Amino Acid Response Research Articles

Transcriptional regulation of the LAT-1/CD98 light chain

LAT-1/CD98 amino acid transporter expression and activity are induced in hepatic cells deprived of arginine. The promoter dependency of this regulation was investigated. LAT-1 expression, in contrast to that of CD98 heavy chain 4F2, was actinomycin D sensitive in cells cultured without arginine. Transient transfection analysis with promoter reporter constructs including the 2 kbp LAT-1 promoter or a sub-sequence containing multiple potential amino acid response elements failed to show significant amino acid sensitivity in various cell types. Chromatin-dependency did not appear to account for this result as hepatic cell clones stably transfected with the promoter constructs showed little or no arginine or leucine responsive promoter activity. These studies suggest that while amino acid sensitivity of LAT-1 expression is transcriptionally regulated, cis elements within the proximal promoter do not directly mediate this regulation. Understanding mechanisms by which this gene responds to amino acid availability will contribute to our knowledge of how eukaryotic cells sense and respond to their environment.

Methylation of the asparagine synthetase promoter in human leukemic cell lines is associated with a specific methyl binding protein.

We have examined the methylation profiles of the asparagine synthetase (ASY) promoter in a number of human leukemic cell lines in relation to their asparagine (ASN) requirements in vitro. Cells in which the promoter is highly methylated are auxotrophs and express ASY at very low levels. Electromobility shift assays (EMSA) of nuclear extracts with oligomers from the promoting region show, in addition to recognized transcription factor binding, a novel methyl binding protein specific for a 12 base consensus sequence, which includes a single methylated CpG. This sequence overlaps that of the amino-acid response unit of the ASY promoter, which is activated byATF4 and C/EBP. Competition by the methyl binding protein could account for the observed failure of the methylated promoter to bind these transcription factors and consequently, although other mechanisms can also be operative, for the specific repression of the gene. The ASY methyl binding protein (ASMB) is present in leukemic lymphoid and myeloid cells irrespective of their methylation status, and in normal lymphocytes after phytohemagglutinin stimulation. It has been purified by affinity chromatography and has a molecular size of 40 kDa in 10% SDS-polyacrylamide gels.

Induction of CHOP Expression by Amino Acid Limitation Requires Both ATF4 Expression and ATF2 Phosphorylation

The CHOP gene is transcriptionally induced by amino acid starvation. We have previously identified a genomic cis-acting element (amino acid response element (AARE)) involved in the transcriptional activation of the human CHOP gene by leucine starvation and shown that it binds the activating transcription factor 2 (ATF2). The present study was designed to identify other transcription factors capable of binding to the CHOP AARE and to establish their role with regard to induction of the gene by amino acid deprivation. Electrophoretic mobility shift assay and transient transfection experiments show that several transcription factors that belong to the C/EBP or ATF families bind the AARE sequence and activate transcription. Among all these transcription factors, only ATF4 and ATF2 are involved in the amino acid control of CHOP expression. We show that inhibition of ATF2 or ATF4 expression impairs the transcriptional activation of CHOP by amino acid starvation. The transacting capacity of ATF4 depends on its expression level and that of ATF2 on its phosphorylation state. In response to leucine starvation, ATF4 expression and ATF2 phosphorylation are increased. However, induction of ATF4 expression by the endoplasmic reticulum stress pathway does not fully activate the AARE-dependent transcription. Taken together our results demonstrate that at least two pathways, one leading to ATF4 induction and one leading to ATF2 phosphorylation, are necessary to induce CHOP expression by amino acid starvation. This work was extended to the regulation of other amino acid regulated genes and suggests that ATF4 and ATF2 are key components of the amino acid control of gene expression.

Proteins and amino acids in enteral nutrition.

