Age modulates liver responses to asparaginase-induced amino acid stress in mice.

Asparaginase is an amino acid-depleting agent used to treat blood cancers. Metabolic complications due to asparaginase affect liver function in humans. To examine how the liver response to asparaginase changes during maturity to adulthood, here we treated juvenile (2-week), young adult (8-week), and mature adult (16-week) mice with drug or excipient for 1 week and conducted RNA-Seq and functional analyses. Asparaginase reduced body growth and liver mass in juveniles but not in the adult animals. Unbiased exploration of the effect of asparaginase on the liver transcriptome revealed that the integrated stress response (ISR) was the only molecular signature shared across the ages, corroborating similar eukaryotic initiation factor 2 phosphorylation responses to asparaginase at all ages. Juvenile livers exhibited steatosis and iron accumulation following asparaginase exposure along with a hepatic gene signature indicating that asparaginase uniquely affects lipid, cholesterol, and iron metabolism in juvenile mice. In contrast, asparaginase-treated adult mice displayed greater variability in liver function, which correlated with an acute-phase inflammatory response gene signature. Asparaginase-exposed adults also had a serine/glycine/one-carbon metabolism gene signature in liver that corresponded with reduced circulating glycine and serine levels. These results establish the ISR as a conserved response to asparaginase-mediated amino acid deprivation and provide new insights into the relationship between the liver transcriptome and hepatic function upon asparaginase exposure.

Insight into the thermostability of thermophilic L-asparaginase and non-thermophilic L-asparaginase II through bioinformatics and structural analysis.

Thermostability plays an important role in the application of L-asparaginase in the pharmaceutical and food industries. Understanding the key residues and structures that influence thermostability in L-asparaginase is necessary to obtain suitable L-asparaginase candidates. In this study, special residues and structures that altered thermostability in thermophilic L-asparaginase and non-thermophilic L-asparaginase II were identified. Interchanging these special residues and structures of L-asparaginases from the four strains, that is, Pyrococcus yayanosii CH1 (PYA), Thermococcus gammatolerans (TGA), Bacillus subtilis (BSA II), and Escherichia coli (ECA II), revealed the 51st and 298th residues of PYA (corresponding to 57th, 305th residues of ECA II) as the key residues responsible for thermal stability of thermophilic L-asparaginase and non-thermophilic L-asparaginase II. Moreover, the C terminal tightness, loop rigidity, and low surface charge around activity sites were of great significance to the thermostability of L-asparaginase. This study therefore revealed the crucial amino acid residues and structures responsible for the difference in thermostability of the thermophilic and non-thermophilic L-asparaginase and provides a reference for engineering thermostability in L-asparaginase II.

Mechanisms of cell death induced by arginase and asparaginase in precursor B-cell lymphoblasts.

Arginase has therapeutic potential as a cytotoxic agent in some cancers, but this is unclear for precursor B acute lymphoblastic leukaemia (pre-B ALL), the commonest form of childhood leukaemia. We compared arginase cytotoxicity with asparaginase, currently used in pre-B ALL treatment, and characterised the forms of cell death induced in a pre-B ALL cell line 697. Arginase and asparaginase both efficiently killed 697 cells and mature B lymphoma cell line Ramos, but neither enzyme killed normal lymphocytes. Arginase depleted cellular arginine, and arginase-treated media induced cell death, blocked by addition of arginine or arginine-precursor citrulline. Asparaginase depleted both asparagine and glutamine, and asparaginase-treated media induced cell death, blocked by asparagine, but not glutamine. Both enzymes induced caspase cleavage and activation, chromatin condensation and phosphatidylserine exposure, indicating apoptosis. Both arginase- and asparaginase-induced death were blocked by caspase inhibitors, but with different sensitivities. BCL-2 overexpression inhibited arginase- and asparaginase-induced cell death, but did not prevent arginase-induced cytostasis, indicating a different mechanism of growth arrest. An autophagy inhibitor, chloroquine, had no effect on the cell death induced by arginase, but doubled the cell death induced by asparaginase. In conclusion, arginase causes death of lymphoblasts by arginine-depletion induced apoptosis, via mechanism distinct from asparaginase. Therapeutic implications for childhood ALL include: arginase might be used as treatment (but antagonised by dietary arginine and citrulline), chloroquine may enhance efficacy of asparaginase treatment, and partial resistance to arginase and asparaginase may develop by BCL-2 expression. Arginase or asparaginase might potentially be used to treat Burkitt lymphoma.

Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming.

Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative L-asparaginase exhibits L-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that L-asparagine is a nutrient important for T cell activation and that L-asparagine deprivation, such as that mediated by the Salmonella Typhimurium L-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and L-lactate secretion. We also show that L-asparagine deprivation mediated by the Salmonella Typhimurium L-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming.

The periplasmic enzyme, AnsB, of Shigella flexneri modulates bacterial adherence to host epithelial cells.

S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB) to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.

Distinct physiological roles for the two L-asparaginase isozymes of Escherichia coli.

