Transcriptional Autoregulation and Inhibition of mRNA Translation of Amino Acid Regulator GenecpcAof Filamentous FungusAspergillus nidulans

The CPCA protein of the filamentous fungus Aspergillus nidulans is a member of the c-Jun-like transcriptional activator family. It acts as central transcription factor of the cross-pathway regulatory network of amino acid biosynthesis and is functionally exchangeable for the general control transcriptional activator Gcn4p of Saccharomyces cerevisiae. In contrast to GCN4, expression of cpcA is strongly regulated by two equally important mechanisms with additive effects that lead to a fivefold increased CPCA protein amount under amino acid starvation conditions. One component of cpcA regulation involves a transcriptional autoregulatory mechanism via a CPCA recognition element (CPRE) in the cpcA promoter that causes a sevenfold increased cpcA mRNA level when cells are starved for amino acids. Point mutations in the CPRE cause a constitutively low mRNA level of cpcA and a halved protein level when amino acids are limited. Moreover, two upstream open reading frames (uORFs) in the 5′ region of thecpcA mRNA are important for a translational regulatory mechanism. Destruction of both short uORFs results in a sixfold increased CPCA protein level under nonstarvation conditions and a 10-fold increase under starvation conditions. Mutations in both the CPRE and uORF regulatory elements lead to an intermediate effect, with a low cpcA mRNA level but a threefold increased CPCA protein level independent of amino acid availability. These data argue for a combined regulation of cpcA that includes a translational regulation like that of yeast GCN4 as well as a transcriptional regulation like that of the mammalianjun and fos genes.

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Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3470 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3470-3480 ◽

Cited By ~ 98

Author(s):

E Moran ◽

B Zerler ◽

T M Harrison ◽

M B Mathews

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Point Mutations ◽

Apparent Molecular Weight ◽

Amino Acid Position ◽

Cysteine Residue ◽

Transformation Function ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

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Extent of damage to amino acid availability of whey protein heated with sugar

Journal of Dairy Research ◽

10.1017/s002202990003003x ◽

1991 ◽

Vol 58 (4) ◽

pp. 431-441 ◽

Cited By ~ 13

Author(s):

Thérèse Desrosiers ◽

Laurent Savoie

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Whey Protein ◽

Significant Proportion ◽

Protein Digestibility ◽

Heating Time ◽

Heat Treatments ◽

Molar Ratio ◽

Amino Acid Availability

SummaryThe effect of heat treatments, at various water activities (αw), on digestibility and on the availabilities of amino acids of whey protein samples in the presence of lactose was estimated by an in vitro digestion method with continuons dialysis. Four αw (0·3, 0·5, 0·7 and 0·97), three temperatures (75, 100 and 121 °C) and three heating periods (50, 500 and 5000 s) were selected. The initial lysine: lactose molar ratio was 1:1. Amino acid profiles showed that excessive heating of whey (121 °C, 5000 s) destroyed a significant proportion of cystine at all αw, lysine at αw 0·3, 0·5 and 0·7, and arginine at αw 0·5 and 0·7. At αw 0·3, 0·5 and 0·7, protein digestibility decreased (P < 0·05) as the temperature increased from 75 to 121 °C for a heating period of 5000 s, and as the heating time was prolonged from 500 to 5000 s at 121 °C. Excessive heating also decreased (P < 0·05) the availabilities of ail amino acids at αw 0·3, 0·5 and 0·7. The availabilities of lysine, proline, aspartic acid, glutamic acid, threonine, alanine, glycine and serine were particularly affected. Severe heating at αw 0·97 did not seem to favour the Maillard reaction, but the availabilities of cystine, tyrosine and arginine were decreased, probably as a result of structural modifications of the protein upon heating. Heating whey protein concentrates in the presence of lactose not only affected lysine, but also impaired enzymic liberation of other amino acids, according to the severity of heat treatments and αw.