This review reports recent findings on the effect of enterally fed protein and amino acids on metabolism, function, and clinical outcome, particularly during the neonatal period. Splanchnic tissues metabolize significant proportions of some enteral amino acids and this likely contributes to the higher requirement for these amino acids when they are provided enterally versus parenterally. Splanchnic tissues are particularly key in the provision of nutrition to preterm infants, who possess an exceedingly high protein anabolic drive, but limited tolerance to aggressive enteral feeding. The protein anabolic response to specific proteins is influenced by the rate of digestion and the pattern of feeding, as well as the amino acid composition of the proteins. The post-prandial rise in amino acids and insulin stimulates neonatal tissue protein synthesis by modulation of the nutrient and insulin signaling pathways that lead to translation initiation. A flurry of investigations into the metabolic response and clinical impact of individual amino acids suggests that leucine, glutamine, and arginine, in particular, have specific roles in regulating protein synthesis and immune function. Recent findings suggest that enteral nutrition support that provides an optimum combination of proteins and amino acids can have a beneficial impact on the clinical outcome of patients.

Transcriptional Control of the Human Sodium-coupled Neutral Amino Acid Transporter System A Gene by Amino Acid Availability Is Mediated by an Intronic Element

System A amino acid transporter (SNAT2) gene expression is up-regulated at the transcriptional level in response to amino acid deprivation. Functional analysis of genomic fragments 5' upstream of the transcription start site, for both human and mouse SNAT2 genes showed that these regions exhibit promoter activity, but were amino acid unresponsive. However, when the human and mouse constructs were extended to include intron 1, it was observed that the rate of transcription was increased following amino acid deprivation. Deletion analysis of the human gene identified an intron 1 sequence spanning 54 nucleotides that was sufficient for conferring amino acid-dependent regulation to a minimal SNAT2 promoter. Alignment of the corresponding region from the human, mouse, and rat genomes revealed three highly conserved sequences. From site-directed mutagenesis, it was concluded that one of these sites functions as an amino acid response element (AARE) to regulate transcription. The core sequence of this site is identical to the AARE in the human CHOP gene. The SNAT2 AARE, along with a nearby conserved CAAT box, has enhancer activity in that it functions in an orientation and position independent manner, and it confers regulated transcription to a heterologous promoter.

Transcriptional Control of the Arginine/Lysine Transporter, Cat-1, by Physiological Stress

Cells respond to physiological stress by phosphorylating the alpha subunit of the translation initiation factor eIF2. This adaptive response inhibits protein synthesis and up-regulates genes essential for cell survival. Cat-1, the transporter for the essential amino acids, arginine and lysine, is one of the up-regulated genes. We previously showed that stress increases cat-1 expression by coordinated stabilization of the mRNA and increased mRNA translation. This induction is triggered by amino acid depletion and the unfolded protein response (UPR), which is caused by unfolded proteins in the endoplasmic reticulum. We show here that cat-1 gene transcription is also increased by cellular stress. Our studies demonstrate that the cat-1 gene promoter/regulatory region is TATA-less and is located in a region that includes 94 bases of the first exon. Transcription from this promoter is stimulated 8-fold by cellular stress. An amino acid response element within the first exon is shown to be required for the response to amino acid depletion but not to the UPR. The stimulation of transcription by amino acid depletion requires activation of GCN2 kinase, which phosphorylates eIF2alpha. This phosphorylation also induces translation of the cat-1 mRNA, demonstrating that stress-induced transcriptional and translational control of cat-1 are downstream targets of a signaling pathway initiating with eIF2alpha phosphorylation. Our studies show that the increase in cat-1 gene expression by cellular stress involves at least three types of coordinate regulation: regulation of transcription, regulation of mRNA stability, and regulation of mRNA translation.

Correlation between Olfactory Receptor Cell Type and Function in the Channel Catfish

