Methionine transport in S37 cells. Substrate-dependent function of amino acid transport system A in exchange processes.

Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine. Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that bothe systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. ALPHA-Aminoisobutyric acid and N-methyl-alpha-aminoisobutyric acid both stimulated efflux of labeled N-methyl-alpha-aminoisobutyric acid from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available.     

Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide

Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO(+/-) mice) were crossed to generate CDO(-/-), CDO(+/-), and CDO(+/+) mice. CDO(-/-) mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO(-/-) mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO(-/-) mice than in CDO(+/-) or CDO(+/+) mice, and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO(-/-) mice. H(2)S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H(2)S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H(2)S/sulfane sulfur levels and facilitate the use of H(2)S as a signaling molecule.     

Effects of tumor necrosis factor-alpha on insulin stimulated amino acid transport in cultured rat hepatocytes

It has been suggested that tumor necrosis factor alpha (TNF-alpha) plays a pivotal role in the pathogenesis of insulin resistance. It could act directly or indirectly in liver. The aim of this study was to determine direct short time (4 h) and long time (24 h) action of TNF-alpha on amino acid transport in cultured rat hepatocytes and possible role of protein kinase C (PKC) in insulin signal pathway and insulin resistance. Hepatocytes were isolated by a modified collagenase perfusion technique and cultured for 24 h in M 199 medium. In the presence of insulin basal alpha-amino isobutyric acid (AIB) uptake was increased 55%. TNF-alpha in short time action did not change basal AIB transport, but significantly (25%) increased insulin stimulated uptake. Short time action of TNF-alpha was ameliorated by phorbol ester treatment. These results indicated that PKC activation is important in insulin signaling and TNF-alpha action. TNF-alpha acting directly did not cause insulin resistance in cultured hepatocytes.     

Methionine transport in S37 cells. Substrate-dependent function of amino acid transport system A in exchange processes

Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine.Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through both systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that both systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. α-Aminoisobutyric acid and $\text{N-}\text{methyl}\text{-α-}\text{aminoisobutyric acid}$ both stimulated efflux of labeled $\text{N-}\text{methyl}\text{-α-}\text{aminoisobutyric acid}$ from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available.     

Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.     

Differential effect of the amino acid cystine in cultured mammalian and bacterial cells exposed to oxidative stress.

The effect of cystine in the cytotoxic response of cultured Chinese hamster ovary and Escherichia coli cells to challenge with hydrogen peroxide has been investigated. It was found that this amino acid could either protect or sensitize cells, depending on the cellular system. In fact, although a reduction in the growth-inhibitory effect of hydrogen peroxide was observed in mammalian cells, a marked increase in the susceptibility to oxidative stress was induced by cystine in bacteria. None of the amino acid precursors of glutathione, e.g., glutamate, glycine or cysteine, afforded protection in the mammalian cell system, whereas cysteine, but not glycine or glutamate, markedly sensitized bacteria to hydrogen peroxide-induced cell killing. In mammalian cells, methionine, an amino acid which is converted to cysteine, was also unable to modify the oxidative response. The results presented indicate that cystine displays differential effects in oxidatively injured mammalian or bacterial cells and suggest that the mechanism whereby the amino acid modulates the lethal action of hydrogen peroxide differs in the two cellular systems.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3)

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.     

Collagenase activity in cultures of rat prostate carcinoma.

A specific collagenase (EC 3.4.24.3) has been found and purified from serum-free culture medium of 11095 epidermoid carcinoma of rat prostate. The molecular weight of this collagenase was estimated at 71 000 and the pH optimum was approx. 7. At 26 degrees C, the collagenase cleaved collagen at a site 3/4 the length from the N-terminus. At 37 degrees C, this collagenase degraded collagen to smaller peptides. The enzyme activity was inhibited by serum, cysteine and EDTA, but not by protease inhibitors. The presence of collagenase in rat tumor tissue suggests that this enzyme might play a significant role in tissue invasion by cancer cells.     

Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low Km) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low Km of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys160 of LAT1 and Cys110 of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl2 with a Ki of 0.56+/-0.11 microM. The inhibitory effect of HgCl2 for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl2 for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys88 and Cys439, altered amino acid transport. The Vmax was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys439 residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo

Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C(26)S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C(26)S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.     

Study of the effect of the administration of Cd(II), cysteine, methionine, and Cd(II) together with cysteine or methionine on the conversion of xanthine dehydrogenase into xanthine oxidase

Cadmium is known as to be a potent pulmonary carcinogen to human beings and to induce prostate tumor. The sequestration of cadmium, an extremely toxic element to living cells, which is performed by biological ligands such as amino acids, peptides, proteins or enzymes is important to minimize its participation in such deleterious processes. The synthesis of metallothionein is induced by a wide range of metals, in which cadmium is a particularly potent inducer. This protein is usually associated with cadmium exposure in man. Because metallothioneins may act as a detoxification agent for cadmium and chelation involves sulfur donor atoms, we administered only cadmium, cysteine, or methionine to rats and also each of these S-amino acids together with cadmium and measured the production of superoxide radicals derived from the conversion of xanthine dehydrogenase to xanthine oxidase. It could be seen in this work that the presence of cadmium enhances this conversion. However, its inoculation with cysteine or methionine almost completely diminishes this effect and this can be the result of the fact that these amino acids complex Cd(II). Thus, these compounds can be a model of the action of metallothionein, removing cadmium from circulation and preventing its deleterious effect.     

Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation

4-Nitrobenzyl [35S]mercaptan S-sulfonic acid ([35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [35S]NBM in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37 degrees C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([35S]NBM)-cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formation of [35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37 degrees C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.     

TA1/LAT-1/CD98 light chain and system L activity, but not 4F2/CD98 heavy chain, respond to arginine availability in rat hepatic cells. Loss Of response in tumor cells

Tumor associated gene-1/L amino acid transporter-1 (TA1/LAT-1) was recently identified as a light chain of the CD98 amino acid transporter and cellular activation marker. Our previous studies with primary rat hepatocyte cultures demonstrated that TA1 RNA levels were responsive to media amino acid concentrations, suggesting adaptive regulation. High level TA1 expression associated with transformed cells also suggested a role in tumor progression. The present study examined the relationship of TA1/CD98 expression, adaptive response, and associated amino acid transport to neoplastic transformation using a panel of well characterized rat hepatic cell lines. We found 1) increased expression of TA1 in response to amino acid depletion, specific for arginine but not glutamine; 2) loss of TA1 response to arginine in gamma-glutamyl transpeptidase-positive transformed and tumorigenic cells; 3) no appreciable response of 4F2/CD98 heavy chain to arginine levels; and 4) correlation of system L amino acid transport activity in response to arginine with changes in TA1/LAT-1 mRNA but not total immunoreacting protein. Our results suggest this CD98 light chain may act as an environmental sensor, responding to amino acid availability and that its regulation is complex. We hypothesize that altered TA1 expression is an early event in hepatocarcinogenesis giving neoplastic cells a growth or survival advantage, particularly under conditions of limited amino acid availability.     

Sulfate transport in cultured tobacco cells

Sulfate transport by tobacco (Nicotiana tabacum L. var. Xanthi) cells cultured on either l-cysteine or sulfate as a sole sulfur source was measured. The transport rate on either sulfur source was low during pre-exponential growth, increased during exponential growth, and was maximal in late exponential cells. The initial increase in transport rate was correlated with a decline in the intracellular sulfate, but was not correlated with the amino acid content of the cells which remained relatively constant before the depletion of the endogenous sulfate pool. The previously reported inhibition of sulfate transport by l-cysteine was shown to be caused by an elevation in intracellular sulfate resulting from the degradation of cysteine to sulfate. It is proposed that the intracellular sulfate pool is the major factor regulating the entry of sulfate into tobacco cells.     

Eicosapentaenoic acid reduces the invasive and metastatic activities of malignant tumor cells

Recent studies have shown that the cyclooxygenase and the 5-lipoxygenase pathways of arachidonic acid, are required for the invasive and metastatic activity of certain tumor cells. We show here that malignant murine melanoma and human fibrosarcoma cells cultured in media supplemented with eicosapentaenoic acid show a dose and time dependent decrease in invasiveness, in collagenase IV production and in the case of the murine cells, a reduced ability to metastasize to the lung after intravenous injection. It was also shown that a metabolite of eicosapentaenoic acid was less potent than the comparable arachidonic acid metabolite in restoring collagenase IV production and invasiveness after inhibition of the lipoxygenase pathway. These studies indicate that such supplements have the potential to reduce the metastasis of certain tumor cells, converting them to benign status.     

Yct1p, a novel, high-affinity, cysteine-specific transporter from the yeast Saccharomyces cerevisiae

Cysteine transport in the yeast Saccharomyces cerevisiae is mediated by at least eight different permeases, none of which are specific for cysteine. We describe a novel, high-affinity, (K(m) = 55 microM), cysteine-specific transporter encoded by the ORF YLL055w that was initially identified by a combined strategy of data mining, bioinformatics, and genetic analysis. Null mutants of YLL055w, but not of the other genes encoding for transporters that mediate cysteine uptake such as GAP1, GNP1, MUP1, or AGP1 in a met15Delta background, resulted in a growth defect when cysteine, at low concentrations, was provided as the sole sulfur source. Transport experiments further revealed that Yll055wp was the major contributor to cysteine transport under these conditions. The contributions of the other transporters became relevant only at higher concentrations of cysteine or when YLL055w was either deleted or repressed. YLL055w expression was repressed by organic sulfur sources and was mediated by the Met4p-dependent sulfur regulatory network. The results reveal that YLL055w encodes the principal cysteine transporter in S. cerevisiae, which we have named YCT1 (yeast cysteine transporter). Interestingly, Yct1p belongs to the Dal5p family of transporters rather than the amino acid permease family to which all the known amino acid transporters belong.     

Unique characteristics of rat spleen lymphocyte, L1210 lymphoma and HeLa cells in glutathione biosynthesis from sulfur-containing amino acids

Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]-cysteine for GSH synthesis. Lymphocytes preferentially utilized cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathway for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including gamma-glutamyl transpeptidase, GSH S-transferase and gamma-cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).