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Michael S Kilberg - One of the best experts on this subject based on the ideXlab platform.
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the role of Asparagine Synthetase on nutrient metabolism in pancreatic disease
Pancreatology, 2020Co-Authors: Chengyu Tsai, Michael S Kilberg, Sohail Z HusainAbstract:The pancreas avidly takes up and synthesizes the amino acid Asparagine (Asn), in part, to maintain an active translational machinery that requires incorporation of the amino acid. The de novo synthesis of Asn in the pancreas occurs through the enzyme Asparagine Synthetase (ASNS). The pancreas has the highest expression of ASNS of any organ, and it can further upregulate ASNS expression in the setting of amino acid depletion. ASNS expression is driven by an intricate feedback network within the integrated stress response (ISR), which includes the amino acid response (AAR) and the unfolded protein response (UPR). Asparaginase is a cancer chemotherapeutic drug that depletes plasma Asn. However, asparaginase-associated pancreatitis (AAP) is a major medical problem and could be related to pancreatic Asn depletion. In this review, we will provide an overview of ASNS and then describe its role in pancreatic health and in the exocrine disorders of pancreatitis and pancreatic cancer. We will offer the overarching perspective that a high abundance of ASNS expression is hardwired in the exocrine pancreas to buffer the high demands of Asn for pancreatic digestive enzyme protein synthesis, that perturbations in the ability to express or upregulate ASNS could tip the balance towards pancreatitis, and that pancreatic cancers exploit ASNS to gain a metabolic survival advantage.
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Asparagine Synthetase is highly expressed at baseline in the pancreas through heightened perk signaling
Cellular and molecular gastroenterology and hepatology, 2020Co-Authors: Amitava Mukherjee, Michael S Kilberg, Nayyar Ahmed, Fateema T Rose, Abraheem N Ahmad, Tanveer A Javed, Li Wen, Rita Bottino, Xiangwei Xiao, Sohail Z HusainAbstract:Asparaginase (ASNase) causes pancreatitis in approximately 10% of leukemia patients, and the mechanisms underlying this painful complication are not known. ASNase primarily depletes circulating Asparagine, and the endogenously expressed enzyme, Asparagine Synthetase (ASNS), replenishes Asparagine. ASNS was suggested previously to be highly expressed in the pancreas. In this study, we determined the expression pattern of ASNS in the pancreas and the mechanism for increased pancreatic ASNS abundance. Compared with other organs, ASNS was highly expressed in both the human and mouse pancreas, and, within the pancreas, ASNS was present primarily in the acinar cells. The high baseline pancreatic ASNS was associated with higher baseline activation of protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in the pancreas, and inhibition of PERK in acinar cells lessened ASNS expression. ASNase exposure, but not the common pancreatitis triggers, uniquely up-regulated ASNS expression, indicating that the increase is mediated by nutrient stress. The up-regulation of acinar ASNS with ASNase exposure was owing to increased transcriptional rather than delayed degradation. Knockdown of ASNS in the 266-6 acinar cells provoked acinar cell injury and worsened ASNase-induced injury, whereas ASNS overexpression protected against ASNase-induced injury. In summary, ASNS is highly expressed in the pancreatic acinar cells through heightened basal activation of PERK, and ASNS appears to be crucial to maintaining acinar cell integrity. The implications are that ASNS is especially hardwired in the pancreas to protect against both baseline perturbations and nutrient deprivation stressors, such as during ASNase exposure.
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Asparagine Synthetase function structure and role in disease
Journal of Biological Chemistry, 2017Co-Authors: Carrie L Lomelino, Jacob T Andring, Robert Mckenna, Michael S KilbergAbstract:Asparagine Synthetase (ASNS) converts aspartate and glutamine to Asparagine and glutamate in an ATP-dependent reaction. ASNS is present in most, if not all, mammalian organs, but varies widely in basal expression. Human ASNS activity is highly responsive to cellular stress, primarily by increased transcription from a single gene located on chromosome 7. Elevated ASNS protein expression is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia. There is evidence that ASNS expression levels may also be inversely correlated with asparaginase efficacy in certain solid tumors as well. Children with mutations in the ASNS gene exhibit developmental delays, intellectual disability, microcephaly, intractable seizures, and progressive brain atrophy. Thus far, 15 unique mutations in the ASNS gene have been clinically associated with Asparagine Synthetase deficiency (ASD). Molecular modeling using the Escherichia coli ASNS-B structure has revealed that most of the reported ASD substitutions are located near catalytic sites or within highly conserved regions of the protein. For some ASD patients, fibroblast cell culture studies have eliminated protein and mRNA synthesis or stability as the basis for decreased proliferation.