Escherichia coli expresses two L-asparaginase (EC 3.5.1.1) isozymes: L-asparaginse I, which is a low affinity, cytoplasmic enzyme that is expressed constitutively, and L-asparaginase II, a high affinity periplasmic enzyme that is under complex co-transcriptional regulation by both Fnr and Crp. The distinct localisation and regulation of these enzymes suggest different roles. To define these roles, a set of isogenic mutants was constructed that lacked either or both enzymes. Evidence is provided that L-asparaginase II, in contrast to L-asparaginase I, can be used in the provision of an anaerobic electron acceptor when using a non-fermentable carbon source in the presence of excess nitrogen.

Coupled amino acid deamidase-transport systems essential for Helicobacter pylori colonization.

In addition to their classical roles as carbon or nitrogen sources, amino acids can be used for bacterial virulence, colonization, or stress resistance. We found that original deamidase-transport systems impact colonization by Helicobacter pylori, a human pathogen associated with gastric pathologies, including adenocarcinoma. We demonstrated that l-asparaginase (Hp-AnsB) and gamma-glutamyltranspeptidase (Hp-gammaGT) are highly active periplasmic deamidases in H. pylori, producing ammonia and aspartate or glutamate from asparagine and glutamine, respectively. Hp-GltS was identified as a sole and specialized transporter for glutamate, while aspartate was exclusively imported by Hp-DcuA. Uptake of Gln and Asn strictly relies on indirect pathways following prior periplasmic deamidation into Glu and Asp. Hence, in H. pylori, the coupled action of periplasmic deamidases with their respective transporters enables the acquisition of Glu and Asp from Gln and Asn, respectively. These systems were active at neutral rather than acidic pH, suggesting their function near the host epithelial cells. We showed that Hp-DcuA, the fourth component of these novel deamidase-transport systems, was as crucial as Hp-gammaGT, Hp-AnsB, and Hp-GltS for animal model colonization. In conclusion, the pH-regulated coupled amino acid deamidase-uptake system represents an original optimized system that is essential for in vivo colonization of the stomach environment by H. pylori. We propose a model in which these two nonredundant systems participate in H. pylori virulence by depleting gastric or immune cells from protective amino acids such as Gln and producing toxic ammonia close to the host cells.

Co-occurrence of both L-asparaginase subtypes in Arabidopsis: At3g16150 encodes a K+-dependent L-asparaginase.

L-asparaginases (EC 3.5.1.1) are hypothesized to play an important role in nitrogen supply to sink tissues, especially in legume-developing seeds. Two plant L-asparaginase subtypes were previously identified according to their K(+)-dependence for catalytic activity. An L-asparaginase homologous to Lupinus K(+)-independent enzymes with activity towards beta-aspartyl dipeptides, At5g08100, has been previously characterized as a member of the N-terminal nucleophile amidohydrolase superfamily in Arabidopsis. In this study, a K(+)-dependent L-asparaginase from Arabidopsis, At3g16150, is characterized. The recombinants At3g16150 and At5g08100 share a similar subunit structure and conserved autoproteolytic pentapeptide cleavage site, commencing with the catalytic Thr nucleophile, as determined by ESI-MS. The catalytic activity of At3g16150 was enhanced approximately tenfold in the presence of K(+). At3g16150 was strictly specific for L-Asn, and had no activity towards beta-aspartyl dipeptides. At3g16150 also had an approximately 80-fold higher catalytic efficiency with L-Asn relative to At5g08100. Among the beta-aspartyl dipeptides tested, At5g08100 had a preference for beta-aspartyl-His, with catalytic efficiency comparable to that with L-Asn. The phylogenetic analysis revealed that At3g16150 and At5g08100 belong to two distinct subfamilies. The transcript levels of At3g16150 and At5g08100 were highest in sink tissues, especially in flowers and siliques, early in development, as determined by quantitative RT-PCR. The overlapping spatial patterns of expression argue for a partially redundant function of the enzymes. However, the high catalytic efficiency suggests that the K(+)-dependent enzyme may metabolize L-Asn more efficiently under conditions of high metabolic demand for N.

Purification of L-asparaginase from a bacteria Erwinia carotovora and effect of a dihydropyrimidine derivative on some of its kinetic parameters.

L-Asparaginase shows antileukemic activity and is generally administered in the body in combination with other anticancer drugs like pyrimidine derivatives. In the present study, L-asparaginase was purified from a bacteria Erwinia carotovora and the effect of a dihydropyrimidine derivative (1-amino-6-methyl-4-phenyl-2-thioxo, 1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester) was studied on the kinetic parameters Km and Vmax of the enzyme using L-asparagine as substrate. The enzyme had optimum activity at pH 8.6 and temperature 35 degrees C, both in the absence and presence of pyrimidine derivative and substrate saturation concentration at 6 mg/ml. For the enzymatic reaction in the absence and presence (1 to 3 mg/ml) of dihydropyrimidine derivative, Km values were 7.14, 5.26, 4.0, and 5.22 M, and Vmax values were 0.05, 0.035, 0.027 and 0.021 mg/ml/min, respectively. The kinetic values suggested that activity of enzyme was enhanced in the presence of dihydropyrimidine derivative.

Transcriptional co-activation at the ansB promoters: involvement of the activating regions of CRP and FNR when bound in tandem.