The olfactory epithelium of fish contains three intermingled types of olfactory receptor neurons (ORNs): ciliated, microvillous, and crypt. The present experiments were undertaken to test whether the different types of ORNs respond to different classes of odorants via different families of receptor molecules and G-proteins corresponding to the morphology of the ORN. In catfish, ciliated ORNs express OR-type receptors and Galpha(olf). Microvillous ORNs are heterogeneous, with many expressing Galpha(q)/11, whereas crypt ORNs express Galpha(o). Retrograde tracing experiments show that ciliated ORNs project predominantly to regions of the olfactory bulb (OB) that respond to bile salts (medial) and amino acids (ventral) (Nikonov and Caprio, 2001). In contrast, microvillous ORNs project almost entirely to the dorsal surface of the OB, where responses to nucleotides (posterior OB) and amino acids (anterior OB) predominate. These anatomical findings are consistent with our pharmacological results showing that forskolin (which interferes with Galpha(olf)/cAMP signaling) blocks responses to bile salts and markedly reduces responses to amino acids. Conversely, U-73122 and U-73343 (which interfere with Galpha(q)/11/phospholipase C signaling) diminish amino acid responses but leave bile salt and nucleotide responses essentially unchanged. In summary, our results indicate that bile salt odorants are detected predominantly by ciliated ORNs relying on the Galpha(olf)/cAMP transduction cascade. Nucleotides are detected by microvillous ORNs using neither Galpha(olf)/cAMP nor Galpha(q)/11/PLC cascades. Finally, amino acid odorants activate both ciliated and microvillous ORNs but via different transduction pathways in the two types of cells.

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

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 (ATF3deltaZip2c and ATF3deltaZip3). Based on sequence analysis, one of the predicted truncated proteins (ATF3deltaZip3) 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.

Dietary Amino Acid and Sodium Bicarbonate Responses in Broilers Reared in Hot Environmental Temperatures

Better environmentally controlled broiler houses have resulted in many integrated broiler operations using the same dietary feed formulations in winter and summer months due to only minor differences in environmental temperature in the broiler house. Much research has addressed broiler nutrient needs in thermoneutral vs. hot temperature conditions, but research evaluating nutrient needs of broilers during moderate temperature conditions (tunnel-ventilated houses during summer months) is lacking. This study evaluates the impact of increasing dietary Lys and other essential amino acids, and the addition of dietary sodium bicarbonate in high and moderate temperature conditions. Experiment 1 was conducted in batteries and evaluated broiler nutritional responses (d 20 to 40) in hot-cyclic temperature conditions (26 to 34°C). Experiment 2 was conducted in floor pens and evaluated broiler nutritional responses (d 37 to 49) in environmental temperature ranges that mimic a tunnel-ventilated broiler house during summer months (26 to 31°C). In both experiments, dietary treatments had minimal impact on live performance and breast meat yield. Lower mortality (P < 0.06), however, was observed in broilers fed the low CP diet in the hot temperature environment.

The appearance and maturation of excitatory and inhibitory neurotransmitter sensitivity during retinal regeneration of the adult newt

Using living slice preparations from newt retinas at different stages of regeneration, we examined the time course of appearance and maturation of neurotransmitter-induced currents with whole-cell patch-clamp methods. Neurons from which currents were recorded were identified by Lucifer Yellow fills. All progenitor cells examined at the regenerating retinas did not express any voltage-gated Na + currents and responsiveness to excitatory amino acid analogues (AMPA and NMDA) and inhibitory amino acids (GABA and glycine). Voltage-gated Na + currents were first detected in premature ganglion cells with round cell body located at the most proximal level of the ‘intermediate-II’ regenerating retina. AMPA- GABA- and glycine-induced currents were simultaneously observed in many premature ganglion cells expressing Na + channels, but not all, suggesting that the onset of the Na + channels is slightly earlier than that of excitatory and inhibitory amino acid receptors in regeneration. NMDA-evoked currents were first observed in the ‘intermediate-III’ regenerating retina just before the synaptogenesis. Pharmacological properties and reversal potential values of the excitatory and inhibitory amino acid responses did not change substantially between regenerating ganglion cells and mature ganglion cells, while rectification properties of current–voltage relations for AMPA and NMDA responses were somewhat different between them.

Characterization of the nutrient-sensing response unit in the human asparagine synthetase promoter.