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Asparagine Synthetase regulation by cell stress and involvement in tumor biology
American Journal of Physiology-endocrinology and Metabolism, 2013Co-Authors: Mukundh N. Balasubramanian, Elizabeth A Butterworth, Michael S KilbergAbstract:Asparagine Synthetase (ASNS) catalyzes the conversion of aspartate and glutamine to Asparagine and glutamate in an ATP-dependent reaction. The enzyme is ubiquitous in its organ distribution in mammals, but basal expression is relatively low in tissues other than the exocrine pancreas. Human ASNS activity is highly regulated in response to cell stress, primarily by increased transcription from a single gene located on chromosome 7. Among the genomic elements that control ASNS transcription is the C/EBP-ATF response element (CARE) within the promoter. Protein limitation or an imbalanced dietary amino acid composition activate the ASNS gene through the amino acid response (AAR), a process that is replicated in cell culture through limitation for any single essential amino acid. Endoplasmic reticulum stress also increases ASNS transcription through the PERK-eIF2-ATF4 arm of the unfolded protein response (UPR). Both the AAR and UPR lead to increased synthesis of ATF4, which binds to the CARE and induces ASNS transcription. Elevated expression of ASNS protein is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia and may be a predictive factor in drug sensitivity for certain solid tumors as well. Activation of the GCN2-eIF2-ATF4 signaling pathway, leading to increased ASNS expression appears to be a component of solid tumor adaptation to nutrient deprivation and/or hypoxia. Identifying the roles of ASNS in fetal development, tissue differentiation, and tumor growth may reveal that ASNS function extends beyond Asparagine biosynthesis.
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transcriptional induction of the human Asparagine Synthetase gene during the unfolded protein response does not require the atf6 and ire1 xbp1 arms of the pathway
Biochemical Journal, 2009Co-Authors: Altin Gjymishka, Michael S KilbergAbstract:The UPR (unfolded protein response) pathway comprises three signalling cascades mediated by the ER (endoplasmic reticulum) stress-sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (Asparagine Synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPbeta (CCAAT/enhancer-binding protein beta) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared with triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box-binding protein 1), ATF6alpha or ATF6beta expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2alpha (phosphorylated eukaryotic initiation factor 2alpha)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, ASNS NSRE-1 and NSRE-2, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian ER-stress-responsive sequences.
Sheldon M Schuster - One of the best experts on this subject based on the ideXlab platform.
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selective apoptosis of natural killer cell tumours by l asparaginase
British Journal of Haematology, 2005Co-Authors: Miki Ando, Sheldon M Schuster, Koichi Sugimoto, Toshiyuki Kitoh, Makoto Sasaki, Kouichi Mukai, Jun Ando, Motoki Egashira, Kazuo OshimiAbstract:We examined the effectiveness of various anti-tumour agents to natural killer (NK)-cell tumour cell lines and samples, which are generally resistant to chemotherapy, using flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay. Although NK-YS and NK-92 were highly resistant to various anti-tumour agents, l-asparaginase induced apoptosis in these two NK-cell lines. NK-cell leukaemia/lymphoma and acute lymphoblastic leukaemia (ALL) samples were selectively sensitive to l-asparaginase and to doxorubicin (DXR) respectively. Samples of chronic NK lymphocytosis, an NK-cell disorder with an indolent clinical course, were resistant to both drugs. Our study clearly separated two major categories of NK-cell disorders and ALL according to the sensitivity to DXR and l-asparaginase. We examined Asparagine Synthetase levels by real-time quantitative polymerase chain reaction (RQ-PCR) and immunostaining in these samples. At least in nasal-type NK-cell lymphoma, there was a good correlation among Asparagine Synthetase expression, in vitro sensitivity and clinical response to l-asparaginase. In aggressive NK-cell leukaemia, although Asparagine Synthetase expression was high at both mRNA and protein levels, l-asparaginase induced considerable apoptosis. Furthermore, samples of each disease entity occupied a distinct area in two-dimensional plotting with Asparagine Synthetase mRNA level (RQ-PCR) and in vitrol-asparaginase sensitivity (TUNEL assay). We confirmed rather specific anti-tumour activity of l-asparaginase against NK-cell tumours in vitro, which provides an experimental background to the clinical use of l-asparaginase for NK-cell tumours.
