Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Specific amino acid binding by aminoacyl-tRNA synthetases (aaRS) is necessary for correct translation of the genetic code. Engineering a modified specificity into aminoacyl-tRNA synthetases has been proposed as a means to incorporate artificial amino acid residues into proteins in vivo. In a previous paper, the binding to aspartyl-tRNA synthetase of the substrate Asp and the analogue Asn were compared by molecular dynamics free energy simulations. Molecular dynamics combined with Poisson-Boltzmann free energy calculations represent a less expensive approach, suitable for examining multiple active site mutations in an engineering effort. Here, Poisson-Boltzmann free energy calculations for aspartyl-tRNA synthetase are first validated by their ability to reproduce selected molecular dynamics binding free energy differences, then used to examine the possibility of Asn binding to native and mutant aspartyl-tRNA synthetase. A component analysis of the Poisson-Boltzmann free energies is employed to identify specific interactions that determine the binding affinities. The combined use of molecular dynamics free energy simulations to study one binding process thoroughly, followed by molecular dynamics and Poisson-Boltzmann free energy calculations to study a series of related ligands or mutations is proposed as a paradigm for protein or ligand design. The binding of Asn in an alternate, "head-to-tail" orientation observed in the homologous asparagine synthetase is analyzed, and found to be more stable than the "Asp-like" orientation studied earlier. The new orientation is probably unsuitable for catalysis. A conserved active site lysine (Lys198 in Escherichia coli) that recognizes the Asp side-chain is changed to a leucine residue, found at the corresponding position in asparaginyl-tRNA synthetase. It is interesting that the binding of Asp is calculated to increase slightly (rather than to decrease), while that of Asn is calculated, as expected, to increase strongly, to the same level as Asp binding. Insight into the origin of these changes is provided by the component analyses. The double mutation (K198L,D233E) has a similar effect, while the triple mutation (K198L,Q199E,D233E) reduces Asp binding strongly. No binding measurements are available, but the three mutants are known to have no ability to adenylate Asn, despite the "Asp-like" binding affinities calculated here. In molecular dynamics simulations of all three mutants, the Asn ligand backbone shifts by 1-2 A compared to the experimental Asp:AspRS complex, and significant side-chain rearrangements occur around the pocket. These could reduce the ATP binding constant and/or the adenylation reaction rate, explaining the lack of catalytic activity in these complexes. Finally, Asn binding to AspRS with neutral K198 or charged H449 is considered, and shown to be less favorable than with the charged K198 and neutral H449 used in the analysis.
J Mol Biol 2001 Feb 16
PMID:Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition by aspartyl-tRNA synthetase. 1123 2

Classical analysis of obligate biotrophic fungi revealed changes of enzyme activities or the concentration of metabolites in infected areas. However, due to the intricate integration of host and parasite metabolism, it was not possible to delineate the individual contributions of the two organisms. Here, we used reverse-transcription-polymerase chain reaction to monitor expression of genes from the rust fungus Uromyces fabae and its host Vicia faba. We focused on genes relevant for amino acid and sugar uptake and metabolism in both organisms. In the fungus, mRNA for plasma membrane ATPase was detected in spores and all infection structures. Two genes for fungal amino acid transporters showed dissimilar regulation. Transcripts for one were detected during all developmental stages, whereas those of the other appeared to be under developmental control. The latter result was also obtained for the so far only hexose transporter known from U. fabae and for one gene of the thiamine biosynthesis pathway. In the host plant, transcripts for two ATPases analyzed generally declined upon infection. Sucrose synthase expression increased in leaves, but decreased in roots. Transcript levels of glucose and sucrose transporter genes appeared unchanged. Markers for amino acid metabolism did not show a uniform trend: transcripts for asparagine synthetase increased, whereas those for two amino acid transporters either decreased or increased. Our analyses revealed that not only expression of genes in the immediate vicinity of the primary infection site is altered, but infection also influences transcription of certain genes in remote organs, like stems and roots. This demonstrates alterations in the source-sink relationships.
Mol Plant Microbe Interact 2001 Nov
PMID:Differential regulation of gene expression in the obligate biotrophic interaction of Uromyces fabae with its host Vicia faba. 1176 30

Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment. Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants. In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. Plants of Lotus corniculatus L. transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor. Nontransgenic plants belonging to the species L. corniculatus L., L. tenuis Waldst. and Kit. ex Willd, and L. pedunculatus Cav., were utilized as recipients. Two experimental fields were established in two areas of central Italy. Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker. No transgene flow between L. corniculatus transformants and the nontransgenic L. tenuis and L. pedunculatus plants was detected. As regards nontransgenic L. corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot. In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.
Mol Ecol 2003 Jun
PMID:Measuring gene flow from two birdsfoot trefoil (Lotus corniculatus) field trials using transgenes as tracer markers. 1275 96

Real-time quantitative polymerase chain reaction (QPCR) using the Roche LightCycler was used to verify the expression of asparagine synthetase (ASNS) identified by microarray analysis as a target of p53 transrepression and mutant p53 transactivation. A p53-null cell line derived from lung carcinoma, H1299, was infected with recombinant adenovirus expressing wild-type (WT) p53, mutant p53-D281G, or beta-galactosidase as a control. After 24 h of infection, RNA was harvested and used for microarray analysis. ASNS was one of several genes whose expression was down-regulated by WT p53 and up-regulated in the presence of mutant p53. Expression levels of ASNS were measured relative to an exogenously applied quality-control nucleic acid template. Real-time PCR product accumulation was monitored using the intercalating dye, SYBR Green I, which exhibits a higher fluorescence upon binding of double-stranded DNA. Relative gene expression was calculated using conditions at the early stages of PCR, when amplification was logarithmic and, thus, could be correlated to initial copy number of gene transcripts. ASNS was found to be down-regulated in the presence of WT p53 and up-regulated by mutant p53.
Methods Mol Biol 2003
PMID:Real-time polymerase chain reaction quantitation of relative expression of genes modulated by p53 using SYBR Green I. 1282 26

Nitrogen assimilation is a vital process controlling plant growth and development. Inorganic nitrogen is assimilated into the amino acids glutamine, glutamate, asparagine, and aspartate, which serve as important nitrogen carriers in plants. The enzymes glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT), and asparagine synthetase (AS) are responsible for the biosynthesis of these nitrogen-carrying amino acids. Biochemical studies have revealed the existence of multiple isoenzymes for each of these enzymes. Recent molecular analyses demonstrate that each enzyme is encoded by a gene family wherein individual members encode distinct isoenzymes that are differentially regulated by environmental stimuli, metabolic control, developmental control, and tissue/cell-type specificity. We review the recent progress in using molecular-genetic approaches to delineate the regulatory mechanisms controlling nitrogen assimilation into amino acids and to define the physiological role of each isoenzyme involved in this metabolic pathway.
Annu Rev Plant Physiol Plant Mol Biol 1996 Jun
PMID:THE MOLECULAR-GENETICS OF NITROGEN ASSIMILATION INTO AMINO ACIDS IN HIGHER PLANTS. 1501 1

We established a real-time quantitative PCR (RQ-PCR) with which to measure abundance of the asparagine synthetase (AS) mRNA. The level of AS mRNA paralleled AS enzyme activity, as well as the AS protein level detected by Western blotting and by in situ immunostaining. Cytotoxicity tests in vitro showed that the AS mRNA level also synchronized with cellular resistance to L-asparaginase in cell lines. Cellular levels of AS enzyme activity correlated with resistance to L-asparaginase. These results indicate that the AS mRNA level is an index of resistance to L-asparaginase. RQ-PCR is superior to enzyme assays, Western blotting, and immunostaining in the following ways: less labor and time, accurate and reproducible quantitativity, and broad dynamic range. In addition, RQ-PCR could evaluate differences in L-asparaginase sensitivity although immunostaining could not. And in clinical samples, we analyzed eight pediatric leukemia cases by this RQ-PCR to evaluate whether this method was applicable to clinical laboratories and the expression level of AS mRNA in each case were predictable for the effectiveness of L-asparaginase treatment. Consequently, this method was useful enough in defining candidates for selective therapy that targets an AS deficiency.
J Mol Diagn 2004 Aug
PMID:Establishment of real-time polymerase chain reaction method for quantitative analysis of asparagine synthetase expression. 1526 98

