when its [2Fe-2S] clusters were reduced by dithionite under anaerobic conditions, and it was rapidly restored by autooxidation. This result is consistent with the hypothesis that induction of the regulon is effected by the univalent oxidation of the Fe-S centers of SoxR. In vivo, this oxidation may be caused by an alteration of the redox balance of electron chain intermediates that normally maintains soxR in an inactive, reduced state. Oxidized SoxR was about twice as effective as reduced SoxR in protecting the soxS operator from endonucleolytic cleavage. However, this difference could not account for a greater than 50-fold difference in their activities and therefore could not support a model in which oxidation activates SoxR by enabling it to bind to DNA. NADPH, ferredoxin, flavodoxin, or ferredoxin (flavodoxin):NADP+ reductase could not reduce SoxR directly in vitro at a measurable rate. The midpoint potential for SoxR was measured at -283 mV.

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Characterization of the osmotic response element of the human aldose reductase gene promoter.

Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer activity. A plasmid construct containing three repeat OREs and a heterologous promoter increased expression 8-fold in isoosmotic media and an additional 4-fold when the transfected cells are subjected to hyperosmotic stress (total approximately 30-fold). These findings will permit future studies to identify the transcription factors involved in the normal regulatory response mechanism to hypertonicity and to identify whether and how this response is altered in a variety of pathologic states, including diabetes.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.

Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

Arrest of endotoxin-induced hypotension by transforming growth factor beta1.

Septic shock is a cytokine-mediated process typically caused by a severe underlying infection. Toxins generated by the infecting organism trigger a cascade of events leading to hypotension, to multiple organ system failure, and frequently to death. Beyond supportive care, no effective therapy is available for the treatment of septic shock. Nitric oxide (NO) is a potent vasodilator generated late in the sepsis pathway leading to hypotension; therefore, NO represents a potential target for therapy. We have previously demonstrated that transforming growth factor (TGF) beta1 inhibits inducible NO synthase (iNOS) mRNA and NO production in vascular smooth muscle cells after its induction by cytokines critical in the sepsis cascade. Thus, we hypothesized that TGF-beta1 may inhibit iNOS gene expression in vivo and be beneficial in the treatment of septic shock. In a conscious rat model of septic shock produced by Salmonella typhosa lipopolysaccharide (LPS), TGF-beta1 markedly reduced iNOS mRNA and protein levels in several organs. In contrast, TGF-beta1 did not decrease endothelium-derived constitutive NOS mRNA in organs of rats receiving LPS. We also performed studies in anesthetized rats to evaluate the effect of TGF-beta1 on the hemodynamic compromise of septic shock; after an initial 25% decrease in mean arterial pressure, TGF-beta1 arrested LPS-induced hypotension and decreased mortality. A decrease in iNOS mRNA and protein levels in vascular smooth muscle cells was demonstrated by in situ hybridization and NADPH diaphorase staining in rats treated with TGF-beta1. Thus these studies suggest that TGF-beta1 inhibits iNOS in vivo and that TGF-beta1 may be of future benefit in the therapy of septic shock.

A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity.

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 microM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent Km values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 microM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.

An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p-hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides.

Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.

Human fatty acid synthase: properties and molecular cloning.

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.

Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1.

The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(II) sepulchrate trichloride] for the electrocatalytically driven omega-hydroxylation of lauric acid. Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 [mRat4A1/mRatOR]L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase. Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor. These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals.