Mode-of-action profiling reveals glutamine synthetase as a collateral metabolic vulnerability of M. tuberculosis to bedaquiline.

Combination chemotherapy can increase treatment efficacy and suppress drug resistance. Knowledge of how to engineer rational, mechanism-based drug combinations, however, remains lacking. Although studies of drug activity have historically focused on the primary drug-target interaction, growing evidence has emphasized the importance of the subsequent consequences of this interaction. Bedaquiline (BDQ) is the first new drug for tuberculosis (TB) approved in more than 40 y, and a species-selective inhibitor of the Mycobacterium tuberculosis (Mtb) ATP synthase. Curiously, BDQ-mediated killing of Mtb lags significantly behind its inhibition of ATP synthase, indicating a mode of action more complex than the isolated reduction of ATP pools. Here, we report that BDQ-mediated inhibition of Mtb's ATP synthase triggers a complex metabolic response indicative of a specific hierarchy of ATP-dependent reactions. We identify glutamine synthetase (GS) as an enzyme whose activity is most responsive to changes in ATP levels. Chemical supplementation with exogenous glutamine failed to affect BDQ's antimycobacterial activity. However, further inhibition of Mtb's GS synergized with and accelerated the onset of BDQ-mediated killing, identifying Mtb's glutamine synthetase as a collateral, rather than directly antimycobacterial, metabolic vulnerability of BDQ. These findings reveal a previously unappreciated physiologic specificity of ATP and a facet of mode-of-action biology we term collateral vulnerability, knowledge of which has the potential to inform the development of rational, mechanism-based drug combinations.

Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani.

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed Km value of 26.3 ± 0.4 mM for l- glutamate and Vmax value of 2.15 ± 0.07 U mg-1. Present study confirms the presence of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target.

Inhibition of glutamine synthetase by phosphinothricin leads to transcriptome reprograming in root nodules of Medicago truncatula.

Glutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.

Excessive ammonium assimilation by plastidic glutamine synthetase causes ammonium toxicity in Arabidopsis thaliana.

Plants use nitrate, ammonium, and organic nitrogen in the soil as nitrogen sources. Since the elevated CO2 environment predicted for the near future will reduce nitrate utilization by C3 species, ammonium is attracting great interest. However, abundant ammonium nutrition impairs growth, i.e., ammonium toxicity, the primary cause of which remains to be determined. Here, we show that ammonium assimilation by GLUTAMINE SYNTHETASE 2 (GLN2) localized in the plastid rather than ammonium accumulation is a primary cause for toxicity, which challenges the textbook knowledge. With exposure to toxic levels of ammonium, the shoot GLN2 reaction produced an abundance of protons within cells, thereby elevating shoot acidity and stimulating expression of acidic stress-responsive genes. Application of an alkaline ammonia solution to the ammonium medium efficiently alleviated the ammonium toxicity with a concomitant reduction in shoot acidity. Consequently, we conclude that a primary cause of ammonium toxicity is acidic stress.

Pretreatment with a glutamine synthetase inhibitor MSO delays the onset of initial seizures induced by pilocarpine in juvenile rats.

The contribution of glutamatergic transmission to generation of initial convulsive seizures (CS) is debated. We tested whether pretreatment with a glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), affects the onset and progression of initial CS by cholinergic stimulus in juvenile rats. Male rats (24 days old, Sprague Dawley) sequentially received i.p. injections of lithium-carbonate, MSO, methyl-scopolamine, and pilocarpine (Pilo). Pilo was given 150 min after MSO. Animals were continuously monitored using the Racine scale, EEG/EMG and intrahippocampal glutamate (Glu) biosensors. GS activity as measured in hippocampal homogenates, was not altered by MSO at 150 min, showed initial, varied inhibition at 165 (15 min post-Pilo), and dropped down to 11% of control at 60 min post-Pilo, whereas GS protein expression remained unaltered throughout. Pilo did neither modulate the effect of MSO on GS activity nor affect GS activity itself, at any time point. MSO reduced from 32% to 4% the number of animals showing CS during the first 12 min post-Pilo, delayed by ~6 min the appearance of electrographic seizures, and tended to decrease EMG power during ~15 min post-Pilo. The results indicate that MSO impairs an aspect of glutamatergic transmission involved in the transition from the first cholinergic stimulus to the onset of seizures. A continuous rise of extracellular Glu lasting 60 min was insignificantly affected by MSO, leaving the nature of the Glu pool(s) involved in altered glutamatergic transmission undefined.