Chromosome rearrangement, a hallmark of cancer, has profound effects on carcinogenesis and tumor phenotype. We used a panel of 60 human cancer cell lines (the NCI-60) as a model system to identify relationships among DNA copy number, mRNA expression level, and drug sensitivity. For each of 64 cancer-relevant genes, we calculated all 4,096 possible Pearson's correlation coefficients relating DNA copy number (assessed by comparative genomic hybridization using bacterial artificial chromosome microarrays) and mRNA expression level (determined using both cDNA and Affymetrix oligonucleotide microarrays). The analysis identified an association of ERBB2 overexpression with 3p copy number, a finding supported by data from human tumors and a mouse model of ERBB2-induced carcinogenesis. When we examined the correlation between DNA copy number for all 353 unique loci on the bacterial artificial chromosome microarray and drug sensitivity for 118 drugs with putatively known mechanisms of action, we found a striking negative correlation (-0.983; 95% bootstrap confidence interval, -0.999 to -0.899) between activity of the enzyme drug L-asparaginase and DNA copy number of genes near asparagine synthetase in the ovarian cancer cells. Previous analysis of drug sensitivity and mRNA expression had suggested an inverse relationship between mRNA levels of asparagine synthetase and L-asparaginase sensitivity in the NCI-60. The concordance of pharmacogenomic findings at the DNA and mRNA levels strongly suggests further study of L-asparaginase for possible treatment of a low-synthetase subset of clinical ovarian cancers. The DNA copy number database presented here will enable other investigators to explore DNA transcript-drug relationships in their own domains of research focus.
Mol Cancer Ther 2006 Apr
PMID:Integrating data on DNA copy number with gene expression levels and drug sensitivities in the NCI-60 cell line panel. 1664 55

L-Asparaginase (l-ASP), a bacterial enzyme used since the 1970s to treat acute lymphoblastic leukemia, selectively starves cells that cannot synthesize sufficient asparagine for their own needs. Molecular profiling of the NCI-60 cancer cell lines using five different microarray platforms showed strong negative correlations of asparagine synthetase (ASNS) expression and DNA copy number with sensitivity to l-ASP in the leukemia and ovarian cancer cell subsets. To assess whether the ovarian relationship is causal, we used RNA interference to silence ASNS in three ovarian lines and observed 4- to 5-fold potentiation of sensitivity to l-ASP with two of the lines. For OVCAR-8, the line that expresses the least ASNS, the potentiation was >500-fold. Significantly, that potentiation was >700-fold in the multidrug-resistant derivative OVCAR-8/ADR, showing that the causal relationship between ASNS expression and l-ASP activity survives development of classical multidrug resistance. Tissue microarrays confirmed low ASNS expression in a subset of clinical ovarian cancers as well as other tumor types. Overall, this pharmacogenomic/pharmacoproteomic study suggests the use of l-ASP for treatment of a subset of ovarian cancers (and perhaps other tumor types), with ASNS as a biomarker for patient selection.
Mol Cancer Ther 2006 Nov
PMID:Asparagine synthetase as a causal, predictive biomarker for L-asparaginase activity in ovarian cancer cells. 1708 36

We recently used RNA interference to show that a negative correlation of L-asparaginase (L-ASP) chemotherapeutic activity with asparagine synthetase (ASNS) expression in the ovarian subset of the NCI-60 cell line panel is causal. To determine whether that relationship would be sustained in a larger, more diverse set of ovarian cell lines, we have now measured ASNS mRNA expression using microarrays and a branched-DNA RNA assay, ASNS protein expression using an electrochemiluminescent immunoassay, and L-ASP activity using an MTS assay on 19 human ovarian cancer cell lines. Contrary to our previous findings, L-ASP activity was only weakly correlated with ASNS mRNA expression; Pearson's correlation coefficients were r = -0.21 for microarray data and r = -0.39 for the branched-DNA RNA assay, with just the latter being marginally statistically significant (P = 0.047, one-tailed). ASNS protein expression measured by liquid-phase immunoassay exhibited a much stronger correlation (r = -0.65; P = 0.0014, one-tailed). We conclude that ASNS protein expression measured by immunoassay is a strong univariate predictor of L-ASP activity in ovarian cancer cell lines. These findings provide rationale for evaluation of ASNS protein expression as a predictive biomarker of clinical L-ASP activity in ovarian cancer.
Mol Cancer Ther 2008 Oct
PMID:Asparagine synthetase is a predictive biomarker of L-asparaginase activity in ovarian cancer cell lines. 1885 15

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of -5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.
Mol Biol Rep 2009 Nov
PMID:PVAS3, a class-II ubiquitous asparagine synthetase from the common bean (Phaseolus vulgaris). 1913 Feb 95


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