Transcription from the human 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 nutrient-sensing pathways converge on the same set of genomic cis -elements, referred to as nutrient sensing-response elements (NSREs) 1 and 2, within the A.S. promoter. The present report uses single-nucleotide mutagenesis to confirm that both NSRE-1 and NSRE-2 are absolutely required for gene activation and to identify the boundaries of each binding site. The core sequence of the NSRE-1 site is contained within nucleotides -68 to -60 and the NSRE-2 core sequence is within nucleotides -48 to -43. Through insertion or deletion of 5-10 nucleotides in the intervening sequence between NSRE-1 and NSRE-2, transient transfection studies with an A.S. promoter/reporter gene construct showed that the 11 bp distance between these two elements is critical. These results document that the optimal configuration is with both binding sites on the same side of the DNA helix, only one helical turn away from each other and the data provide support for the hypothesis that a larger multi-protein complex exists between the binding proteins for NSRE-1 and NSRE-2. The data also illustrate that the combination of NSRE-1 and NSRE-2, referred to as the nutrient-sensing response unit (NSRU), has enhancer activity in that it functions in an orientation- and position-independent manner, and conveys nutrient-dependent transcriptional control to a heterologous promoter.

Leucine: a key amino acid in ageing-associated sarcopenia?

During ageing, a progressive loss of muscle mass has been well described in both man and rodents. This loss of proteins results from an imbalance between protein synthesis and degradation rates. Although some authors have shown a decrease of myofibrillar protein synthesis rates in human volunteers, this imbalance is not clearly apparent when basal rates of protein turnover are measured. A decrease in muscle protein synthesis stimulation was detected nevertheless in ageing rats during the postprandial period, suggesting that the 'meal signal' was altered during ageing. Many results now suggest that aged muscle is less sensitive to the stimulatory effect of amino acids at physiological concentrations but is still able to respond if the increase in aminoacidaemia is sufficiently large. Indeed amino acids play an important role in regulating muscle protein turnover both in vitro and in vivo. At the molecular level, amino acids modulate gene expression. Amino acid response elements have been characterised in the promoter of transcriptional factor CCAAT-enhancer binding protein homologous protein and asparagine synthetase genes. Among amino acids, leucine seems to play the major role in regulating the metabolic function. It inhibits proteolysis and stimulates muscle protein synthesis independently of insulin. Leucine has been shown to act as a real mediator by modulating specifically the activities of intracellular kinases linked to the translation of proteins such as phosphatidylinosinol 3' kinase and mammalian target of rapamycin-70 kDa ribosomal protein S6 (p70S6K) kinases. We recently demonstrated in vitro that protein synthesis of ageing rat muscles becomes resistant to the stimulatory effect of leucine in its physiological concentration range. However, when leucine concentration was increased greatly above its postprandial level, protein synthesis was stimulated normally. Moreover, we studied the effect of meal leucine supplementation on in vivo protein synthesis in adult and ageing rats. Leucine supplementation had no additional effect on muscle protein synthesis in adults but totally restored its stimulation in ageing rats. Whether chronic oral leucine supplementation would be beneficial for maintaining muscle protein mass in elderly men and women remains to be studied.

Differences in the Molecular Mechanisms Involved in the Transcriptional Activation of the CHOP and Asparagine Synthetase Genes in Response to Amino Acid Deprivation or Activation of the Unfolded Protein Response

A promoter element called the amino acid response element (AARE), which is essential for the induction of CHOP (a CCAAT/enhancer-binding protein-related gene) transcription by amino acid depletion, has been previously characterized. Conversely, the human asparagine synthetase (AS) promoter contains two cis-acting elements termed nutrient-sensing response elements (NSRE-1 and NSRE-2) that are required to activate the gene by either amino acid deprivation or the endoplasmic reticulum stress response. The results reported here document the comparison between CHOP and AS transcriptional control elements used by the amino acid pathway. We first establish that the AS NSRE-1 sequence shares nucleotide sequence and functional similarities with the CHOP AARE. However, we demonstrate that the CHOP AARE can function independently, whereas AS NSRE-1 is functionally weak by itself and instead requires the presence of NSRE-2. Furthermore, AS NSRE-2 can confer endoplasmic reticulum stress responsiveness to the CHOP AARE. Using activating transcription factor-2-deficient mouse embryonic fibroblasts, we also show that lack of this transcription factor does not abolish the amino acid inducibility of AS transcription, but this transcription factor is necessary to obtain the full AS response to amino acid starvation. Collectively, these results document that there are significant differences in the molecular mechanisms involved in the transcriptional activation of CHOP and AS by amino acid limitation.