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Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis

Cell Death and Disease ◽

10.1038/s41419-021-04417-w ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Hyeon-Ok Jin ◽

Sung-Eun Hong ◽

Ji-Young Kim ◽

Se-Kyeong Jang ◽

In-Chul Park

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cell Survival ◽

Amino Acid Deprivation ◽

General Control ◽

Akt Activation ◽

Amino Acid Availability ◽

Survival Signal ◽

Cancer Cell Survival ◽

Glutamine Deprivation

AbstractAmino acid availability is sensed by various signaling molecules, including general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). However, it is unclear how these sensors are associated with cancer cell survival under low amino acid availability. In the present study, we investigated AKT activation in non-small cell lung cancer (NSCLC) cells deprived of each one of 20 amino acids. Among the 20 amino acids, deprivation of glutamine, arginine, methionine, and lysine induced AKT activation. AKT activation was induced by GCN2/ATF4/REDD1 axis-mediated mTORC2 activation under amino acid deprivation. In CRISPR-Cas9-mediated REDD1-knockout cells, AKT activation was not induced by amino acid deprivation, indicating that REDD1 plays a major role in AKT activation under amino acid deprivation. Knockout of REDD1 sensitized cells cultured under glutamine deprivation conditions to radiotherapy. Taken together, GCN2/ATF4/REDD1 axis induced by amino acid deprivation promotes cell survival signal, which might be a potential target for cancer therapy.

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Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein

Biochemical Journal ◽

10.1042/bj3070457 ◽

1995 ◽

Vol 307 (2) ◽

pp. 457-463 ◽

Cited By ~ 24

Author(s):

D A Brooks ◽

D A Robertson ◽

C Bindloss ◽

T Litjens ◽

D S Anson ◽

...

Keyword(s):

Amino Acids ◽

Enzyme Activity ◽

Amino Acid ◽

Cell Line ◽

Protein Level ◽

Structural Modification ◽

Wild Type ◽

Cellular Content ◽

Control Protein ◽

Wild Type Control

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.

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Molecular analysis of GCN3, a translational activator of GCN4: evidence for posttranslational control of GCN3 regulatory function

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4808-4820.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4808-4820

Author(s):

E M Hannig ◽

A G Hinnebusch

Keyword(s):

Amino Acid ◽

Normal Growth ◽

Growth Conditions ◽

Regulatory Function ◽

Regulatory Sequences ◽

Noncoding Dna ◽

Physical Description ◽

Amino Acid Availability ◽

Translational Activator ◽

Starvation Conditions

GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. The GCN3 product is a positive regulator required for increased synthesis of GCN4 protein in amino acid-starved cells. GCN3 appears to act indirectly by antagonizing GCD-encoded negative regulators of GCN4 expression under starvation conditions; however, GCN3 can also suppress the effects of gcd12 mutations under nonstarvation conditions. These results imply that the GCN3 product can promote either repression or activation of GCN4 expression depending on amino acid availability. We present a complete physical description of the GCN3 gene and its transcript, plus measurements of GCN3 expression at the transcriptional and translational levels under different growth conditions. GCN3 encodes a 305-amino-acid polypeptide with no significant homology to any other known protein sequence. GCN3 mRNA contains no leader AUG codons, and no potential GCN4 binding sites were found in GCN3 5' noncoding DNA. In accord with the absence of these regulatory sequences found at other genes in the general control system, GCN3 mRNA and a GCN3-lacZ fusion enzyme are present at similar levels under both starvation and nonstarvation conditions. These data suggest that modulation of GCN3 regulatory function in response to amino acid availability occurs posttranslationally. A gcn3 deletion leads to unconditional lethality in a gcd1-101 mutant, supporting the idea that GCN3 is expressed under normal growth conditions and cooperates with the GCD1 product under these circumstances to carry out an essential cellular function. We describe a point mutation that adds three amino acids to the carboxyl terminus of GCN3, which inactivates its positive regulatory function required under starvation conditions without impairing its ability to promote functions carried out by GCD12 under nonstarvation conditions.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa055 ◽

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.

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Amino acid availability: aspects of chemical analysis and bioassay methodology

Nutrition Research Reviews ◽

10.1079/nrr200365 ◽

2003 ◽

Vol 16 (2) ◽

pp. 127-141 ◽

Cited By ~ 66

Author(s):

