Effect Of Amino Acid Starvation Research Articles

Investigation of the Effects of Autophagy Signaling on the Transcription of Yeast Retrotransposon Ty2-917

Objective: Ty2-917 is a low copy retrotransposon found in the Saccharomyces cerevisiae genome. It has structural similarities to metazoan retroviruses in terms of genome organization and propagation mechanisms in the host cells. The objective of this study is to analyze the effects of autophagy signaling on the transcriptional regulation of Ty2 in yeast cells. Materials and Methods: Ty2-LacZ gene fusions on the YEp vectors have been used as reporter genes to analyze the effects of amino acid starvation, nitrogen source, and autophagy signals on the transcription of Ty2. These reporter gene fusions have been transformed into the wild type and also isogenic mutant yeast strains that are defective for one of the regulatory factors involved in nutrient sensing and signaling. To activate autophagy signaling, yeast transformants were treated with caffeine or 3-amino 1-2-3 triazole. Transcription levels of Ty2-LacZ gene fusions in treated and untreated yeast cells were analyzed by β-galactosidase assays. Results: Results of this study show that transcription of Ty2 decreases up to eightfold in response to amino acid starvation. Caffeine treatment of the yeast cells also represses Ty2 transcription, independent of the TOR1 pathway. In addition, our results suggest that Ty2 transcription is also regulated in a nitrogen source-dependent manner through the GATA factors. Conclusions: Our results suggest that activation of autophagy signal results in significant level repression of Ty2 transcription. We have found that the GATA class of transcription factors is involved in the regulation of Ty2 transcription in response to autophagy signaling.

Circulating Metabolomic Signature in Generalized Pustular Psoriasis Blunts Monocyte Hyperinflammation by Triggering Amino Acid Response.

Generalized pustular psoriasis (GPP), the most grievous variant of psoriasis, is featured by dysregulated systemic inflammatory response. The cellular and molecular basis of GPP is poorly understood. Blood monocytes are key players of host defense and producers of inflammatory cytokines including IL-1β. How the immune response of monocytes is affected by metabolic internal environment in GPP remains unclear. Here, we performed a metabolomic and functional investigation of GPP serum and monocytes. We demonstrated a significant increase in IL-1β production from GPP monocytes. In GPP circulation, serum amyloid A (SAA), an acute-phase reactant, was dramatically increased, which induced the release of IL-1β from monocytes in a NLRP3-dependent manner. Using metabolomic analysis, we showed that GPP serum exhibited an amino acid starvation signature, with glycine, histidine, asparagine, methionine, threonine, lysine, valine, isoleucine, tryptophan, tyrosine, alanine, proline, taurine and cystathionine being markedly downregulated. In functional assay, under amino acid starvation condition, SAA-stimulated mature IL-1β secretion was suppressed. Mechanistically, at post-transcriptional level, amino acid starvation inhibited the SAA-mediated reactive oxygen species (ROS) formation and NLRP3 inflammasome activation. Moreover, the immune-modulatory effect of amino acid starvation was blocked by silencing general control nonderepressible 2 kinase (GCN2), suggesting the involvement of amino acid response (AAR) pathway. Collectively, our results suggested that decreased serum amino acids in GPP blunted the innate immune response in blood monocytes through AAR pathway, serving as a feedback mechanism preventing excessive inflammation in GPP.

Amino Acid Starvation Sensitizes Resistant Breast Cancer to Doxorubicin-Induced Cell Death.

Many clinical trials are beginning to assess the effectiveness of compounds known to regulate autophagy in patients receiving anti-cancer chemotherapy. However, autophagy inhibition, through exogenous inhibitors, or activation, through starvation, has revealed conflicting roles in cancer management and chemotherapeutic outcome. This study aimed to assess the effect of amino acid starvation on doxorubicin-treated breast cancer cells by assessing the roles of autophagy and apoptosis. An in vitro breast cancer model consisting of the normal breast epithelial MCF12A and the metastatic breast cancer MDAMB231 cells was used. Apoptotic and autophagic parameters were assessed following doxorubicin treatments, alone or in combination with bafilomycin, ATG5 siRNA or amino acid starvation. Inhibition of autophagy, through ATG5 siRNA or bafilomycin treatment, increased caspase activity and intracellular doxorubicin concentrations in MCF12A and MDAMB231 cells during doxorubicin treatment. While amino acid starvation increased autophagic activity and decreased caspase activity and intracellular doxorubicin concentrations in MCF12A cells, no changes in autophagic parameters or caspase activity were observed in MDAMB231 cells. Our in vivo data showed that 24 h protein starvation during high dose doxorubicin treatment resulted in increased survival of tumor-bearing GFP-LC3 mice. Results from this study suggest that short term starvation during doxorubicin chemotherapy may be a realistic avenue for adjuvant therapy, especially with regards to the protection of non-cancerous cells. More research is however, needed to fully understand the regulation of autophagic flux during starvation.

