Autophagy is essential for the maintenance of amino acids and ATP levels during acute amino acid starvation in MDAMB231 cells

2018 ◽  
Vol 36 (2) ◽  
pp. 65-79 ◽  
Author(s):  
Mark Thomas ◽  
Tanja Davis ◽  
Ben Loos ◽  
Balindiwe Sishi ◽  
Barbara Huisamen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Starvation ◽  
Atp Levels

scholarly journals The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels

2020 ◽  
Vol 48 (6) ◽  
pp. 3071-3088
Author(s):  
Matthew R McFarland ◽  
Corina D Keller ◽  
Brandon M Childers ◽  
Stephen A Adeniyi ◽  
Holly Corrigall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurological Disorders ◽  
Trna Synthetase ◽  
Northern Blotting ◽  
Amino Acid Starvation ◽  
Starvation Response ◽  
Kinase Activation ◽  
Trna Synthetases ◽  
Starvation Conditions

Abstract During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

scholarly journals Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown

1976 ◽  
Vol 154 (2) ◽  
pp. 541-552
Author(s):  
J E. M. Midgley
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Turnover Rate ◽  
Amino Acid Starvation ◽  
High Rate ◽  
Glucose Medium ◽  
Amino Acid Deprivation ◽  
Acid Accumulation ◽  
Associated Proteins ◽  
Kinetics Of

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing “stepdown” in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active β-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in “stepped-down” rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of β-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.

Control of RNA Synthesis by Amino Acids in Landschutz Ascites Tumor Cells

1974 ◽  
Vol 52 (10) ◽  
pp. 867-876 ◽  
Author(s):  
Paul Jolicoeur ◽  
Fernand Labrie
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Inhibitory Effect ◽  
Amino Acid Starvation ◽  
Cytoplasmic Rna ◽  
Polysomal Mrna ◽  
Deficient Medium ◽  
Synthesis And Processing

Landschutz cells incubated in amino acid-deficient medium for 2.5 h show a markedly reduced incorporation of [3H]uridine into 18 S and 28 S cytoplasmic ribosomal RNA (rRNA) and into 28 S, 32 S, and 36 S nuclear RNA measured during the last 90 min of incubation, whereas the radioactivity associated with 45 S pre-rRNA is not affected. Ten-minute pulse-labeling and 15-min pulse-chase experiments show that amino acid starvation inhibits both the synthesis and processing of 45 S pre-rRNA. Amino acid starvation has no significant effect on the labeling of the nucleotide pools. This effect of amino acids was specific for rRNA since the synthesis of 4 S and 5 S cytoplasmic RNA separated on polyacrylamide gels and of polysomal mRNA analyzed on sucrose gradients was not significantly affected during amino acid starvation. These data also indicate that RNA synthesis is non-coordinated in Landschutz cells. Among the 13 amino acids essential for growth of these cells, arginine and glutamine appear to be mainly responsible for the inhibition of synthesis of 18 S and 28 S rRNA measured during incubation in complete amino acid-deficient medium. The removal of any one of the other amino acids has a small inhibitory effect on the incorporation of [3H]uridine into rRNA and their effect on the synthesis of 18 S rRNA is more pronounced than on that of 28 S rRNA. Such effect results in an unbalanced production of these two ribosomal RNA species.

scholarly journals Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells

2017 ◽  
Vol 42 (6) ◽  
pp. 2418-2429 ◽  
Author(s):  
Huiying Shi ◽  
Xinyan Zhao ◽  
Zhen Ding ◽  
Chaoqun Han ◽  
Ye Jiang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Amino Acid Starvation ◽  
Autophagic Flux ◽  
Intestinal Epithelial ◽  
Expression Levels ◽  
Sequestosome 1

Background/Aims: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs). Na+/H+-exchanger 3 (NHE3) has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. Methods: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3) levels, transmission electron microscopy (TEM), tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62) mRNA and protein levels, and autophagy-related gene (ATG) 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes) to red (autolysosomes) LC3 puncta. Results: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. Conclusions: These results demonstrate that NHE (most likely NHE3) may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

scholarly journals The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids.

1995 ◽  
Vol 15 (8) ◽  
pp. 4497-4506 ◽  
Author(s):  
S A Wek ◽  
S Zhu ◽  
R C Wek
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Trna Synthetase ◽  
Amino Acid Starvation ◽  
Related Sequence ◽  
General Amino Acid Control ◽  
Trna Synthetases ◽  
Acid Control ◽  
Direct Regulator

Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety. Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control. It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation. In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations. Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals. We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation. Cells containing a defective aminoacyl-tRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase. Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2. Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response. These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.

scholarly journals Ammonium-Dependent Shortening of CLS in Yeast Cells Starved for Essential Amino Acids Is Determined by the Specific Amino Acid Deprived, through Different Signaling Pathways

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Júlia Santos ◽  
Cecília Leão ◽  
Maria João Sousa
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Essential Amino Acid ◽  
Essential Amino Acids ◽  
Amino Acid Starvation ◽  
Yeast Cells ◽  
Environmental Interventions ◽  
Specific Amino Acid ◽  
Chronological Life Span ◽  
In Cells