Astroglial glutamate transporter 1 and glutamine synthetase of the nucleus accumbens are involved in the antidepressant-like effects of allopregnanolone in learned helplessness rats.

Clinical studies have demonstrated that allopregnanolone (3α5α-tetrahydroprogesterone, ALLO) has antidepressant-like effects on patients with depression. Previous studies have shown alteration of the astroglial glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) in depression, and ALLO is known to modulate glutamate release. The present study aimed to investigate whether astroglial GLT-1 and GS are indeed involved in the antidepressant-like effects of ALLO in learned helplessness (LH) rats, a validated animal model of depression. The results of this study showed that bilateral microinjection of ALLO into the lateral ventricles could normalize the levels of GLT-1 and GS in the nucleus accumbens (NAc) and of GS in the hippocampal CA1 region of LH rats. These results suggest a certain connection between the antidepressant-like effects of ALLO and the astroglial GLT-1/GS system of the NAc in LH rats.

Colonic luminal ammonia and portal blood L-glutamine and L-arginine concentrations: a possible link between colon mucosa and liver ureagenesis.

The highest ammonia concentration in the body is found in the colon lumen and although there is evidence that this metabolite can be absorbed through the colonic epithelium, there is little information on the capacity of the colonic mucosa to transfer and metabolize this compound. In the present study, we used a model of conscious pig with a canula implanted into the proximal colon to inject endoluminally increasing amounts of ammonium chloride and to measure during 5 h the kinetics of ammonia and amino acid concentration changes in the portal and arterial blood. By injecting as a single dose from 1 to 5 g ammonia into the colonic lumen, a dose-related increase in ammonia concentration in the portal blood was recorded. Ammonia concentration remained unchanged in the arterial blood except for the highest dose tested, i.e. 5 g which thus apparently exceeds the hepatic ureagenesis capacity. By calculating the apparent net ammonia absorption, it was determined that the pig colonic epithelium has the capacity to absorb 4 g ammonia. Ammonia absorption through the colonic epithelium was concomitant with increase of L-glutamine and L-arginine concentrations in the portal blood. This coincided with the expression of both glutamate dehydrogenase and glutamine synthetase in isolated colonic epithelial cells. Since L-glutamine and L-arginine are known to represent activators for liver ureagenesis, we propose that increased portal concentrations of these amino acids following increased ammonia colonic luminal concentration represent a metabolic link between colon mucosa and liver urea biosynthesis.

Pathophysiology of hepatic encephalopathy and future treatment options. / Fisiopatología y opciones de tratamiento a futuro en la encefalopatía hepática.

Understanding of the pathophysiology of hepatic encephalopathy has conditioned new treatment options. Ammonia detoxification in hepatic encephalopathy is regulated by two enzymes: glutaminase or glutamine synthetase. The first produces ammonia and the second detoxifies the ammonia, which is why treatments are aimed at glutaminase inhibition or glutamine synthetase activation. At present, we know that both enzymes are found not only in the liver, but also in the muscle, intestine, kidney, and brain. Therefore, current treatments can be directed at each enzyme at different sites. Awareness of those potential treatment sites makes different options of approach possible in the patient with hepatic encephalopathy, and each approach should be personalized.

Lithium and glutamine synthetase: Protective effects following stress.

Even though lithium is widely used as treatment for mood disorders, the exact mechanisms of lithium in the brain remain unknown. A potential mechanism affects the downstream target of the Wnt/ß-catenin signaling pathway, specifically glutamine synthetase (GS). Here, we investigate the effect of lithium on GS-promoter activity in the brain. Over seven days, B6C3H-Glultm(T2A-LacZ) mice that carry LacZ as a reporter gene fused to the GS-promotor received either daily intraperitoneal injections of lithium carbonate (25 mg/kg) or NaCl, or no treatment. Following histochemical staining of ß-galactosidase relative GS-promotor activity was measured by analyzing the intensity of the staining. Furthermore cell counts were conducted. GS-promotor activity was significantly decreased in female compared to male mice. Treatment group differences were only found in male hippocampi, with increased activity after NaCl treatment compared to both the lithium treatment and no treatment. Lithium treatment increased the overall number of cells in the CA1 region in males. Daily injections of NaCl might have been sufficient to induce stress-related GS-promotor activity changes in male mice; however, lithium was able to reverse the effect. Taken together, the current study indicates that lithium acts to prevent stress, rather affecting general GS-promoter activity.