Previous work with semi-synthetic promoters containing a single CRP binding site centred at 41.5 bp from the transcription start site has demonstrated enhanced transcription (synergism) when a second binding site, for CRP or FNR, is placed upstream at around -91 bp. The ansB promoter in Escherichia coli is co-activated in a co-dependent manner by one dimer each of CRP and FNR protein whose binding sites are at around -91 and -41 bp, respectively, from the transcription start site. Similarly, the homologous ansB promoter in Salmonella is co-activated by two dimers of CRP which function synergistically. The binding sites at the E. coli promoter have been changed by mutation to provide a number of active promoter derivatives carrying other combinations of FNR and CRP binding sites. The co-dependent versus synergistic interaction of these activators and their requirement for known activating regions have been examined. The results demonstrate that FNR can co-activate when located upstream at around -91 bp in combination with either FNR or CRP downstream. When FNR occupies the downstream site the promoter is co-dependent on an upstream activator, but not when CRP occupies this site. Activating region 1 in CRP (defined by substitutions at residue H159) and its putative equivalent in FNR (defined by substitutions at S73) are mainly required in the upstream activator; the putative equivalent in FNR of activating region 3 of CRP (defined by substitutions at G85 and K52, respectively) is mainly required in the dimer which binds downstream. Activating region 1 of FNR is required only in the downstream subunit of the upstream activator in a promoter which is co-dependent on two FNR dimers. These data suggest that both bound upstream and downstream activators interact with RNA polymerase to promote transcription, and that co-dependence is determined by the nature of the activator plus the promoter context.

In vivo effect of asparagine in the hydrophobic region of the signal sequence.

On the basis of the biophysical studies on the synthetic mutant (Ile-8----Asn) OmpA signal peptide in the preceding paper (Hoyt, D. C., and Gierasch, L.M. (1991) J. Biol. Chem. 266, 14406-14412), the in vivo effects of the same mutation were examined by fusing the mutant OmpA signal sequence to Staphylococcus aureus nuclease or TEM beta-lactamase. The mutation in which the isoleucine residue at position 8 of the OmpA signal sequence of Escherichia coli was replaced with a neutral polar residue, asparagine, resulted in a defective signal peptide. The mutant signal sequence was unable to be processed, and the precursor molecule accumulated in the cytoplasmic as well as in the membrane fractions, indicating that the Ile-8----Asn OmpA signal sequence is not competent for translocating nuclease A or beta-lactamase across the membrane. This result is consistent with the in vitro studies on the Ile-8----Asn OmpA signal peptide, which indicated that the mutant signal peptide was unable to penetrate into the hydrophobic core of the lipid bilayer. Other asparagine or glutamine substitution mutations in the hydrophobic region of the OmpA signal sequence were also examined. Interestingly, the OmpA signal sequence with either Ile-8----Gln, Val-10----Asn, or Leu-12----Asn mutation was completely defective as the Ile-8----Asn OmpA signal sequence, while the Ile-6----Asn and Ala-9----Asn OmpA nucleases were able to be processed to secrete nuclease, although the processing occurred at a much slower rate than the wild-type OmpA nuclease. These results indicate that the defects depend on the position of the lesion in the hydrophobic core of the OmpA signal sequence.

Staphylococcal L-asparaginase: antilymphoma and immunosuppressive action.

Staphylococcal L-asparaginase inhibits blastic transformation of human lymphocytes and growth of mice leukemia lymphoblasts L5178Y-R. The enzyme is removed from blood stream of DBA/2 mice very rapidly.

Tumor inhibitory and non-tumor inhibitory L-asparaginases from Pseudomonas geniculata.

Two enzymes that catalyze the hydrolysis of l-asparagine have been isolated from extracts of Pseudomonas geniculata. After initial salt fractionation, the enzymes were separated by chromatography on diethylaminoethyl-Sephadex and purified to homogeneity by gel filtration, ion-exchange chromatography, and preparative polyacrylamide electrophoresis. The enzymes differ markedly in physicochemical properties. One enzyme, termed asparaginase A, has a molecular weight of approximately 96,000 whereas the other, termed asparaginase AG, has a molecular weight of approximately 135,000. Both enzymes are tetrameric. The asparaginase A shows activity only with l-asparagine as substrate, whereas the asparaginase AG hydrolyzes l-asparagine and l-glutamine at approximately equal rates and it is also active with d-asparagine and d-glutamine as substrates. The asparaginase A was found to be devoid of antitumor activity in mice, whereas the asparaginase AG was effective in increasing the mean survival times of both C3H mice carrying the asparagine-requiring Gardner 6C3HED tumor line and Swiss mice bearing the glutamine-requiring Ehrlich ascites tumor line. These differences in antitumor activity were related to differences in the K(m) values for l-asparagine for the two enzymes. The asparaginase A has a K(m) value of 1 x 10(-3) M for this substrate whereas the corresponding value for the AG enzyme is 1.5 x 10(-5) M. Thus the concentration of asparagine necessary for maximal activity of the asparaginase A is very high compared with that of the normal plasma level of asparagine, which is approximately 50 muM.