The Tuberous Sclerosis Protein TSC2 Is Not Required for the Regulation of the Mammalian Target of Rapamycin by Amino Acids and Certain Cellular Stresses

Amino acids positively regulate signaling through the mammalian target of rapamycin (mTOR). Recent work demonstrated the importance of the tuberous sclerosis protein TSC2 for regulation of mTOR by insulin. TSC2 contains a GTPase-activator domain that promotes hydrolysis of GTP bound to Rheb, which positively regulates mTOR signaling. Some studies have suggested that TSC2 also mediates the control of mTOR by amino acids. In cells lacking TSC2, amino acid withdrawal still results in dephosphorylation of S6K1, ribosomal protein S6, the eukaryotic initiation factor 4E-binding protein, and elongation factor-2 kinase. The effects of amino acid withdrawal are diminished by inhibiting protein synthesis or adding back amino acids. These studies demonstrate that amino acid signaling to mTOR occurs independently of TSC2 and involves additional unidentified inputs. Although TSC2 is not required for amino acid control of mTOR, amino acid withdrawal does decrease the proportion of Rheb in the active GTP-bound state. Here we also show that Rheb and mTOR form stable complexes, which are not, however, disrupted by amino acid withdrawal. Mutants of Rheb that cannot bind GTP or GDP can interact with mTOR complexes. We also show that the effects of hydrogen peroxide and sorbitol, cell stresses that impair mTOR signaling, are independent of TSC2. Finally, we show that the ability of energy depletion (which impairs mTOR signaling in TSC2+/+ cells) to increase the phosphorylation of eukaryotic elongation factor 2 is also independent of TSC2. This likely involves the phosphorylation of the elongation factor-2 kinase by the AMP-activated protein kinase.

Evidence of a new role for the high-osmolarity glycerol mitogen-activated protein kinase pathway in yeast: regulating adaptation to citric acid stress.

Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite minor activation of glycerol biosynthesis, the inhibitory effect of citric acid was not due to an osmotic shock. HOG1 negatively regulated the expression of a number of proteins in response to citric acid stress, including Bmh1p. Evidence suggests that BMH1 is induced by citric acid to counteract the effect of amino acid starvation. In addition, deletion of BMH2 rendered cells sensitive to citric acid. Deletion of the transcription factor MSN4, which is known to be regulated by Bmh1p and Hog1p, had a similar effect. HOG1 was also required for citric acid-induced up-regulation of Ssa1p and Eno2p. To counteract the cation chelating activity of citric acid, the plasma membrane Ca(2+) channel, CCH1, and a functional vacuolar membrane H(+)-ATPase were found to be essential for optimal adaptation. Also, the transcriptional regulator CYC8, which mediates glucose derepression, was required for adaptation to citric acid to allow cells to metabolize excess citrate via the tricarboxylic acid (TCA) cycle. Supporting this, Mdh1p and Idh1p, both TCA cycle enzymes, were up-regulated in response to citric acid.

Effects of ploidy, growth conditions and the mitochondrial nucleoid-associated protein Ilv5p on the rate of mutation of mitochondrial DNA in Saccharomyces cerevisiae.