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activation of the human Asparagine Synthetase gene by the amino acid response and the endoplasmic reticulum stress response pathways occurs by common genomic elements
Journal of Biological Chemistry, 2000Co-Authors: Ione Parra Barbosatessmann, Can Zhong, Chin Chen, Fai Siu, Sheldon M Schuster, Harry S Nick, Michael S KilbergAbstract:Abstract The human Asparagine Synthetase(AS) gene is transcriptionally regulated by amino acid deprivation (amino acid response, AAR) and the endoplasmic reticulum stress response (ERSR), also known as the unfolded protein response pathway. The results reported here document the novel observation that induction of the AS gene by the AAR and ERSR pathways occurs via the same set of genomic elements. Data supporting this conclusion include transient transfection of AS promoter/reporter gene constructs that illustrate that the transcriptional control elements used by both pathways are contained with nucleotides −111 to −34 of the AS promoter. In vivofootprinting analysis of this region identified six specific protein-binding sites. Within two of these sites, altered footprinting was observed following amino acid or glucose deprivation, but the patterns were identical for both the AAR and the ERSR pathway. Site-directed mutation of individual nucleotides within these two binding sites confirmed their importance for regulated transcription, and none of the mutations resulted in loss of response of only one pathway. Neither of these two sites corresponds to a recently identified ERSR cis-element, nor do they contain consensus sequences for known transcription factors. Collectively, the data document that there are at least two independent transcriptional mechanisms for gene activation by the ERSR pathway, one of which terminates at the same genomic elements used by the AAR pathway.
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three dimensional structure of escherichia coli Asparagine Synthetase b a short journey from substrate to product
Biochemistry, 1999Co-Authors: Todd M Larsen, Sheldon M Schuster, Susan K Boehlein, Nigel G J Richards, James B Thoden, Hazel M Holden, Ivan RaymentAbstract:Asparagine Synthetase B catalyzes the assembly of Asparagine from aspartate, Mg2+ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia coli determined and refined to 2.0 A resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP. Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel β-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg2+ATP and aspartate, is dominated by a five-stranded parallel β-sheet flanked on either side by α-helices. The AMP moiety is anchored to the protein via hydrogen b...
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mutagenesis and chemical rescue indicate residues involved in beta aspartyl amp formation by escherichia coli Asparagine Synthetase b
Journal of Biological Chemistry, 1997Co-Authors: Susan K Boehlein, Nigel G J Richards, Ellen S Walworth, Sheldon M SchusterAbstract:Abstract Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of β-aspartyl-AMP in the reaction mechanism ofEscherichia coli Asparagine Synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317–330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable Asparagine Synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation β-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediatesen route to β-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in Asparagine synthesis.
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glutamine dependent nitrogen transfer in escherichia coli Asparagine Synthetase b searching for the catalytic triad
Journal of Biological Chemistry, 1994Co-Authors: Susan K Boehlein, Nigel G J Richards, Sheldon M SchusterAbstract:Abstract The mechanism of nitrogen transfer in glutamine-dependent amidotransferases remains to be unambiguously established. We now report the overexpression, purification, and kinetic characterization of both the glutamine- and ammonia-dependent activities of Escherichia coli Asparagine Synthetase B (AS-B) and a series of mutants. In common with other members of the purF family of amidotransferases, the recombinant enzyme possesses an NH2-terminal cysteine residue. Replacement of Cys-1 by either alanine or serine results in a loss of glutaminase and glutamine-dependent activity, without out any significant effect upon ammonia-dependent Asparagine synthesis. As previously observed for human AS (Sheng, S., Moraga-Amador, D., Van Heeke, G., Allison, R. D., Richards, N. G. J., and Schuster, S. M. (1993) J. Biol. Chem. 268, 16771-16780), glutamine is an inhibitor of the ammonia-dependent reaction catalyzed by both the Cys-1-->Ala (C1A) and Cys-1-->Ser (C1S) mutants of AS-B. In the case of C1A, the inhibition pattern suggests that an abortive complex is formed. This is consistent with a recent proposal implicating the formation of an imide intermediate in the nitrogen transfer reaction (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). In contrast, glutamine appears to be only a competitive inhibitor of the ammonia-dependent activity of C1S. Cys-1 does not appear to be required for glutamine binding. Replacement of Asp-33 by either Asparagine or glutamic acid has little effect on the kinetic properties of the mutant enzymes when compared to wild-type AS-B. Cys-1 and Asp-33 are cognate to residues Cys-1 and Asp-29 in glutamine phosphoribosylpyrophosphate amidotransferase which have been proposed to be members of a catalytic triad responsible for mediating nitrogen transfer in this enzyme (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619). In the case of AS-B, although Cys-1 is essential for glutamine-dependent activity, Asp-33 does not appear to participate in mediating nitrogen transfer. In an effort to locate other residues which might form part of a "catalytic triad" in the glutamine amidotransferase domain of AS-B, we have expressed and characterized mutant proteins in which His-29 and His-80, which are conserved within the glutamine amidotransferase domain of purF amidotransferases, are replaced by alanine (H29A and H80A).(ABSTRACT TRUNCATED AT 400 WORDS)
Hong Chen - One of the best experts on this subject based on the ideXlab platform.
