N-acetylcysteine modulates glutamatergic dysfunction and depressive behavior in Huntington's disease.

Glutamatergic dysfunction has been implicated in the pathogenesis of depressive disorders and Huntington's disease (HD), in which depression is the most common psychiatric symptom. Synaptic glutamate homeostasis is regulated by cystine-dependent glutamate transporters, including GLT-1 and system xc- In HD, the enzyme regulating cysteine (and subsequently cystine) production, cystathionine-γ-lygase, has recently been shown to be lowered. The aim of the present study was to establish whether cysteine supplementation, using N-acetylcysteine (NAC) could ameliorate glutamate pathology through the cystine-dependent transporters, system xc- and GLT-1. We demonstrate that the R6/1 transgenic mouse model of HD has lower basal levels of cystine, and showed depressive-like behaviors in the forced-swim test. Administration of NAC reversed these behaviors. This effect was blocked by co-administration of the system xc- and GLT-1 inhibitors CPG and DHK, showing that glutamate transporter activity was required for the antidepressant effects of NAC. NAC was also able to specifically increase glutamate in HD mice, in a glutamate transporter-dependent manner. These in vivo changes reflect changes in glutamate transporter protein in HD mice and human HD post-mortem tissue. Furthermore, NAC was able to rescue changes in key glutamate receptor proteins related to excitotoxicity in HD, including NMDAR2B. Thus, we have shown that baseline reductions in cysteine underlie glutamatergic dysfunction and depressive-like behavior in HD and these changes can be rescued by treatment with NAC. These findings have implications for the development of new therapeutic approaches for depressive disorders.

Genetic Variation of the Serine Acetyltransferase Gene Family for Sulfur Assimilation in Maize.

Improving sulfur assimilation in maize kernels is essential due to humans and animals' inability to synthesize methionine. Serine acetyltransferase (SAT) is a critical enzyme that controls cystine biosynthesis in plants. In this study, all SAT gene members were genome-wide characterized by using a sequence homology search. The RNA-seq quantification indicates that they are highly expressed in leaves, other than root and seeds, consistent with their biological functions in sulfur assimilation. With the recently released 25 genomes of nested association mapping (NAM) founders representing the diverse maize stock, we had the opportunity to investigate the SAT genetic variation comprehensively. The abundant transposon insertions into SAT genes indicate their driving power in terms of gene structure and genome evolution. We found that the transposon insertion into exons could change SAT gene transcription, whereas there was no significant correlation between transposable element (TE) insertion into introns and their gene expression, indicating that other regulatory elements such as promoters could also be involved. Understanding the SAT gene structure, gene expression and genetic variation involved in natural selection and species adaption could precisely guide genetic engineering to manipulate sulfur assimilation in maize and to improve nutritional quality.

Monitoring disulfide bond formation in the eukaryotic cytosol.

Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein-based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be -289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.

Promoter elements and regulation of expression of the cysD gene of Escherichia coli K-12.

The cysD gene, involved in cysteine biosynthesis in Escherichia coli and Salmonella typhimurium, is positively regulated by the CysB regulatory protein. The cysD promoter of E. coli K-12 in a 492-bp PstI-Eco RI fragment was sequenced. The in vivo transcription start point (tsp) for the cysD gene was determined by the methods of T4 DNA polymerase mapping and mung-bean nuclease mapping. The -10 region of the cysD promoter (TATAGT) is closely homologous to the -10 consensus sequence (TATAAT) for E. coli promoters. The -35 region of this promoter (TTCATT) is less closely related to the -35 consensus sequence (TTGACA). Several mutants were obtained by using a chain-termination method for generating unidirectional deletions. Evidence is presented for a possible CysB protein binding site around -89, thought to be involved in regulation of expression of the cysD gene.

PCR random mutagenesis into Escherichia coli serine acetyltransferase: isolation of the mutant enzymes that cause overproduction of L-cysteine and L-cystine due to the desensitization to feedback inhibition.

PCR random mutagenesis in the cysE gene encoding Escherichia coli serine acetyltransferase was employed to isolate the mutant enzymes that, due to a much less feedback inhibition by L-cysteine, cause overproduction of L-cysteine and L-cystine in the recombinant strains. The L-cysteine auxotrophic and non-utilizing E. coli strain was transformed with plasmids having the altered cysE genes. Then, several transformants overproducing L-cysteine were selected by detecting the halo formation of the L-cysteine auxotroph. The production test of amino acids and analysis of the catalytic property on the mutant enzymes suggest that the carboxy-terminal region of serine acetyltransferase plays an important role in the desensitization to feedback inhibition and the high level production of L-cysteine and L-cystine.

Selenoamino acids in tissues of rats administered inorganic selenium.

There are conflicting reports in the literature concerning the synthesis of selenoamino acids from inorganic selenium in animals, and this work was undertaken to further investigate this. Pronase digests of acetone powders of liver and kidney tissue from rats administered 75SeO3= were subjected to fractionation by cation exchange chromatography using current methods for separating the various amino acids. Very little, if any, selenocystine was found in the digests. However, good evidence was obtained for the occurrence of 2,7-diamino-4-thia-5-selenaoctanedioic acid. It is suggested that the selenocysteine portion of this compound was formed by the reduction of the selenite to selenide with its subsequent incorporation into the amino acid by the action of serine hydrolase (E C 4.2.1.22). No selenomethionine was found under the conditions of this study.

Overproduction of L-cysteine and L-cystine by expression of genes for feedback inhibition-insensitive serine acetyltransferase from Arabidopsis thaliana in Escherichia coli.

