Recent Advances in the Understanding of Amino Acid Regulation of Gene Expression

Julien Averous, Alain Bruhat, Sylvie Mordier and Pierre Fafournoux

Unite´ de Nutrition et Me´tabolisme Prote´ique, Institut National de la Recherche Agronomique de Theix, 63122 Saint Gene`s Champanelle, France

ABSTRACT

In mammals, the impact of nutrients on gene expression has become an important area of research. Because amino acids have multiple and important functions, their homeostasis has to be finely maintained. However, amino acidemia can be affected by certain nutritional conditions or various forms of stress. Consequently, mammals must adjust several of the physiological functions involved in the adaptation to amino acid availability by regulating expression of numerous genes. It has been shown that amino acids alone can modify the expression of target genes. However, understanding of amino acid-dependent control of gene expression has just started to emerge. This review focuses on recent advances in the understanding of mechanisms involved in the amino acid control of gene expression.

Mammals have to adjust their metabolism to the intermittent intake of food. In addition, they must adapt to internal metabolic changes such as the menstrual cycle or pregnancy in females and growth of tissues in the young. All of these external and internal factors demand metabolic responses and associated regulatory mechanisms. Regulation of metabolism is achieved by coordinated actions between tissues and cells. These mechanisms involve the conditional regulation of specific genes in the presence or absence of appropriate nutrients. In multicellular organisms, the control of gene expression involves complex interactions of hormonal, neuronal and nutritional factors.

Control of gene expression by nutrient availability has been well documented in prokaryotes and lower eukaryotes. These organisms are able to adjust their metabolic capacity to variations in the nutrient supply by altering their pattern of gene expression. For example, the lac operon of Escherichia coli and the gal regulon of accharomyces cerevisiae are among the best-understood regulatory pathways of gene expression (1). Although not as widely appreciated, nutritional signals play an important role in controlling gene expression in mammals. It was shown (2-7) that major (carbohydrates, fatty acids, sterols) or minor (minerals, vitamins) dietary constituents participate in the regulation of gene expression. However, the mechanisms involved in the amino acid control of gene expression have just begun to be understood for mammalian cells (8-11).

This review summarizes recent work on the effect of amino acid availability in the regulation of biological functions. On the basis of the physiological concepts of amino acid homeostasis, we discuss specific examples of the role of amino acids in the regulation of physiological functions. Particularly, we focus on the mechanisms involved in the amino acid regulation of gene expression.

Regulation of amino acid metabolism and homeostasis in the whole animal

Mammals must precisely regulate amino acid homeostasis while taking into account two important characteristics of amino acid metabolism: 1) multicellular organisms are unable to synthesize all amino acids, and 2) there are no important dispensable amino acid stores (in contrast with lipids or glucose).

The size of the pool of each amino acid is the result of a balance between input and removal. The metabolic outlets for amino acids are protein synthesis and amino acid degradation, whereas the inputs are de novo synthesis (for nonessential amino acids), protein breakdown and dietary intake. Changes in the rates of these systems lead to an adjustment in nitrogen balance. For example, amino acid homeostasis and protein metabolism can be altered in response to malnutrition (12,13) and/or various forms of trauma such as sepsis, fevers, thermal burns, etc. (14-19) with two major consequences: a large variation in the blood amino acid concentration and a negative nitrogen balance. In these situations, individuals have to adjust several physiological functions that are involved in the defense of and adaptation to amino acid limitation by regulating numerous genes. The specific role of amino acids in the adaptation to two different amino acid-deficient diets is considered.

Protein undernutrition

Prolonged feeding of a low-protein diet causes a decrease in the plasma level of most essential amino acids. For example, in plasma of children affected by kwashiorkor, leucine and methionine concentrations can be reduced from ;100-150 and 18-30 mM to ;20 and 5 mM, respectively (13,20). Consequently, individuals must adjust several physiological functions to adapt to this amino acid deficiency. In both children and young animals, the main consequence of feeding a low-protein diet is a dramatic inhibition of growth. Straus et al. (21) demonstrated that growth inhibition was due to a striking overexpression of insulin-like growth factor binding protein-1 (IGFBP-1), which binds insulin-like growth factors-1 and -2 and modulates their mitogenic and metabolic properties (22). According to the literature (22), IGFBP-1 expression is regulated by growth hormone, insulin or glucose. However, the high IGFBP-1 levels associated with feeding a protein-deficient diet cannot be explained by only these three factors. It was demonstrated (21,23) that a decrease in amino acid concentration is directly responsible for IGFBP-1 induction. Therefore, amino acid limitation as occurs during dietary protein deficiency participates in the downregulation of growth through the induction of IGFBP-1 expression.

Imbalanced diet.