Does concomitant glucose and glutamine supplementation change the response of glutamine synthetase to fasting in healthy adult rats?

BACKGROUND & AIMS: Glutamine synthetase (GS), a key enzyme in the glutamine synthesis, is thus crucial in glutamine homeostasis. GS is known to be up-regulated by fasting and inhibited by glutamine supplementation. The aim of this study was to determine whether the presence of glucose in glutamine supplementation with refeeding differently affects up-regulation of muscle GS by fasting in vivo in adult female rats than glutamine alone. METHODS: Muscle GS activities were assessed in 5-day-fasted female Wistar adult rats refed and supplemented with glutamine or glycine in the presence or not of glucose. RESULTS: After 5-day-fasting, the up-regulated GS activity was decreased whatever the type of amino acid supplementation (glutamine or glycine), whereas it was more decreased by supplementation with a mixture glutamine/glucose. In glycine/glucose supplemented rats, no effect of glucose supplementation was observed on GS activity. CONCLUSION: These results demonstrated that intramuscular glutamine was spared when glucose was added to glutamine supplementation in adult rats. Consequently, the role of glucose consisted in slowing down the glutamine synthesis. By contrast, glucose has no role when it was associated with glycine whose degradation does not produce energy.

Regulation of the mGluR5, EAAT1 and GS expression by glucocorticoids in MG-63 osteoblast-like osteosarcoma cells.

INTRODUCTION: Growth factors, cytokines, sex steroid hormones and glucocorticoids have differential and complex effects on skeletal metabolism. Recently, the presence of the glutamatergic (Glu) system in bone cells has provided new evidence for its possible role in bone physiology. Consequently, we have investigated the regulation of certain components of the Glu system by glucocorticoids in MG-63 osteoblast-like osteosarcoma cells, in vitro. MATERIALS AND METHODS: We characterized the effects of dexamethasone on the expression of the mGluR5, EAAT1 and GS, at mRNA and protein level, using relative quantitative RTPCR and Western blot analysis, respectively. RESULTS: We confirmed the induction of GS expression by dexamethasone published previously. In addition, we documented for the first time the expression of the mGluR5 and EAAT1 in MG-63 cells, as well as the ability of dexamethasone to upregulate the expression of the mGluR5 and EAAT1 in the MG-63 cells. CONCLUSIONS: Components of the glutamatergic system may play a role in bone pathophysiology.

The 3'-UTR of the glutamine-synthetase gene interacts specifically with upstream regulatory elements, contains mRNA-instability elements and is involved in glutamine sensing.

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine.

GC-MS based metabolite profiling implies three interdependent ways of ammonium assimilation in Medicago truncatula root nodules.

In symbiotic interaction with legume plants, bacteria termed Rhizobia can fix massive amounts of atmospheric nitrogen which is primarily provided in the form of ammonium to the host plants. Therefore, legume root nodules that house the symbiotic bacteria are ideally suited to study the process of primary ammonium assimilation. Here, we present a GC-MS based metabolite profiling analysis of Medicago truncatula root nodules (induced by the bacterium Sinorhizobium meliloti) before and after inhibition of glutamine synthetase (GS) by the chemical herbicide phosphinotricine. The primary role of GS in ammonium assimilation was revealed by drastically reduced levels of glutamine in phosphinotricine treated root nodules. In comparison to previous results of increased asparagine synthetase transcript and protein abundances in GS inhibited nodules the metabolic data revealed that decreased amounts of aspartate might preclude taking advantage of this elevated enzymatic activity. A potential role of glutamate dehydrogenase in ammonium assimilation was metabolically indicated 24 and 48 h after GS inhibition. Therefore, nodule ammonium assimilation might in principle involve three interdependent metabolic pathways which are adjusted to control basic nitrogen metabolism.

Limitations to the development of humanized antibody producing Chinese hamster ovary cells using glutamine synthetase-mediated gene amplification.