Two cDNAs encoding feedback inhibition-insensitive serine acetyltransferases of Arabidopsis thaliana were expressed in the chromosomal serine acetyltransferase-deficient and L-cysteine non-utilizing Escherichia coli strain JM39-8. The transformants produced 1600 to 1700 mg l(-1) of L-cysteine and L-cystine from glucose. The amount of these amino acids produced per cell was 30 to 60% higher than that of an E. coli strain carrying mutant serine acetyltransferase less sensitive to feedback inhibition.

Purification, characterization and identification of cysteine desulfhydrase of Corynebacterium glutamicum, and its relationship to cysteine production.

We highly purified the enzyme having L-cysteine desulfhydrase activity from Corynebacterium glutamicum. According to its partial amino acid sequence, the enzyme was identified as the aecD gene product, a C-S lyase with alpha, beta-elimination activity [I. Rossol and A. Pühler (1992) J. Bacteriol. 174, 2968-2977]. To produce L-cysteine in C. glutamicum, the Escherichia coli-altered cysE gene encoding Met256Ile mutant serine acetyltransferase, which is desensitized to feedback inhibition by L-cysteine, was introduced into C. glutamicum. When the altered cysE gene was expressed in the aecD-disrupted strain, the transformants produced approximately 290 mg of L-cysteine plus L-cystine per liter from glucose. The produced amount of these amino acids was about two-fold higher than that of the wild-type strain.

Overproduction of L-cysteine and L-cystine by Escherichia coli strains with a genetically altered serine acetyltransferase.

Organisms that overproduced L-cysteine and L-cystine from glucose were constructed by using Escherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by L-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by L-cysteine. At the same time, these transformants produced approximately 200 mg of L-cysteine plus L-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetytransferase gene. However, the production of L-cysteine and L-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production of L-cysteine plus L-cystine was markedly increased compared to production in JM39.

Assembly of MHC class I peptide complexes from the perspective of disulfide bond formation.

Assembly of functional major histocompatibility complex (MHC) class I peptide complexes within the endoplasmic reticulum is critically important for the development of an adaptive immune response. The highly regulated loading of peptides onto MHC class I molecules is controlled by a multi-component chaperone system called the MHC class I peptide loading complex. The recent identification of the thioredoxin family member ERp57 as a component of the loading complex led to an interesting question: Why is there a thiol-disulfide oxidoreductase inside a complex dedicated to inserting peptides into a receptor binding site? Most recently, specific ERp57-mediated disulfide bond rearrangements have been identified inside the loading complex. What these biochemical events mean for the peptide loading process remains a matter of conjecture. While several important questions wait to be answered, this review intends to summarize our current view of the oxidative folding of MHC class I molecules and addresses the question of how the receptor ligand interaction might be regulated by thiol-based redox reactions.

Genetic studies on SCP1-prime strains of Streptomyces coelicolor A3(2).

SCP1'-cysB carries a short chromosomal insertion: cysB was the only chromosomal locus identified on this plasmid; the plasmid was seldom lost; and a comparatively low frequency of crossing-over between the exo- and endogenote in a cysB+/cysB+ heterozygote. SCP1'-argA, uraB carries a longer chromosomal insertion: five chromosomal loci have been detected on the plasmid; it was very often lost; and there was frequent crossing-over between exo- and endogenote in heterozygous strains. Both SCP1' plasmids gave rise to plasmids closely resembling, or perhaps identical with, the original SCP1 by deletion of the chromosomal insertion. Markers argA and uraB were always deleted together from SCP1'-argA, uraB, whereas random deletion should more often have removed uraB alone. This suggests that special DNA sequences are involved in SCP1' formation. An analysis of recombination between exo- and endogenote within a double heterozygote for argA and uraB supports the hypothesis that single copies of plasmid and chromosome assort from a pool of interacting molecules at, or immediately before, spore formation. A strain carrying the SCP1'-cysB plasmid gave rise to a 'bidirectional' donor of chromosomal markers in which the plasmid had apparently integrated into the chromosome at or close to the cysB locus.

Inter-domain redox communication in flavoenzymes of the quiescin/sulfhydryl oxidase family: role of a thioredoxin domain in disulfide bond formation.

Flavoproteins of the quiescin/sulfhydryl oxidase (QSOX) family catalyze oxidation of peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX family members contain several domains, including an N-terminal thioredoxin domain (Trx) and an FAD-binding-domain (ERV) toward the C-terminus. Partial proteolysis of avian QSOX leads to two fragments, designated 30 and 60 kDa from their apparent mobilities on SDS-PAGE. The 30 kDa fragment is a monomer under nondenaturing conditions and contains a Trx domain with a CxxC sequence typical of protein disulfide isomerase (WCGHC). This QSOX fragment is not detectably glycosylated, contains no detectable FAD, and shows undetectable sulfhydryl oxidase activity. In contrast, the 60 kDa fragment is a dimeric glycoprotein that binds FAD tightly and oxidizes dithiothreitol about 1000-fold slower than intact QSOX. Reduced RNase is not a significant substrate of the 60 kDa fragment. The redox behavior of the 60 kDa flavoprotein fragment is profoundly different from that of intact QSOX. Thus, dithionite or photochemical reduction of the 60 kDa fragment leads to two-electron reduction of the FAD without subsequent reduction of the other two CxxC motifs or the appearance of a thiolate to flavin charge-transfer complex. Further characterization of the fragments and insights gained from the crystal structure of yeast ERV2p (Gross, E., Sevier, C. S., Vala, A., Kaiser, C. A., and Fass, D. (2002) Nat. Struct. Biol. 9, 61-67) suggest that the flow of reducing equivalents in intact avian QSOX is dithiol substrate --> C80/83 --> C519/522 --> C459/462 --> FAD --> oxygen. The ancient fusion of thioredoxin domains to a catalytically more limited ERV domain has produced an efficient catalyst for the direct introduction of disulfide bonds into a wide range of proteins and peptides in multicellular organisms.