Ammonium (NH4+) leads to chronological life span (CLS) shortening inSaccharomyces cerevisiaeBY4742 cells, particularly evident in cells starved for auxotrophy-complementing amino acids (leucine, lysine, and histidine) simultaneously. Here, we report that the effect ofNH4+on aging yeast depends on the specific amino acid they are deprived of. Compared with no amino acid starvation, starvation for leucine alone or in combination with histidine resulted in the most pronouncedNH4+-induced CLS shortening, whereas starvation for lysine, alone or in combination with histidine resulted in the least sensitivity toNH4+. We also show thatNH4+-induced CLS shortening is mainly mediated by Tor1p in cells starved for leucine or histidine but by Ras2p in cells starved for lysine, and in nonstarved cells. Sch9p protected cells from the effect ofNH4+under all conditions tested (starved or nonstarved cells), which was associated with Sch9p-dependent Hog1p phosphorylation. Our data show thatNH4+toxicity can be modulated through manipulation of the specific essential amino acid supplied to cells and of the conserved Ras2p, Tor1p, and Sch9p regulators, thus providing new clues to the development of environmental interventions for CLS extension and to the identification of new therapeutic targets for diseases associated with hyperammonemia.

scholarly journals Targeting the Proline–Glutamine–Asparagine–Arginine Metabolic Axis in Amino Acid Starvation Cancer Therapy

2021 ◽  
Vol 14 (1) ◽  
pp. 72
Author(s):  
Macus Kuo ◽  
Helen Chen ◽  
Lynn Feun ◽  
Niramol Savaraj
Keyword(s):  
Amino Acids ◽  
Drug Resistance ◽  
Amino Acid ◽  
Lymphoblastic Leukemia ◽  
Essential Amino Acids ◽  
Amino Acid Starvation ◽  
Preclinical Development ◽  
Key Enzymes ◽  
Survival Pathways ◽  
Translational Mechanisms

Proline, glutamine, asparagine, and arginine are conditionally non-essential amino acids that can be produced in our body. However, they are essential for the growth of highly proliferative cells such as cancers. Many cancers express reduced levels of these amino acids and thus require import from the environment. Meanwhile, the biosynthesis of these amino acids is inter-connected but can be intervened individually through the inhibition of key enzymes of the biosynthesis of these amino acids, resulting in amino acid starvation and cell death. Amino acid starvation strategies have been in various stages of clinical applications. Targeting asparagine using asparaginase has been approved for treating acute lymphoblastic leukemia. Targeting glutamine and arginine starvations are in various stages of clinical trials, and targeting proline starvation is in preclinical development. The most important obstacle of these therapies is drug resistance, which is mostly due to reactivation of the key enzymes involved in biosynthesis of the targeted amino acids and reprogramming of compensatory survival pathways via transcriptional, epigenetic, and post-translational mechanisms. Here, we review the interactive regulatory mechanisms that control cellular levels of these amino acids for amino acid starvation therapy and how drug resistance is evolved underlying treatment failure.

scholarly journals Inducible Phase Separation of GSK3α As a Mechanism for Asparaginase Resistance in Acute Leukemias

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 169-169
Author(s):  
Laura Hinze ◽  
Sabine Schreek ◽  
Andre Zeug ◽  
Evgeni Ponimaskin ◽  
Roxane Labrosse ◽  
...  
Keyword(s):  
Amino Acids ◽  
Phase Separation ◽  
Amino Acid ◽  
Heat Shock ◽  
Research Funding ◽  
Amino Acid Starvation ◽  
Alternative Source ◽  
Terminal Domain ◽  
N Terminus ◽  
Equity Ownership