Microsatellites, or simple repetitive sequences, are abundant in eukaryotic genomes and in the mitochondrial genome of Saccharomyces cerevisiae. These sequences alter at rates significantly higher than non-repetitive sequences of comparable size. The stability of a mitochondrial microsatellite is nearly 100-fold greater in diploid yeast cells than in isogenic haploid cells. We were able to demonstrate that this effect is likely due to ploidy alone, rather than mating-type-specific gene expression. In addition, we demonstrated that amino acid starvation affects the organization of the mitochondrial DNA and its segregation into the bud. We also tested the effect of amino acid starvation on the copy number and the mutation rate of mitochondrial DNA in both haploid and diploid yeast cells. Yeast cells grown in rich medium have a lower mitochondrial DNA content than cells starved for amino acids and have a correspondingly higher mutation rate for both frameshift mutations and point mutations in mitochondrial DNA. These effects appear to be dependent on the mitochondrial nucleoid-associated protein Ilv5p.

Effect of amino acid starvation on glucose transport in two archaeal organisms.

When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes. One of the events under SC is transport of metabolites. Thus, under amino acid starvation, E. coli fails to accumulate the non-metabolizable glucose analog alpha-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog. Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified. In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius. The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly. Unlike E. coli, H. volcanii and S. acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation. Thus, in both Archaea glucose analog transport is not under SC, as it is in E. coli.

Impact of the cross-pathway control on the regulation of lysine and penicillin biosynthesis in Aspergillus nidulans.

The non-proteinogenic amino acid, alpha-aminoadipate, defines the biosynthetic branch-point of lysine and penicillin biosynthesis in the filamentous fungus, Aspergillus nidulans. Regulation of both pathways was analysed in response to amino acid limitation. The lysF-encoded homoaconitase acts upstream of the alpha-aminoadipate branch point, whereas the lysA gene product, saccharopine dehydrogenase, catalyses the ultimate step of the lysine-specific branch. The lysA gene from A. nidulans was identified and isolated. Amino acid starvation resulted in significantly increased transcription of lysA but not lysF. Starvation-dependent changes in transcription levels of lysA were dependent on the presence of the central transcriptional activator of the cross-pathway control (CPCA). The effect of amino acid starvation under penicillin-producing conditions was analysed in A. nidulans strains with reporter genes for the penicillin-biosynthesis genes, acvA and ipnA, and genetically altered activity of the cross-pathway control. Overproduction of CPCA decreased expression of ipnAand acvA reporter genes and even more drastically reduced penicillin production. This work suggests that, upon amino acid starvation, the cross-pathway control overrules secondary metabolite biosynthesis and favours the metabolic flux towards amino acids instead of penicillin in A. nidulans.

Effect of amino acid starvation on glucose transport in two archaeal organisms

When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes. One of the events under SC is transport of metabolites. Thus, under amino acid starvation, E. coli fails to accumulate the non-metabolizable glucose analog α-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog. Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified. In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius. The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly. Unlike E. coli, H. volcanii and S. acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation. Thus, in both Archaea glucose analog transport is not under SC, as it is in E. coli.

Relaxed rrn expression and amino acid requirement of a Corynebacterium glutamicum rel mutant defective in (p)ppGpp metabolism

The stringent response in Corynebacterium glutamicum was investigated. Sets of rrn– cat fusions were constructed in their native chromosomal position to examine the effects of amino acid starvation in a rel + strain and a Δ rel mutant defective in (p)ppGpp metabolism. The expression of the six rrn operons in the rel + control was stringently regulated and reduced to 79% upon induction of amino acid starvation. The Δ rel mutant displayed a relaxed regulation and was unable to reduce the rrn expression under amino acid depletion conditions. In addition, the Δ rel mutant grew more slowly in minimal medium than a rel + control. This growth effect was restored by a plasmid-encoded copy of rel or, alternatively, by supplementation of the minimal medium with the amino acid mixture casamino acids. In particular, the Δ rel strain of C. glutamicum displayed a requirement for the amino acids histidine and serine.

Relaxedrrnexpression and amino acid requirement of aCorynebacterium glutamicum relmutant defective in (p)ppGpp metabolism

The stringent response in Corynebacterium glutamicum was investigated. Sets of rrn-cat fusions were constructed in their native chromosomal position to examine the effects of amino acid starvation in a rel(+) strain and a Deltarel mutant defective in (p)ppGpp metabolism. The expression of the six rrn operons in the rel(+) control was stringently regulated and reduced to 79% upon induction of amino acid starvation. The Deltarel mutant displayed a relaxed regulation and was unable to reduce the rrn expression under amino acid depletion conditions. In addition, the Deltarel mutant grew more slowly in minimal medium than a rel(+) control. This growth effect was restored by a plasmid-encoded copy of rel or, alternatively, by supplementation of the minimal medium with the amino acid mixture casamino acids. In particular, the Deltarel strain of C. glutamicum displayed a requirement for the amino acids histidine and serine.