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amino acid deprivation induces the transcription rate of the human Asparagine Synthetase gene through a timed program of expression and promoter binding of nutrient responsive basic region leucine zipper transcription factors as well as localized his
Journal of Biological Chemistry, 2004Co-Authors: Hong Chen, Elizabeth E. Dudenhausen, Yuan Xiang Pan, Michael S KilbergAbstract:Expression of human Asparagine Synthetase (ASNS), which catalyzes Asparagine and glutamate biosynthesis, is transcriptionally induced following amino acid deprivation. Previous overexpression and electrophoresis mobility shift analysis showed the involvement of the transcription factors ATF4, C/EBPbeta, and ATF3-FL through the nutrient-sensing response element-1 (NSRE-1) within the ASNS promoter. Amino acid deprivation caused an elevated mRNA level for ATF4, C/EBPbeta, and ATF3-FL, and the present study established that the nuclear protein content for ATF4 and ATF3-FL were increased during amino acid limitation, whereas C/EBPbeta-LIP declined slightly. The total amount of C/EBPbeta-LAP protein was unchanged, but changes in the distribution among multiple C/EBPbeta-LAP forms were observed. Overexpression studies established that ATF4, ATF3-FL, and C/EBPbeta-LAP could coordinately modulate the transcription from the human ASNS promoter. Chromatin immunoprecipitation demonstrated that amino acid deprivation increased ATF3-FL, ATF4, and C/EBPbeta binding to the ASNS promoter and enhanced promoter association of RNA polymerase II, TATA-binding protein, and TFIIB of the general transcription machinery. A time course revealed a markedly different temporal order of interaction between these transcription factors and the ASNS promoter. During the initial 2 h, there was a 20-fold increase in ATF4 binding and a rapid increase in histone H3 and H4 acetylation, which closely paralleled the increased transcription rate of the ASNS gene, whereas the increase in ATF3-FL and C/EBPbeta binding was considerably slower and more closely correlated with the decline in transcription rate between 2 and 6 h. The data suggest that ATF3-FL and C/EBPbeta act as transcriptional suppressors for the ASNS gene to counterbalance the transcription rate activated by ATF4 following amino acid deprivation.
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amino acid deprivation induces the transcription rate of the human Asparagine Synthetase gene through a timed program of expression and promoter binding of nutrient responsive basic region leucine zipper transcription factors as well as localized histone acetylation
Journal of Biological Chemistry, 2004Co-Authors: Hong Chen, Elizabeth E. Dudenhausen, Yuan Xiang Pan, Michael S KilbergAbstract:Expression of human Asparagine Synthetase (ASNS), which catalyzes Asparagine and glutamate biosynthesis, is transcriptionally induced following amino acid deprivation. Previous overexpression and electrophoresis mobility shift analysis showed the involvement of the transcription factors ATF4, C/EBPβ, and ATF3-FL through the nutrient-sensing response element-1 (NSRE-1) within the ASNS promoter. Amino acid deprivation caused an elevated mRNA level for ATF4, C/EBPβ, and ATF3-FL, and the present study established that the nuclear protein content for ATF4 and ATF3-FL were increased during amino acid limitation, whereas C/EBPβ-LIP declined slightly. The total amount of C/EBPβ-LAP protein was unchanged, but changes in the distribution among multiple C/EBPβ-LAP forms were observed. Overexpression studies established that ATF4, ATF3-FL, and C/EBPβ-LAP could coordinately modulate the transcription from the human ASNS promoter. Chromatin immunoprecipitation demonstrated that amino acid deprivation increased ATF3-FL, ATF4, and C/EBPβ binding to the ASNS promoter and enhanced promoter association of RNA polymerase II, TATA-binding protein, and TFIIB of the general transcription machinery. A time course revealed a markedly different temporal order of interaction between these transcription factors and the ASNS promoter. During the initial 2 h, there was a 20-fold increase in ATF4 binding and a rapid increase in histone H3 and H4 acetylation, which closely paralleled the increased transcription rate of the ASNS gene, whereas the increase in ATF3-FL and C/EBPβ binding was considerably slower and more closely correlated with the decline in transcription rate between 2 and 6 h. The data suggest that ATF3-FL and C/EBPβ act as transcriptional suppressors for the ASNS gene to counterbalance the transcription rate activated by ATF4 following amino acid deprivation.
