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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.

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 x c - 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 x c - 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 x c - 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., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

The flavoenzyme Ero1p produces disulfide bonds for oxidative protein folding in the endoplasmic reticulum. Disulfides generated de novo within Ero1p are transferred to protein disulfide isomerase and then to substrate proteins by dithiol-disulfide exchange reactions. Despite this key role of Ero1p, little is known about the mechanism by which this enzyme catalyzes thiol oxidation. Here, we present the X-ray crystallographic structure of Ero1p, which reveals the molecular details of the catalytic center, the role of a CXXCXXC motif, and the spatial relationship between functionally significant cysteines and the bound cofactor. Remarkably, the Ero1p active site closely resembles that of the versatile thiol oxidase module of Erv2p, a protein with no sequence homology to Ero1p. Furthermore, both Ero1p and Erv2p display essential dicysteine motifs on mobile polypeptide segments, suggesting that shuttling electrons to a rigid active site using a flexible strand is a fundamental feature of disulfide-generating flavoenzymes.

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.

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.

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.

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.

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.

COS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel alphaE chain and those of the more abundant subclass whose alpha chains lack alphaE's globular C-terminus. That region, referred to as the alphaEC domain, is closely related to the ends of beta and gamma chains of fibrinogen (betaC and gammaC). Transfection of COS cells with alphaE, beta, and gamma cDNAs alone results in secretion of the symmetrical molecule (alphaEbetagamma)2, also known as Fib420. Cotransfection with cDNA for the shorter alpha chain yielded secretion of both (alphabetagamma)2 and (alphaEbetagamma)2 but no mixed molecules of the structure alphaalphaE(betagamma)2. Exploiting the COS cells' fidelity with regard to Fib420 production, identification was made of the highly conserved Asn667 as the sole site of N-linked glycosylation in the alphaE chain. No evidence from Cys --> Ser replacements was found for interchain disulfide bridges involving the four cysteines of the alphaEC domain. However, for fibrinogen secretion, the alphaE, beta, and gamma subunits do exhibit different requirements for integrity of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dissimilar structural roles in the process of fibrinogen assembly., (Copyright 1998 by The American Society of Hematology)

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.

Heparin-binding neurite-promoting factor (HBNF) is a highly basic, cysteine-rich 136-residue protein, and a member of a new class of heparin-binding proteins. It exhibits a neurite-outgrowth promoting activity and its expression is both temporally and spacially regulated during fetal and postnatal development. A high interspecies sequence conservation suggests important, presently unknown, biological functions. HBNF is structurally and most likely functionally related to the product of a developmentally regulated gene, MK (midkine). To elucidate biological roles of these proteins, recombinant forms of the proteins were produced. Expression of human recombinant HBNF and MK in Escherichia coli lead to the formation of insoluble aggregated protein that accounted for about 25% of the total cellular protein. Homogeneous, monomeric forms of each protein were recovered from inclusion bodies by reduction with dithiothreitol and solubilization in 8 M urea. Refolding of the reduced and denatured protein occurred upon dialysis at pH 7.4. Human recombinant (hr) HBNF and hrMK prepared in this manner were further purified by heparin affinity chromatography. Chromatographic evidence demonstrates that refolding and concomitant disulfide bond formation in hrHBNF proceeds in high yield with minimal formation of stable nonnative disulfides. Studies on the redox status of the 10 cysteine residues of bovine brain HBNF and the refolded recombinant protein indicate that all cysteines are engaged in disulfide bond formation. The disulfide arrangements for the recombinant protein were found to be identical to those in the native protein isolated from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)

The formation of disulfide bridges in membrane and secretory proteins occurs during the protein folding process in the endoplasmic reticulum of eukaryotic cells and the periplasm of prokaryotes. The formation of disulfide bridges is facilitated by the thiol-disulfide oxidoreductases, protein disulfide isomerase (PDI) in eukaryotes and dsbA in prokaryotes. Structure-function analysis of these soluble proteins demonstrates that their active sites are sequences with similarity to the active site of the redox protein thioredoxin. Although these active sites share homology with thioredoxin, it is evident that other structural determinants change the redox characteristics of these proteins to enable them to facilitate the formation of correct disulfide bridging in the nascent protein. The analysis of structure-function relationships of PDI and dsbA have indicated that these thiol-disulfide oxidoreductases act as protein oxidants to facilitate the formation of disulfides during the folding process. The ability of PDI and dsbA to catalyze the formation and/or isomerization of disulfide bridging establishes their usefulness in facilitating the in vitro and in vivo folding of proteins. Protocols for the purification and assay of PDI activity have been described. Systems for the expression of PDI in Escherichia coli and Spodoptera frugiperda cells have been developed which may prove useful in the expression of recombinant proteins.

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.

A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recA1 early and recipients carrying wild-type or mutant alleles. It was found that recA+ but not recA1 complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA- strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for lambda. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recArut not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process.

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.