Life in protein-rich environments: the relA-independent response of Streptococcus pyogenes to amino acid starvation.

Considering that group A streptococci are multiple auxotrophs that may encounter shortage of amino acids during specific stages of the infectious process, we studied their adaptive response to amino acid deprivation. We found that, in addition to the (p)ppGpp-mediated stringent response characterized previously, Streptococcus pyogenes exhibits a relA-independent response comprising transcriptional modulation of a specific subset of genes involved in pathogenesis. Genes/operons transcriptionally upregulated during starvation of both wild type and relA mutants included the two-component signal transduction system covRS, the positive regulator (ropB) of the pyrogenic exotoxin B gene, speB, the oligopeptide (opp) and dipeptide (dpp) permease systems and the pepB gene putatively involved in the intracellular processing of oligopeptides. Upregulation of covRS was accompanied by downregulation of ska, one of the target genes of the negative CovR regulator, and the net effect of amino acid starvation also favoured repression of speB. A significant feature of upregulated opp expression was stimulated readthrough transcription of the operon-internal oppA terminator, leading to increased mRNA levels for synthesis of the translocator complex relative to the substrate-binding protein. Based on these and previous results, a stimulus-response network is proposed that counteracts the stringent response and may enable the pathogen to mount a dynamic response to the protein-rich environment provided by its human host.

Erroneous synthesis of ribosomal proteins in amino acid starved E. coli

The effect of amino acid starvation on the accuracy of translation of ribosomal proteins was analyzed in a stringent ( relA + )/relaxed ( relA) pair of E. coli strains. The degree of misreading was estimated from the amount of cysteine erroneously incorporated into individual proteins during arginine starvation of bacteria. Illegitimate incorporation of cysteine was found to occur to a significant extent in several proteins from both the small and the large subunits of ribosomes, in either type of strain.

Conformational changes in bacterial polysomes induced by amino acid starvation

1. 1. The effects of amino acid starvation on polysome conformation were analyzed comparatively in stringent (relA +) and relaxed (relA) bacteria by measuring the accessibility in vitro of ribosomal proteins to reductive methylation. 2. 2. In polysomes of stringent cells, the conformational state of two proteins (L13 and L29) appeared significantly changed by starvation. 3. 3. In polysomes isolated from relaxed mutants, the accessibility of five proteins (L5, L13, L29, L31 and L32) was found modified.

Computer simulation of ribosome editing

A stochastic model of protein synthesis was modified by including the process of dissociating peptidyl-tRNA from ribosomes. To simulate ribosome editing, the probability of dissociation was assumed to be high if the peptidyl-tRNA was erroneous; that is, if it resulted from transfer of a peptide to an aminoacyl-tRNA that was inappropriate relative to the mRNA codon. The effects of amino acid starvation on protein synthesis were simulated both by increasing the probability of such erring at and by reducing the conditional probability of elongation at “hungry” codons, those whose correct amino acid was in short supply. These probabilities were varied systematically to simulate tryptophan limitation during synthesis of coat protein from bacteriophage MS2. Significant reduction, during starvation, in the synthesis of complete coat protein required large reductions in the probability of elongation at hungry codons but only small increases in the probability of erring. Enhanced dissociation of peptidyl-tRNA during starvation, followed rapidly by dissociation of ribosomes from mRNA, led to reductions in mean polysome size, a result that had been interpreted by others as due to some effect of starvation on the initiation of protein synthesis. Results from experiments by Goldman (1982) on the cell-free synthesis of MS2 coat protein during tryptophan starvation could be mimicked in detail by the computer simulations. A simple competition between correct and erroneous amino acids was sufficient to explain the tryptophan dependence of complete coat protein and internal peptide syntheses. Values for the Michaelis constants were derived from the computer simulations.