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amino acid deprivation and endoplasmic reticulum stress induce expression of multiple activating transcription factor 3 mrna species that when overexpressed in hepg2 cells modulate transcription by the human Asparagine Synthetase promoter
Journal of Biological Chemistry, 2003Co-Authors: Yuan Xiang Pan, Hong Chen, Fai Siu, Michael S KilbergAbstract:Abstract Transcription from the ASNS (Asparagine Synthetase) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the ASNS promoter, referred to as nutrient-sensing response element-1 and -2. Chromatin immunoprecipitation analysis provides the first in vivo evidence for activating transcription factor (ATF)-3 binding to the proximal ASNS promoter containing the nutrient-sensing response element-1 sequence. Overexpression of the full-length ATF3 protein caused a concentration-dependent biphasic response in ASNS promoter-driven transcription. Both amino acid limitation and activation of the endoplasmic reticulum stress response by glucose deprivation caused an increase in ATF3 mRNA content. However, reverse transcriptase-PCR analysis revealed that the increase in the ATF3 mRNA species detected by Northern analysis actually encoded both full-length ATF3 and two predicted truncated ATF3 isoforms (ATF3ΔZip2c and ATF3ΔZip3). Based on sequence analysis, one of the predicted truncated proteins (ATF3ΔZip3) is likely incapable of binding DNA; and yet, exogenous expression of the cDNA enhanced starvation-induced or ATF4-activated ASNS transcription, possibly by sequestering corepressor proteins. Collectively, the results provide evidence for a potential role of multiple predicted ATF3 isoforms in the transcriptional regulation of the ASNS gene in response to nutrient deprivation.
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atf4 is a mediator of the nutrient sensing response pathway that activates the human Asparagine Synthetase gene
Journal of Biological Chemistry, 2002Co-Authors: Fai Siu, Hong Chen, Perry J Bain, Rene Leblancchaffin, Michael S KilbergAbstract:Transcription from the Asparagine Synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the A.S. promoter referred to as nutrient-sensing response elements (NSRE) 1 and 2, both of which are necessary for gene activation. The NSRE-1 sequence was used to screen ATF/CREB family members by electrophoresis mobility shift assays and supershift by specific antibodies. The results indicated that ATF4 binds to the NSRE-1 sequence and that the amount of the ATF4 complex was increased when extracts from amino acid-deprived or glucose-deprived cells were tested. Using electrophoresis mobility shift assay experiments and a probe that contained both NSRE-1 and NSRE-2, mutation of the NSRE-1 sequence completely prevented formation of the ATF4-containing complexes, whereas mutation of the NSRE-2 sequence did not. Overexpression of ATF4 increased A.S. promoter-driven transcription, whereas an inhibitory dominant negative ATF4 mutant blocked both basal and starvation-enhanced transcription. Collectively, the results provide both in vitro and in vivo evidence for a role of ATF4 in the transcriptional activation of the A.S. gene in response to nutrient deprivation.
Fai Siu - One of the best experts on this subject based on the ideXlab platform.