Long term human lymphoid cells from normal subjects were able to convert [35S]homocysteine to [35S]-cystine. The labeled cystine was found incorporated into proteins. Cells cultured from a patient with vitamin B6-unresponsive cystathioninuria, which contain no measurable cystathionase activity, were unable to perform this conversion. This is the first direct demonstration of transsulfuration by diploid cells in culture.

Induction of a heat-inducible abnormal lambda prophage inserted between lysA and thyA in Escherichia coli resulted in a number of auxotrophic mutants in the surviving cured-cell populations. These mutants could not be accounted for by deletions arising on formation of lambda hybrid particles carrying regions adjacent to the insertion site. The properties of these mutants, which were almost all spontaneously revertable, have been described and mapped by F' episome complementation. Tentatively, it was suggested that induction of the lambda lysogen leads to a mutagenic state.

Six ethionine-resistant (Etr) regulatory mutants of Saccharomycopsis lipolytica Sl/1 overproducing methionine have been isolated. Five of them are also resistant to seleno-methionine. The activity of homocysteine synthase (O-acetyl-L-hormoserine-acetate lyase, adding hydrogen sulfide) is derepressed in these mutants and is not susceptible to the methionine-mediated repression. The pool of free methionine in Etr mutants is enhanced 1.5 to 18 times, and incorporation of 35S into methionine is 1.5 to 50 times higher than that in the wild strain. Neither accumulation of endogenous free methionine in Etr mutants nor the uptake of exogenous methionine is accompanied by an increase in the S-adenosylmethionine pool. This implies compartmentation of methionine metabolism in S. lipolytica.

Certain continuous lymphoid and myeloid tumor cell lines of rodent origin are unable to grow in tissue culture in the absence of pre-formed L-cystine (CYS). In contrast, three NZB murine lymphoid cell lines obtained from NZB mice free of hematopoietic neoplasm can grow as well in cystine-deficient media containing L-cystathionine (CSN), the immediate precursor of CYS in the biosynthetic pathway, as in cystine sufficient medium. The former class of cells is, therefore, CYS auxotrophs (CYS-) and the latter CYS prototrophs (CYS+). Compared to CYS+ cells, the CYS- lines appear to be relatively deficient in the enzyme cystathionase, which catalyzes the cleavage of CSN to CYS and alpha-ketobutyrate. Using protein synthetic capacity as a criterion, normal thymocytes from mixed-bred Swiss mice behave like CYS prototrophs, while those from littermates bearing Moloney type C virus-induced thymic tumors behave like CYS auxotrophs. The former are also characterized by substantially higher levels of cystathionase than the latter. Extracts of thymocytes from tumor-free AKR mouse thymus are also characterized by higher levels of cystathionase activity than extracts of spontaneous AKR thymomas. Exogenous in vitro type C virus infection of a CYS+ cell results in vigorous virus production but no concomitant reduction in cystathionase activity. Thus viral replication alone in any random lymphoid cell is not sufficient to alter the enzyme level. The data therefore suggests that CYS auxotrophy may closely accompany neoplastic transformation of certain hematopoietic cells in vivo, including that induced by certain "thymic" type C viruses.

Microorganisms able to form L-cysteine from DL-2-amino-delta2-thiazoline-4-carboxylic acid (DL-ATC), a chemical intermediate in the synthesis of DL-cysteine, were isolated from soil samples and classified as Pseudomonas sp., Pseudomonas cohaerens, P. desmolytica, and P. ovalis. Thirteen L-cysteine-producing bacteria were also found in among 463 stock cultures representing 37 genera. These were Achromobacter delmarvae. Alcaligenes denitrificans, Bacillus brevis, Brevibacterium flavum, Enterobacter aerogenes, Erwinia carotovora, Escherichia coli, Micrococcus sodonensis, Myocoplana dimorpha, Sarcina lutea, Serratia marcescens, Flavobacterium acidoficum, and Pseudomonas ovalis. In the presence of intact cells of Pseudomonas sp. AJ 3854, 6.1 mg of L-cysteine and/or L-cystine per ml was produced from 10 mg of DL-ATC-3H2O per ml in a molar yield of 100%. This finding suggests that racemization and asymmetric hydrolysis occurred simultaneously in this incubation mixture. After the complete oxidation of cysteine to cystine by aeration in the presence of ferrous ion, crystalline cystine was isolated; its configuration was the L isomer based on data from X-ray diffraction, microbioassay, and optical rotation studies.

Cyst(e)ine synthesized by microorganisms in the caecum of sheep was labelled following the infusion of Na2 35SO4 into the terminal ileum. [35S]Cyst(e)ine activity was detected in the faeces, but not in plasma or wool. Appreciable absorption of 35S, presumably in the form of sulphide, into the circulation occurred, and its presence in saliva was demonstrated. It was concluded that nutritionally negligible quantities of cyst(e)ine are likely to be absorbed from the ovine large intestine.

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.