Cell volume change of Escherichia coli under stringent control apparent increase of K + in rel− cells

With several pairs of rel + and rel − strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K + and the rate of uptake of 42K + were investigated. In the early phase of the experiments, the intracellular concentration of K + was estimated by the conventional method in which the cell volume per A 660 value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K + concentration of rel + cells was kept almost constant, while that of rel − cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A 660 changed only slightly in CP78 ( rel +) cells, while it increased markedly in CP79 ( rel −) cells after the exposure to amino acid starvation. Reestimation of the K + concentrations based on the estimated respective values of cell volumes per A 660 revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel + cells was arrested upon amino acid starvation whereas that in the rel − cells was not. The 42K + uptake by the rel + cells was depressed upon amino acid starvation, whereas that by the rel − cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K + concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A 660 of the culture, was pointed out.

Functional aspects of bacterial polysomes during limited protein synthesis

The effects of amino acid starvation on the metabolic behavior of polysomes and the size distribution of proteins have been studied in an otherwise isogenic pair of stringent ( relA +) and relaxed ( relA) strains of Escherichia coli. The stability of polysomes has been analyzed by using two different approaches. First, the process of their degradation has been followed after treating the cells with rifampicin, an inhibitor of the synthesis of all classes of RNA including messenger RNA. Secondly, the process of their assembly has been studied after their previous conversion to monosomes, as induced by glucose deprivation of cells. It is shown that, in either type of bacterial strain, polysomes are continually broken down and re-synthesized during amino acid starvation. However, such polysome turnover is then less rapid than in normally growing bacteria, and, moreover, it seems amino acid specific since it occurs at a lower rate during arginine starvation than during histidine starvation, namely, in the relaxed strain. The molecular weight distribution of proteins has been determined after labeling of cells with radioactive methionine and separation of polypeptides by one-dimensional polyacrylamide gel electrophoresis. The average size of polypeptides synthesized in the stringent strain during starvation is quite similar to that measured during normal growth. By contrast, a significant shift towards smaller molecules is observed in the relaxed strain deprived of an essential amino acid. Here again, this reduction of the size of polypeptides seems amino acid specific since it is especially marked during arginine starvation. These results are discussed in terms of ribosomes translocation and premature peptide chain termination in connection with the accuracy of the translational process.

The effect of amino acid starvation on a major, acid soluble compound on Saccharomyces cerevisiae.

The effect of amino acid starvation on a major, acid soluble compound on Saccharomyces cerevisiae.

Inefficiency of the stringent response in the fungus [formula omitted

Removal of a required amino acid from the growth medium or addition of cycloheximide caused an immediate stoppage of growth and protein synthesis in the fungus Mucor racemosus . However, RNA synthesis persisted for several hours at rates that only gradually decreased under the same circumstances. An analysis of the major classes of RNA synthesized during the first hour of treatment showed that cycloheximide preferentially inhibited rRNA synthesis, whereas amino acid starvation slowed synthesis of all RNA species uniformly. Neither treatment affected the percentage of mRNA synthesized. The partial and delayed effects of amino acid starvation and cycloheximide treatment on RNA synthesis reported here suggest the absence of or the gross inefficiency of a classical stringent response in M. racemosus .

The effect of amino acid starvation on nucleoside uptake and RNA synthesis in Tetrahymena.

The uptake of nucleosides and the synthesis of RNA in Tetrahymena thermophila were examined following amino acid starvation. Omission of leucine, phenylalanine, or arginine from the medium resulted in a rapid decrease in the incorporation of [3H]uridine into the acid-soluble pool and acid-insoluble material (RNA). Amino acid starvation inhibited the uptake of all ribo- and deoxyribonucleosides tested but did not affect the uptake of amino acids or glucose. In addition, under the conditions used, the omission of an amino acid did not result in a large decrease in amino acid incorporation into total protein. Treatment of cells with cycloheximide or emetine gave results similar to the effects of amino acid starvation, but in these experiments the inhibition of protein synthesis was essentially complete. Nucleotide pool sizes were also measured following amino acid starvation. ATP and UTP levels were essentially unchanged, but the dTTP pool size was decreased by 40%. The decrease in RNA synthesis in vivo in the absence of an essential amino acid was reflected in the endogenous RNA synthetic activity of isolated nuclei. However, when solubilized RNA polymerase activity was measured with calf thymus DNA as template, no significant difference was observed between control and amino acid-starved cells.