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amino acid deprivation and endoplasmic reticulum stress induce expression of multiple activating transcription factor 3 mrna species that when overexpressed in hepg2 cells modulate transcription by the human Asparagine Synthetase promoter
Journal of Biological Chemistry, 2003Co-Authors: Yuan Xiang Pan, Hong Chen, Fai Siu, Michael S KilbergAbstract:Abstract Transcription from the ASNS (Asparagine Synthetase) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the ASNS promoter, referred to as nutrient-sensing response element-1 and -2. Chromatin immunoprecipitation analysis provides the first in vivo evidence for activating transcription factor (ATF)-3 binding to the proximal ASNS promoter containing the nutrient-sensing response element-1 sequence. Overexpression of the full-length ATF3 protein caused a concentration-dependent biphasic response in ASNS promoter-driven transcription. Both amino acid limitation and activation of the endoplasmic reticulum stress response by glucose deprivation caused an increase in ATF3 mRNA content. However, reverse transcriptase-PCR analysis revealed that the increase in the ATF3 mRNA species detected by Northern analysis actually encoded both full-length ATF3 and two predicted truncated ATF3 isoforms (ATF3ΔZip2c and ATF3ΔZip3). Based on sequence analysis, one of the predicted truncated proteins (ATF3ΔZip3) is likely incapable of binding DNA; and yet, exogenous expression of the cDNA enhanced starvation-induced or ATF4-activated ASNS transcription, possibly by sequestering corepressor proteins. Collectively, the results provide evidence for a potential role of multiple predicted ATF3 isoforms in the transcriptional regulation of the ASNS gene in response to nutrient deprivation.
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atf4 is a mediator of the nutrient sensing response pathway that activates the human Asparagine Synthetase gene
Journal of Biological Chemistry, 2002Co-Authors: Fai Siu, Hong Chen, Perry J Bain, Rene Leblancchaffin, Michael S KilbergAbstract:Transcription from the Asparagine Synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the A.S. promoter referred to as nutrient-sensing response elements (NSRE) 1 and 2, both of which are necessary for gene activation. The NSRE-1 sequence was used to screen ATF/CREB family members by electrophoresis mobility shift assays and supershift by specific antibodies. The results indicated that ATF4 binds to the NSRE-1 sequence and that the amount of the ATF4 complex was increased when extracts from amino acid-deprived or glucose-deprived cells were tested. Using electrophoresis mobility shift assay experiments and a probe that contained both NSRE-1 and NSRE-2, mutation of the NSRE-1 sequence completely prevented formation of the ATF4-containing complexes, whereas mutation of the NSRE-2 sequence did not. Overexpression of ATF4 increased A.S. promoter-driven transcription, whereas an inhibitory dominant negative ATF4 mutant blocked both basal and starvation-enhanced transcription. Collectively, the results provide both in vitro and in vivo evidence for a role of ATF4 in the transcriptional activation of the A.S. gene in response to nutrient deprivation.
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ccaat enhancer binding protein β is a mediator of the nutrient sensing response pathway that activates the human Asparagine Synthetase gene
Journal of Biological Chemistry, 2001Co-Authors: Fai Siu, Chin Chen, Can Zhong, Michael S KilbergAbstract:Abstract Transcription from the human Asparagine Synthetase (AS) gene is increased in response to either amino acid (amino acid response) or glucose (unfolded protein response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the AS promoter, which are referred to as nutrient-sensing response element (NSRE)-1 and -2, both of which are absolutely necessary for gene activation. The NSRE-1 sequence was used to identify the corresponding transcription factor by yeast one-hybrid screening. Based on those results, electrophoretic mobility shift assays for individual CCAAT/enhancer-binding protein-β (C/EBP) family members were performed to test for supershifting of complexes by specific antibodies. The results indicated that of all the family members, C/EBPβ bound to the NSRE-1 sequence to the greatest extent and that the absolute amount of this complex was increased when extracts from amino acid- or glucose-deprived cells were tested. Using electrophoretic mobility shift assays, mutation of the NSRE-1 sequence completely prevented formation of the C/EBPβ-containing complexes. In contrast, mutation of the NSRE-2 sequence did not block C/EBPβ binding. Overexpression in HepG2 hepatoma cells of the activating isoform of C/EBPβ increased AS promoter-driven transcription, whereas the inhibitory dominant-negative isoform of C/EBPβ blocked enhanced transcription following amino acid or glucose deprivation. Collectively, the results provide both in vitro and in vivo evidence for a role of C/EBPβ in the transcriptional activation of the AS gene in response to nutrient deprivation.