Recombinant Chinese hamster ovary (CHO) cells expressing a humanized antibody were obtained by transfection of an antibody expression vector (pKC-GS-HC-huS) into CHO-K1 cells and subsequent glutamine synthetase (GS)-mediated gene amplification in media containing different concentrations of methionine sulfoximine (MSX). Concentrations consisted of 25, 200, 500, and 1000 microM of MSX. The highest producer (HP) subclones were isolated from each MSX level by the limiting dilution method and were characterized with respect to antibody production. No positive relationship was observed between specific antibody productivity (q(Ab)) and MSX concentration. Furthermore, it was found that the antibody production stability of these subclones was very poor even in the presence of selection pressure. During long-term cultures in the presence of the corresponding concentrations of MSX, q(Ab) of all HP subclones significantly decreased for the first six passages and thereafter stabilized. Southern and slot blot analyses showed that the loss of antibody gene copies was only partially responsible for the decreased q(Ab). Fluorescence in situ hybridization (FISH) analysis revealed some cytogenetic features indicative of antibody production instability. Unstable chromosomal structures including dicentrics, rings, and extremely long chromosomes were observed. Amplified sequences enclosed in nuclear projections were often observed. The telomeric repeat sequence, which may be involved in the stabilization of amplified arrays, was found to be absent at the ends of most marker chromosomes. Furthermore, FISH analysis revealed that the overall chromosome content was duplicated in some HP subclones. When metaphase of 12 high producing parental clones was examined, the frequency of occurrence of the polyploidy was 25%. Taken together, the data obtained here suggests that instability could be a concern in the development of CHO cells with GS-mediated gene amplification.

Effect of olanzapine treatment on platelet glutamine synthetase-like protein and glutamate dehydrogenase immunoreactivity in schizophrenia.

According to contemporary views, the glutamatergic system is implicated in the pathogenesis of schizophrenia, and atypical neuroleptics exert their effects (at least partially) through the glutamatergic system. Immunoreactive glutamate-metabolising enzymes, such as glutamine synthetase-like protein (GSLP) and two glutamate dehydrogenase isoenzymes (GDH), have been discovered in human platelets. The amount of GSLP in the platelets of 40 chronic patients with schizophrenia was found to be significantly higher than in 33 controls (consistent with our previous finding of increased amounts of GSLP in the prefrontal cortex of chronic schizophrenia patients). Moreover, survival analysis of the group of patients treated with olanzapine for 28 weeks showed that the larger amount of GSLP measured in platelets before treatment, the shorter the treatment time needed to achieve a positive clinical response (defined a priori as > or = 20% reduction in PANSS total score from the initial level before the treatment). Hence, GSLP level may serve as a predictor of the treatment duration to achieve a positive outcome with olanzapine. Both GSLP and GDH were found significantly changed in the course of treatment; hence, treatment with olanzapine influences the amounts of glutamate-metabolising enzymes in the platelets of chronic schizophrenia patients.

Low- and high-activity forms of glutamine synthetase from Rhodospirillum rubrum: sensitivity to feed-back effectors and activation of the low-activity form.

Glutamine synthetase from Rhodospirillum rubrum can be isolated in two forms, with low and high activity, respectively, depending on the concentration of combined nitrogen in the medium before harvest. The two forms have been studied with respect to their dependence on Mn2+ and Mg2+ in both the transferase and the biosynthetic assay. There is no difference in pH optimum between the forms in the biosynthetic assay. In addition the pH-optima for the two cations studied are very close, 7.4 (Mg2+) and 7.2 (Mn2+). It also shows that the activity of the low-activity form is higher than that of the high-activity form in the Mn(2+)-dependent biosynthetic assay. The two forms of Rsp. rubrum glutamine synthetase have also been studied with respect to their sensitivity towards feed-back effectors. In the transferase assay both forms are inhibited to essentially the same degree by alanine, glycine, histidine, AMP, CTP and UTP, CTP being the most effective of the nucleotides and of the amino acids alanine causes the highest inhibition. In the biosynthetic assay these effectors show different degrees of inhibition on the two different forms; the high-activity form being the most sensitive. The results are discussed in relation to properties of glutamine synthetase from Escherichia coli and other phototropic bacteria in which regulation of glutamine synthetase is known to be due to adenylylation. It is also shown that the low-activity form of Rsp. rubrum glutamine synthetase can be activated in crude extracts in a reaction that is inhibited by glutamine.