Paul J. Moughan

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Chemical Analysis ◽

Structural Damage ◽

Human Subjects ◽

Scientific Evidence ◽

Hydrolysis Time ◽

Amino Acid Availability

AbstractIt is important to be able to characterise foods and feedstuffs according to their available amino acid contents. This involves being able to determine amino acids chemically and the conduct of bioassays to determine amino acid digestibility and availability. The chemical analysis of amino acids is not straightforward and meticulousness is required to achieve consistent results. In particular and for accuracy, the effect of hydrolysis time needs to be accounted for. Some amino acids (for example, lysine) can undergo chemical modification during the processing and storage of foods, which interferes with amino acid analysis. Furthermore, the modified amino acids may also interfere with the determination of digestibility. A new approach to the determination of available lysine using a modifiedin vivodigestibility assay is discussed. Research is required into other amino acids susceptible to structural damage. There is recent compelling scientific evidence that bacterial activity in the small intestine of animals and man leads to the synthesis and uptake of dietary essential amino acids. This has implications for the accuracy of the ileal-based amino acid digestibility assay and further research is required to determine the extent of this synthesis, the source of nitrogenous material used for the synthesis and the degree of synthesis net of amino acid catabolism. Although there may be potential shortcomings in digestibility assays based on the determination of amino acids remaining undigested at the terminal ileum, there is abundant evidence in simple-stomached animals and growing evidence in human subjects that faecal-based amino acid digestibility coefficients are misleading. Hindgut microbial metabolism significantly alters the undigested dietary amino acid profile. The ileal amino acid digestibility bioassay is expected to be more accurate than its faecal-based counterpart, but correction of the ileal amino acid flow for amino acids of endogenous origin is necessary. Approaches to correcting for the endogenous component are discussed.

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Amino acids suppress proteolysis independent of insulin throughout the neonatal period

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1997.272.4.e592 ◽

1997 ◽

Vol 272 (4) ◽

pp. E592-E599 ◽

Cited By ~ 10

Author(s):

B. B. Poindexter ◽

C. A. Karn ◽

J. A. Ahlrichs ◽

J. Wang ◽

C. A. Leitch ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neonatal Period ◽

Essential Amino Acids ◽

Whole Body ◽

Amino Acid Availability ◽

Dose Dependent Fashion ◽

Term Newborns ◽

Dose Dependent ◽

Acid Administration

To determine how increased amino acid availability alters rates of whole body proteolysis and the irreversible catabolism of the essential amino acids leucine and phenylalanine throughout the neonatal period, leucine and phenylalanine kinetics were measured under basal conditions and in response to intravenous amino acids in two separate groups of healthy, full-term newborns (at 3 days and 3 wk of age). The endogenous rates of appearance of leucine and phenylalanine (reflecting proteolysis) were suppressed equally in both groups and in a dose-dependent fashion (by approximately 10% with 1.2 g x kg(-1) x day(-1) and by approximately 20% with 2.4 g x kg(-1) x day(-1)) in response to intravenous amino acid delivery. Insulin concentrations remained unchanged from basal values during amino acid administration. The irreversible catabolism of leucine and phenylalanine increased in a stepwise fashion in response to intravenous amino acids; again, no differences were observed between the two groups. This study clearly demonstrates that the capacity to acutely increase rates of leucine oxidation and phenylalanine hydroxylation is fully present early in the neonatal period in normal newborns. Furthermore, these data suggest that amino acid availability is a primary regulator of proteolysis in normal newborns throughout the neonatal period.

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Regulation of type I collagen mRNA in lung fibroblasts by cystine availability

Biochemical Journal ◽

10.1042/bj3310417 ◽

1998 ◽

Vol 331 (2) ◽

pp. 417-422 ◽

Cited By ~ 13

Author(s):

David C. RISHIKOF ◽

Ping-Ping KUANG ◽

Christine POLIKS ◽

Ronald H. GOLDSTEIN

Keyword(s):

Amino Acids ◽

Steady State ◽

Amino Acid ◽

Type I Collagen ◽

State Level ◽

Lung Fibroblasts ◽

Type I ◽

Mrna Levels ◽

Collagen Mrna ◽

Amino Acid Availability

The steady-state level of α1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases α1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of α1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of α1(I) collagen mRNA. We found that re-expression of α1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of α1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased α1(I) collagen mRNA levels. The combination of glutamine and cystine increased α1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase α1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of α1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of γ-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of α1(I) collagen mRNA.

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Minireview: Nutrient Sensing by G Protein-Coupled Receptors

Molecular Endocrinology ◽

10.1210/me.2013-1100 ◽

2013 ◽

Vol 27 (8) ◽

pp. 1188-1197 ◽

Cited By ~ 49

Author(s):

Eric M. Wauson ◽

Andrés Lorente-Rodríguez ◽

Melanie H. Cobb

Keyword(s):

Amino Acids ◽

Amino Acid ◽

G Protein ◽

Taste Receptor ◽

Sweet Taste ◽

Nutrient Sensing ◽

G Protein Coupled Receptors ◽

Amino Acid Availability ◽

Class C ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them.

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