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activation of the human Asparagine Synthetase gene by the amino acid response and the endoplasmic reticulum stress response pathways occurs by common genomic elements
Journal of Biological Chemistry, 2000Co-Authors: Ione Parra Barbosatessmann, Can Zhong, Chin Chen, Fai Siu, Sheldon M Schuster, Harry S Nick, Michael S KilbergAbstract:Abstract The human Asparagine Synthetase(AS) gene is transcriptionally regulated by amino acid deprivation (amino acid response, AAR) and the endoplasmic reticulum stress response (ERSR), also known as the unfolded protein response pathway. The results reported here document the novel observation that induction of the AS gene by the AAR and ERSR pathways occurs via the same set of genomic elements. Data supporting this conclusion include transient transfection of AS promoter/reporter gene constructs that illustrate that the transcriptional control elements used by both pathways are contained with nucleotides −111 to −34 of the AS promoter. In vivofootprinting analysis of this region identified six specific protein-binding sites. Within two of these sites, altered footprinting was observed following amino acid or glucose deprivation, but the patterns were identical for both the AAR and the ERSR pathway. Site-directed mutation of individual nucleotides within these two binding sites confirmed their importance for regulated transcription, and none of the mutations resulted in loss of response of only one pathway. Neither of these two sites corresponds to a recently identified ERSR cis-element, nor do they contain consensus sequences for known transcription factors. Collectively, the data document that there are at least two independent transcriptional mechanisms for gene activation by the ERSR pathway, one of which terminates at the same genomic elements used by the AAR pathway.
Yuan Xiang Pan - One of the best experts on this subject based on the ideXlab platform.
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correlation between asparaginase sensitivity and Asparagine Synthetase protein content but not mrna in acute lymphoblastic leukemia cell lines
Pediatric Blood & Cancer, 2008Co-Authors: Yuan Xiang Pan, Mi Zhou, Richard C Harvey, Stephen P Hunger, Michael S KilbergAbstract:Background Asparaginase (ASNase) is an essential component of most treatment protocols for childhood acute lymphoblastic leukemia (ALL). Although increased Asparagine Synthetase (ASNS) expression may contribute to ASNase resistance, there is conflicting data from patient samples with regard to correlation between ASNS mRNA content and ASNase sensitivity. Procedure Both T-cell and B-cell derived ALL cell lines were treated with ASNase and then monitored for cell proliferation, cell death, and ASNS mRNA and protein expression. Results Despite elevated ASNS mRNA following ASNase treatment, different ALL cell lines varied widely in translation to ASNS protein. Although ASNS mRNA levels did not consistently reflect ASNase sensitivity, there was an inverse correlation between ASNS protein and ASNase-induced cell death. Expression of ASNS in an ASNase-sensitive cell line resulted in enhanced ASNase resistance, and conversely, siRNA-mediated inhibition of ASNS expression promoted increased drug sensitivity. Conclusions These observations provide an explanation for the ASNase sensitivity of ALL cells and demonstrate the importance of measuring ASNS protein rather than mRNA in predicting ASNase responsiveness. Pediatr Blood Cancer 2008;50:274–279. © 2007 Wiley-Liss, Inc.
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amino acid deprivation induces the transcription rate of the human Asparagine Synthetase gene through a timed program of expression and promoter binding of nutrient responsive basic region leucine zipper transcription factors as well as localized his
Journal of Biological Chemistry, 2004Co-Authors: Hong Chen, Elizabeth E. Dudenhausen, Yuan Xiang Pan, Michael S KilbergAbstract:Expression of human Asparagine Synthetase (ASNS), which catalyzes Asparagine and glutamate biosynthesis, is transcriptionally induced following amino acid deprivation. Previous overexpression and electrophoresis mobility shift analysis showed the involvement of the transcription factors ATF4, C/EBPbeta, and ATF3-FL through the nutrient-sensing response element-1 (NSRE-1) within the ASNS promoter. Amino acid deprivation caused an elevated mRNA level for ATF4, C/EBPbeta, and ATF3-FL, and the present study established that the nuclear protein content for ATF4 and ATF3-FL were increased during amino acid limitation, whereas C/EBPbeta-LIP declined slightly. The total amount of C/EBPbeta-LAP protein was unchanged, but changes in the distribution among multiple C/EBPbeta-LAP forms were observed. Overexpression studies established that ATF4, ATF3-FL, and C/EBPbeta-LAP could coordinately modulate the transcription from the human ASNS promoter. Chromatin immunoprecipitation demonstrated that amino acid deprivation increased ATF3-FL, ATF4, and C/EBPbeta binding to the ASNS promoter and enhanced promoter association of RNA polymerase II, TATA-binding protein, and TFIIB of the general transcription machinery. A time course revealed a markedly different temporal order of interaction between these transcription factors and the ASNS promoter. During the initial 2 h, there was a 20-fold increase in ATF4 binding and a rapid increase in histone H3 and H4 acetylation, which closely paralleled the increased transcription rate of the ASNS gene, whereas the increase in ATF3-FL and C/EBPbeta binding was considerably slower and more closely correlated with the decline in transcription rate between 2 and 6 h. The data suggest that ATF3-FL and C/EBPbeta act as transcriptional suppressors for the ASNS gene to counterbalance the transcription rate activated by ATF4 following amino acid deprivation.