Disruption of glucocorticoid receptor exon 2 yields a ligand-responsive C-terminal fragment that regulates gene expression.

Mice in which exon 2 of the glucocorticoid receptor (GR) has been disrupted [GR exon 2 knockout (GR2KO)] have been used as a model to study the requirement for this receptor in a number of biological systems. A recent report showed that these mice actually express a truncated ligand-binding GR fragment, prompting us to ask whether this mutation truly results in a glucocorticoid-insensitive phenotype. Based on cDNA microarray analysis of fetal thymocytes, we found that glucocorticoids were able to enhance or repress activation-induced gene expression in GR2KO and wild-type thymocytes to a similar degree. Moreover, although changes in gene expression induced by glucocorticoids alone were blunted, the expression of a substantial number of genes in GR2KO thymocytes was modulated by stimulation with glucocorticoids. Among these genes, as confirmed by quantitative real-time PCR, was the classic glucocorticoid-responsive gene glutamine synthetase as well as genes implicated in T cell development and function such as IL-7 receptor alpha-chain and glucocorticoid-induced leucine zipper (GIL2). Thus, the truncated C-terminal GR2KO product, which lacks the major transactivation domain, retains, to a large extent, the ability to regulate gene expression both positively and negatively in a ligand-responsive manner when expressed in vivo.

Cyclic adenosine-3',5'-monophosphate-mediated activation of a glutamine synthetase composite glucocorticoid response element.

The glutamate synthetase gene (GS) contains a composite glucocorticoid response element (cGRE) comprised of a GRE and an adjacent element with features of both a cAMP-response element (CRE) and a 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE). The CRE/TRE element of the cGRE contributed to two modes of transcriptional activation: 1) enhancement of the response to cortisol and 2) a synergistic response to cortisol and increased cAMP. COS-7 cells transfected with a cGRE-luciferase construct show minimal expression under basal conditions or forskolin treatment. After cortisol treatment, luciferase activity from the cGRE is enhanced 4- to 8-fold greater than the GRE portion of the cGRE or a GRE from the tyrosine aminotransferase gene. Treatment with both forskolin and cortisol produced a 2- to 4-fold synergistic response over cortisol alone. Synergy is also seen with 8-bromo-cAMP, is specific for the cGRE, and occurs in a number of established cell lines. Elimination of the GRE or CRE/TRE reduces the synergy by 70-100%. Altering the CRE/TRE to GRE spacing changed both enhancement and synergy. Moving the elements 3 bp closer or extending 15 bp reduced enhancement. Synergy was markedly reduced when elements were one half of a helical turn out of phase. Western blots verified that CREB (cAMP-responsive binding protein) and ATF-1 (activating transcription factor-1) binds to the cGRE sequence. A specific dominant negative inhibitor of the CREB family, A-CREB, reduced synergy by 50%. These results suggest that the GS cGRE can potentially integrate signaling from both the cAMP and glucocorticoid receptor transduction pathways and that CREB/ATF-1 may play an important role in this process.

Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity does not determine the co-chaperonin requirement.

One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.

Peroxidase activity of a TSA-like antioxidant protein from a pathogenic amoeba.

The 29 kDa surface protein of Entamoeba histolytica is an abundant antigenic protein expressed by pathogenic strains of this organism. The protein is a member of a widely-dispersed group of homologues which includes at least two cysteinyl peroxidases, Salmonella typhimurium alkyl hydroperoxidase C-22 protein (AhpC) and Saccharomyces cerevisiae thiol-specific antioxidant protein (TSA). Here, for the first time in a pathogenic eukaryote, we have demonstrated that the amoebic protein also possesses peroxidatic and antioxidant activities in the presence of reductants such as dithiothreitol or thioredoxin reductase plus thioredoxin. Although the S. typhimurium AhpF flavoprotein was not an effective reductant of the amoebic TSA protein, one inhibitory monoclonal antibody directed toward amoebic TSA was also partially inhibitory toward reduced but not oxidized bacterial AhpC. These antioxidant proteins are likely to be important not only in general cell protection, but also in the promotion of infection and invasion by these pathogenic organisms through protection against oxidative attack by activated host phagocytic cells.