Because mammals cannot synthesize all of the amino acids, the diet must provide those that cannot be synthesized. Thus, in the event of a deficiency of one of the indispensable amino acids, the remaining amino acids are catabolized and lost, and body proteins are broken down to provide the limiting amino acid (24). The ability to distinguish balance from imbalance among the amino acids in the diet and to select for the growth-limiting essential amino acid provides adaptive advantage to animals. After feeding on an amino acid-imbalanced diet, an animal first recognizes the amino acid deficiency and then develops a conditioned taste aversion. Recognition and anorexia that result from an amino acid-imbalanced diet take place very rapidly (25,26). The mechanisms that underlie the recognition of protein quality must act through the free amino acids that result from the intestinal digestion of proteins. It was observed that a marked decrease in the blood concentration of the limiting amino acid becomes apparent as early as a few hours after feeding an imbalanced diet. The anorectic response is well correlated with a decreased concentration of the limiting amino acid in the plasma (27,28). Several lines of evidence suggest that the decrease in the limiting amino acid concentration is detected in the brain. Gietzen’s laboratory demonstrated that a specific brain area, the anterior pyriform cortex, can sense the amino acid concentration [for review, see (28)]. This recognition phase is associated with localized decreases in the concentration of the limiting amino acid and with important changes in protein synthesis rate and gene expression. Subsequent to recognition of the deficiency, the second step, development ofanorexia, involves another part of the brain (27).

Amino acid control of gene expression

Although the molecular mechanisms involved in the control of gene expression by amino acid availability have just begun to be investigated in mammals, these mechanisms have been extensively studied for yeasts. After a summary of these processes, we focus on the control of gene expression in mammalian cells.

Control of gene expression in yeasts.

In yeasts, several amino acid-sensing systems have been described (Fig. 1).

Specific control processes

It is well documented (29) that numerous operons are regulated by the specific end products of the corresponding enzymes. A small effector molecule can induce the transition of transcriptional activators from the inactive to the active form. For example, leucine biosynthesis is controlled by the transcriptional activator Leu3p in response to leucine availability. Leu3p is activated by the levels of the metabolic intermediate a-isopropylmalate, which serves as a sensor of leucine availability (30). This type of regulation has also been described (31) for the control of amino acid catabolism (proline, for example).

General control process

TheGCN2protein kinase pathway. In addition to specific control, yeasts use a general control process whereby a subset of genes is coordinately induced by starvation of the cell for one single amino acid. Free tRNA accumulate and thus stimulate the activity of the GCN2 protein kinase, which phosphorylates the a-subunit of eukaryotic initiation factor-2 (eIF2) and in turn impairs the synthesis of the 43S preinitiation complex (Met-tRNAdGTPdeIF2). Despite the strong inhibition

of protein synthesis, the transcription factor GCN4 is translationally upregulated. This control is due to the particular structure of the 59 untranslated region (UTR) of the GCN4 mRNA. As a result, GCN4 induces.30 different genes that are involved in several different biosynthetic pathways (32-34).

The target of rapamycin pathway.

The target of rapamycin (TOR) pathway is regulated by amino acid availability and is involved in the regulation of several cellular processes such as translation, transcription and protein degradation (35). The molecular mechanisms involved in the amino acid control of TOR activity remain to be identified.

The Ssy1pdPtr3pdSsy5p complex.

Recent advances in our understanding of nutrient sensing indicate that yeast cells possess an amino acid-sensing system that is localized at the plasma membrane that transduces information regarding the presence of extracellular amino acids. The primary amino acid sensor is a multimeric three-protein complex, Ssy1pdPtr3pd Ssy5p (called the SPS complex). The Ssy1p component closely resembles an amino acid permease, which is a family of proteins that normally catalyzes the transport of amino acids into a cell (36-38). In response to a change in amino acid availability, a complex network of regulatory processes is activated by Ssy1p to modify the expression of target genes. The SPS complex is required for induction of a set of target genes (BAP3, TAT2, CHA1, etc.) by amino acids and is also required for the amino acid repression of another set of target genes (DAL4, MET3, MMP1, etc.) (39).

Control of gene expression in mammalian cells.

Genes upregulated by amino acids. Genes that are specifically upregulated in response to supraphysiological concentrations of amino acids have been described. For example, a high concentration of L-tryptophan enhances the expression of collagenase and tissue inhibitors of metalloproteinase. In rat hepatocytes, Na1-cotransported amino acids such as glutamine, alanine and proline stimulate acetyl-coenzyme A carboxylase, glycogen synthetase and arginino succinate synthetase activity. It was demonstrated (40-42) that the swelling that resulted from the addition of amino acid could be involved in the regulation of gene expression; however, the molecular mechanisms involved in these processes are poorly understood.

Genes upregulated by amino acid starvation. In mammalian cells, a few examples of specific mRNA that are induced after amino acid deprivation have been reported (43). Most of the molecular mechanisms involved in the amino acid regulation of gene expression have been obtained by studying the upregulation of CCAAT / enhancer binding protein (C/EBP) homologous protein (CHOP), asparagine synthetase (AS) and the cationic amino acid transporter (Cat-1) genes.

Molecular mechanisms involved in regulation of gene expression by amino acid limitation

Amino acid regulation of most of the amino acid-regulated genes has both transcriptional and/or posttranscriptional components (44-46).