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amino acid deprivation induces the transcription rate of the human Asparagine Synthetase gene through a timed program of expression and promoter binding of nutrient responsive basic region leucine zipper transcription factors as well as localized histone acetylation
Journal of Biological Chemistry, 2004Co-Authors: Hong Chen, Elizabeth E. Dudenhausen, Yuan Xiang Pan, Michael S KilbergAbstract:Expression of human Asparagine Synthetase (ASNS), which catalyzes Asparagine and glutamate biosynthesis, is transcriptionally induced following amino acid deprivation. Previous overexpression and electrophoresis mobility shift analysis showed the involvement of the transcription factors ATF4, C/EBPβ, and ATF3-FL through the nutrient-sensing response element-1 (NSRE-1) within the ASNS promoter. Amino acid deprivation caused an elevated mRNA level for ATF4, C/EBPβ, and ATF3-FL, and the present study established that the nuclear protein content for ATF4 and ATF3-FL were increased during amino acid limitation, whereas C/EBPβ-LIP declined slightly. The total amount of C/EBPβ-LAP protein was unchanged, but changes in the distribution among multiple C/EBPβ-LAP forms were observed. Overexpression studies established that ATF4, ATF3-FL, and C/EBPβ-LAP could coordinately modulate the transcription from the human ASNS promoter. Chromatin immunoprecipitation demonstrated that amino acid deprivation increased ATF3-FL, ATF4, and C/EBPβ binding to the ASNS promoter and enhanced promoter association of RNA polymerase II, TATA-binding protein, and TFIIB of the general transcription machinery. A time course revealed a markedly different temporal order of interaction between these transcription factors and the ASNS promoter. During the initial 2 h, there was a 20-fold increase in ATF4 binding and a rapid increase in histone H3 and H4 acetylation, which closely paralleled the increased transcription rate of the ASNS gene, whereas the increase in ATF3-FL and C/EBPβ binding was considerably slower and more closely correlated with the decline in transcription rate between 2 and 6 h. The data suggest that ATF3-FL and C/EBPβ act as transcriptional suppressors for the ASNS gene to counterbalance the transcription rate activated by ATF4 following amino acid deprivation.
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amino acid deprivation and endoplasmic reticulum stress induce expression of multiple activating transcription factor 3 mrna species that when overexpressed in hepg2 cells modulate transcription by the human Asparagine Synthetase promoter
Journal of Biological Chemistry, 2003Co-Authors: Yuan Xiang Pan, Hong Chen, Fai Siu, Michael S KilbergAbstract:Abstract Transcription from the ASNS (Asparagine Synthetase) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the ASNS promoter, referred to as nutrient-sensing response element-1 and -2. Chromatin immunoprecipitation analysis provides the first in vivo evidence for activating transcription factor (ATF)-3 binding to the proximal ASNS promoter containing the nutrient-sensing response element-1 sequence. Overexpression of the full-length ATF3 protein caused a concentration-dependent biphasic response in ASNS promoter-driven transcription. Both amino acid limitation and activation of the endoplasmic reticulum stress response by glucose deprivation caused an increase in ATF3 mRNA content. However, reverse transcriptase-PCR analysis revealed that the increase in the ATF3 mRNA species detected by Northern analysis actually encoded both full-length ATF3 and two predicted truncated ATF3 isoforms (ATF3ΔZip2c and ATF3ΔZip3). Based on sequence analysis, one of the predicted truncated proteins (ATF3ΔZip3) is likely incapable of binding DNA; and yet, exogenous expression of the cDNA enhanced starvation-induced or ATF4-activated ASNS transcription, possibly by sequestering corepressor proteins. Collectively, the results provide evidence for a potential role of multiple predicted ATF3 isoforms in the transcriptional regulation of the ASNS gene in response to nutrient deprivation.