Probing Src homology 2 domain ligand interactions by differential line broadening.

Few techniques for probing the role of individual amino acids in interactions of a protein with ligands are available. Chemical shift perturbations in NMR spectra provide qualitative information about the response of individual amino acids of a protein to its interactions with ligands. Line shapes derived from (15)N-HSQC spectra recorded for different steps of a ligand titration yield both kinetic constants and insight into mechanisms by which the ligand binds. Here we have analyzed line shapes for 37 signals of amino acids of the N-terminal src homology 2 domain (N-SH2) of the 85 kDa subunit of phosphatidylinositol 3-kinase (PI3-K) upon binding of phosphotyrosine (ptyr)-containing peptides. Kinetic rates at individual amino acids of the SH2 varied throughout the structure. For a subset of SH2 residues, the fine structure of the NMR line shapes indicated slow motions induced by the presence of small amounts of the ligand. These complex line shapes require one or more additional conformational states on the kinetic pathway. Modeling of the observed ligand interactions suggests a quasi-allosteric initial binding step. N-SH2 mutants with altered ligand affinity or specificity were also examined. Analysis of their line shapes revealed three distinct classes of mutants with different kinetic behaviors.

Effect of an endurance race on muscle amino acids, pro- and macroglycogen and triglycerides.

Glycogenolysis and lipolysis are essential for energy production in muscle during prolonged exercise but less is known about the role of amino acid metabolism. The aim was to study the effect of an endurance race on pro- and macroglycogen, triglycerides and amino acid concentrations in muscle and on selected blood parameters, especially the amino acid response in the blood during the recovery phase. Seven endurance-trained horses completed a race covering 100-120 km. Blood samples were collected one day before the race, after the finish and 1 and 8 days after the race. Needle biopsy specimens from the gluteus medius muscle were taken on completion of the race and 8 days postrace. The pro- and macroglycogen concentrations were lower and most amino acid concentrations were higher in the muscle after the finish of the race, than 8 days postrace, whereas the triglyceride concentrations did not differ. The concentrations of several amino acids in serum were higher during the recovery phase than before the race. The observed changes suggest that an endurance race influences amino acid metabolism and a net protein degradation may occur that persists during the recovery phase. Furthermore, the results suggest that macroglycogen is utilised to a greater extent than proglycogen during an endurance race. Further research is needed concerning amino acid metabolism during an endurance race as it seems to play an important role both for performance and recovery after exercise.

ATF4 Is a Mediator of the Nutrient-sensing Response Pathway That Activates the Human Asparagine Synthetase Gene

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.

Effects of Drought on the Competitive Interference of an Early Successional Species (Rubus fruticosus) on Fagus sylvatica L. Seedlings: 15N Uptake and Partitioning, Responses of Amino Acids and other N Compounds

Abstract: We assessed the role of water availability as a factor regulating the ability of beech seedlings to cope with competitive interference for nitrogen resources by an early successional species (Rubus fruticosus). A glasshouse experiment was performed with two levels of interference (beech with and without R. fruticosus) and three levels of irrigation (high, intermediate, none). 15N uptake and partitioning of both species, and composition of N pools in leaves, roots and phloem of beech, were determined. Under all irrigation regimes, 15N uptake by beech seedlings decreased when grown together with R. fruticosus. R. fruticosus had higher 15N uptake rates than beech, under all water supply levels. When irrigation was reduced, a substantial decrease in 15N uptake of beech seedlings and a concurrent increase in 15N uptake by R. fruticosus were observed. Interference by R. fruticosus and low irrigation also affected the 15N partitioning in beech seedlings and resulted in reduced allocation of 15N to the roots. The combination of competitive interference and lack of irrigation led to an increase in soluble non‐protein N in roots and leaves of beech, due to protein degradation. This response was attributed to an increase in levels of amino acids serving as osmoprotectants under these conditions. The concentration of proline in leaves of beech was negatively correlated to shoot water potential. A competition‐induced reduction of total N in leaves of beech under high and intermediate irrigation was found. These results illustrate (1) the advantage of R. fruticosus in terms of N uptake when compared to young beech, particularly under inadequate water supply, and (2) the changes in N composition of beech seedlings in order to cope with reduced soil water and interference by R. fruticosus.