Asparaginase is an antileukemic enzyme that depletes the nonessential amino acid asparagine, but resistance is a common clinical problem whose biologic basis is poorly understood. We recently found that Wnt-induced inhibition of glycogen synthase kinase 3 (GSK3) profoundly sensitizes drug-resistant leukemias to asparaginase (Hinze et al, Cancer Cell, 2019;35:664). This effect is mediated by a β-catenin independent branch of Wnt signaling termed Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein ubiquitination and proteasomal degradation (Acebron et al, Mol Cell, 2014;54:663). Thus, asparaginase-resistant leukemias rely on catabolic protein degradation as an alternative source of amino acids to survive asparaginase therapy. Asparaginase resistance is selectively mediated by GSK3α, because its genetic or pharmacologic inhibition fully phenocopied Wnt-induced sensitization to asparaginase (p < 0.0001), whereas selective inhibition of GSK3β had no effect. This is surprising because GSK3α and GSK3β are closely related paralogs thought to be redundant for many of their biologic functions. Thus, our objective was to define why asparaginase resistance is selectively dependent on GSK3α activity. To define the GSK3 domains responsible for asparaginase resistance, we leveraged the fact that selective depletion of GSK3α induces profound sensitization to asparaginase, and this effect is rescued by expression of a cDNA encoding GSK3α, but not GSK3β. We thus tested whether asparaginase resistance could be restored by expression of a series of GSK3 alleles in which the N-terminal, kinase, and C-terminal domains of GSK3α and GSK3β were swapped in various configurations. This revealed that asparaginase resistance is dependent on the N-terminal domain of GSK3α, whereas the kinase and the C-terminal domain were interchangeable. Fusing the N-terminus of GSK3α to the kinase and C-terminal domains of GSK3β fully restored asparaginase resistance in GSK3α depleted T-ALL (p < 0.0001) and AML cells (p < 0.0001). By contrast, fusing the N-terminus of GSK3β to the kinase and C-terminus of GSK3α had no discernible effect on response to asparaginase (p = n.s.). To investigate how the N-terminus of GSK3α regulates asparaginase response, we first applied structural prediction algorithms. This revealed that the N-terminal domain of GSK3α is a low-complexity (or prion-like) domain predicted to be intrinsically disordered, features associated with liquid-liquid phase separation. Phase separation is an increasingly recognized feature of cell biology that allows cells to concentrate components of important biochemical reactions in so-called membraneless organelles, thus promoting high-catalytic efficiency. Indeed, immunofluorescence confocal microscopy revealed that GSK3α, but not GSK3β, translocates into cytoplasmic bodies in response to asparagine depletion (p < 0.0001). The cytoplasmic GSK3a bodies were membraneless, as assessed by a proteinase K protection assay, and appeared to be distinct from known phase-separated compartments such as stress granules, P-bodies and aggresomes. However, cytoplasmic GSK3α bodies colocalized with the heat shock protein 70 (HSP70), K48-linked ubiquitin and the proteasome, suggesting that these bodies function in protein unfolding, ubiquitination and degradation. Indeed, genetic depletion of either GSK3α or HSP70 blocked formation of phase-separated GSK3α bodies (p < 0.0001) and induced asparaginase sensitivity (p < 0.0001). To explore the clinical relevance of GSK3α body formation in response to asparagine starvation, we tested a panel of matched asparaginase resistant vs. sensitive T-ALL, AML and B-ALL patient-derived xenografts (PDXs), and found that the ability of GSK3α to undergo phase separation significantly correlated with resistance to asparaginase in T-ALL, B-ALL and AML (Figure 1, p < 0.0001). Our data support a model in which inducible phase separation of GSK3α and heat shock proteins represents a previously unrecognized response to amino acid starvation that concentrates the cellular machinery for protein degradation, thus allowing efficient catalysis of this alternative source of amino acids in response to amino acid starvation. Disclosures Chiosis: Samus Therapeutics: Equity Ownership, Patents & Royalties: Intellectual rights to the PU-FITC assay. Stevenson:Celgene: Research Funding. Neuberg:Celgene: Research Funding; Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership. Bourquin:Servier: Other: Travel support.

scholarly journals Structure-seq2 probing of RNA structure upon amino acid starvation reveals both known and novel RNA switches in Bacillus subtilis

2020 ◽  
Author(s):  
Laura E. Ritchey ◽  
David C. Tack ◽  
Helen Yakhnin ◽  
Elizabeth A. Jolley ◽  
Sarah M. Assmann ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Protein Binding ◽  
Rna Structure ◽  
Transcription Termination ◽  
Dimethyl Sulfate ◽  
Transcript Abundance ◽  
Amino Acid Starvation ◽  
Leader Peptide

ABSTRACTRNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of Bacillus subtilis grown in the presence and absence of amino acids. Our results reveal that amino acid starvation results in lower overall dimethyl sulfate (DMS) reactivity of the transcriptome, indicating enhanced protection owing to protein binding or RNA structure. Starvation-induced changes in DMS reactivity correlated inversely with transcript abundance changes. This correlation was particularly pronounced in genes associated with the stringent response and CodY regulons, which are involved in adaptation to nutritional stress, suggesting that RNA structure contributes to transcript abundance change in regulons involved in amino acid metabolism. Structure-seq2 accurately reported on four known amino acid-responsive riboswitches: T-box, SAM, glycine, and lysine riboswitches. Additionally, we discovered a transcription attenuation mechanism that reduces yfmG expression when amino acids are added to the growth medium. We also found that translation of a leader peptide (YfmH) encoded just upstream of yfmG regulates yfmG expression. Our results are consistent with a model in which a slow rate of yfmH translation caused by limitation of the amino acids encoded in YfmH prevents transcription termination in the yfmG leader region by favoring formation of an overlapping antiterminator structure. This novel RNA switch offers a way to simultaneously monitor the levels of multiple amino acids.

scholarly journals The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels

2019 ◽  
Author(s):  
Matthew R. McFarland ◽  
Corina D. Keller ◽  
Brandon M. Childers ◽  
Stephen A. Adeniyi ◽  
Holly Corrigall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurological Disorders ◽  
Trna Synthetase ◽  
Northern Blotting ◽  
Amino Acid Starvation ◽  
Starvation Response ◽  
Kinase Activation ◽  
Trna Synthetases ◽  
Starvation Conditions

ABSTRACTDuring protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

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