Response of Feeding Amino Acids Supplemented Cottonseed Meal on Growth Performance and Digestibility of Early Weaned Cow Calves

This study was designed to examine the response of feeding early weaning diets without and with amino acids supplemented cottonseed meal on growth performance and digestibility of early weaned cow calves. Fifteen 14-21 days old cross bred cow calves were randomly allotted to three experimental diets. Diet A comprised of milk replacer and concentrate feed whereas two iso- caloric and iso-nitrogenous early weaning diets viz., B and C were prepared without and with lysine and methionine supplemented cottonseed meal, respectively. These early weaning diets were used as substitute of milk replacer for calves. Calves were placed in individual cages and fed twice daily for a period of 60 days. Daily feed intake, feed refused and weekly weight gain was recorded. Two digestibility trials I and II were performed at 5th and 9th week of the experiment, respectively. During the digestibility trial I, calves were fed on ad libitum basis whereas in trial II, calves were fed at 90% of their voluntary DMI. Feed, orts and feces samples were collected, weighed daily, composited, subsampled and analysed for DM and CP. Results indicated that weight gain of calves was (p<0.05) higher on diets A (0.63 kg/d/calf) and C (0.64 kg/d/calf) compared to calves on diet B (0.57 kg/d/calf). Significantly (p<0.05) less daily DMI was observed on diet A (1.48 kg) compared to early weaning diets B (1.70 kg) and C (1.72 kg). The feed efficiency was (p<0.05) better on diet A (2.33) compared to diets B (2.95) and C (2.65). The economic efficiency was noticed to be better on diets B and C compared to diet A. In trial I, digestibility of DM and CP of diet A was (p<0.05) higher than diets B and C. Whereas in trial II, digestibility of DM and CP of diet A was (p<0.05) less than diets B and C. It was concluded that early weaning diet based on lysine and methionine supplemented cottonseed meal produced better weight gain and feed efficiency compared to non-supplemented cottonseed meal based diet. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 2 : 184-187)

CCAAT/Enhancer-binding Protein-β Is a Mediator of the Nutrient-sensing Response Pathway That Activates the Human Asparagine Synthetase Gene

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-beta (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/EBPbeta 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/EBPbeta-containing complexes. In contrast, mutation of the NSRE-2 sequence did not block C/EBPbeta binding. Overexpression in HepG2 hepatoma cells of the activating isoform of C/EBPbeta increased AS promoter-driven transcription, whereas the inhibitory dominant-negative isoform of C/EBPbeta 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/EBPbeta in the transcriptional activation of the AS gene in response to nutrient deprivation.

Up-regulation of upstream stimulatory factors by protein malnutrition and its possible role in regulation of the IGF-binding protein-1 gene.

Protein malnutrition drastically induces the expression of the IGF-binding protein-1 (IGFBP-1) gene. We have previously shown that the region between -77 and -112 bp upstream of the rat IGFBP-1 gene contributes to the response of this gene to amino acid limitation. In an attempt to elucidate the basis of the responsiveness of this putative amino acid response unit (AARU), we searched the nucleus of the rat liver for a trans-acting factor whose binding to AARU was dependent on protein nutrition. Liver nuclear extracts of rats fed a protein-free diet and of those fed a control diet were compared by EMSA using the AARU as probe. One of the protein-probe complexes underwent a drastic increase after dietary protein deprivation. Assays using specific antibodies and several competitor oligonucleotides led to identification of the protein composing the complex as upstream stimulatory factor-1 (USF) and USF-2. The binding site of the USF proteins in the AARU turned out to be a CACGGG sequence that was homologous to the consensus USF-binding sequence (E box; CANNTG). Further, Western blot analyses showed that a protein-free diet caused significant increases in USF-1 and USF-2 levels. Thus, elevated expression of the IGFBP-1 gene under protein malnutrition can be attributable to increased binding of USF to its promoter, which results from increased USF levels. The data suggest that the changes in these ubiquitously distributed transcription factors play an important role in the nutritional regulation of expression of mammalian genes.