Posttranscriptional regulation of gene expression by amino acid availability.

Recently it was shown that the translation rate of specific genes could be regulated by amino acid availability. Hatzoglou and collaborators have demonstrated that amino acid depletion initiates molecular events that specifically activate translation of the Cat-1 gene. They have shown the presence of an internal ribosome entry site (IRES) located within the 59 UTR of the Cat-1 mRNA. This IRES is involved in the amino acid control of translation of the Cat-1 transcript (47,48). Under conditions of amino acid starvation, translation from this IRES is stimulated, whereas the capdependent protein synthesis is decreased. Another example of translation induced by amino acid starvation was reported (49) for the branched-chain a-ketoacid dehydrogenase kinase, but the mechanism of translational control was not studied. This mechanism of compensatory response allows translation of major proteins despite the inhibition of the cap-dependent translational apparatus.

Transcriptional activation of mammalian genes by amino acid starvation.

It was established that the increase in CHOP or AS mRNA after amino acid starvation is mainly due to an increased transcription (44,50). By first identifying the genomic cis-elements and then the corresponding transcription factors responsible for regulation of these specific target genes, it is anticipated that one can progress backward up the signal transduction pathway to understand the individual steps required.

Regulation of human CHOP gene by amino acid starvation.

CHOP encodes a ubiquitous transcription factor that heterodimerizes avidly with the other members of the C/EBP (51) and Jun/Fos (52) families. The CHOP gene is tightly regulated by a wide variety of stresses in mammalian cells (53-55). Leucine limitation in human cell lines leads to induction of CHOP mRNA and protein in a dose-dependent manner (43).

We have identified (56) in the CHOP promoter a cispositive element located between 2313 and 2295 that is essential for amino acid regulation of the CHOP promoter (Fig. 2). This short sequence can regulate a basal promoter in response to starvation of several individual amino acids and then can be called an amino acid regulatory element (AARE). The sequence of the CHOP AARE region shows some homology with the specific binding sites of the C/EBP and activating transcription factor (ATF)/cyclic adenosine 59 monophosphate response-element binding-protein transcription-factor families. We have shown that many transcription factors that belong to the ATF or C/EBP family have the ability to bind in vitro to the CHOP AARE. Among these factors, at least ATF-2 and -4 are involved in the amino acid control of CHOP expression: when knockout cell lines for these two proteins were tested, amino acid regulation of CHOP expression was abolished (56; J. Averous, unpublished data). This work was enlarged to the regulation of other amino acid-regulated genes and confirms that ATF-2 and -4 are key components of the amino acid control of gene expression (J. Averous, unpublished data).

Regulation of AS by amino acid availability.

AS is expressed in most mammalian cells as a housekeeping enzyme responsible for the biosynthesis of asparagine from aspartate and glutamine (57). The levels of AS mRNA increase not only in response to asparagine starvation but also after deprivation of leucine, isoleucine or glutamine (45,50,58). Kilberg’s group (59) has analyzed the regulation of the AS promoter by amino acid availability. They have characterized (Fig. 2) a nutrient-sensing regulatory unit that includes two cis-acting elements termed nutrient-sensing response elements (NSRE-1 and -2) that are required to induce the AS expression level by either amino acid deprivation or the endoplasmic reticulum (ER) stress response. Gel-shift experiments and overexpression of dominant-negative 2042S SUPPLEMENT mutants suggest that activation of the AS gene by either amino acid limitation or ER stress response involves ATF-4 and C/EBP-b binding to the NSRE-1 site (60,61). The comparison between CHOP and AS transcriptional control elements shows that AS NSRE-1 and CHOP AARE share nucleotide sequences and functional similarities (Fig. 2A). However, the CHOP AARE can function alone, whereas AS NSRE-1 is functionally weak by itself and requires the presence of NSRE-2 (62). The AS NSRE-2 has two properties: 1) it amplifies the NSRE-1 activity in response to amino acid starvation, and 2) it confers a response to ER stress. For example, when cloned downstream of the CHOP AARE, it can confer ER stress responsiveness to the CHOP AARE.

Amino acid signaling pathway.

It appears that mammalian cells have more than one amino acid signaling pathway independent of the ER stress pathway (63,64). However, the individual steps required for these pathways are not well understood.

ATF-4 and amino acid signaling pathways.

The group of Ron has revealed a signaling pathway for regulating gene expression in mammals that is homologous to the well-characterized yeast general-control response to amino acid deprivation (65). Its components include (Fig. 3) the mammalian homolog of the GCN2 kinase, eIF2a and ATF-4. Like the GCN4 transcript, the ATF-4 mRNA contains an upstream open reading frame in its 59 UTR that allows translation when the cap-dependent translation is inhibited. The authors showed that GCN2 activation, phosphorylation of eIF2a and translational activation of ATF-4 are necessary but not sufficient for the induction of CHOP expression in response to leucine starvation (see Fig. 2). These data are in good agreement with the analysis of the CHOP and AS promoter in showing that ATF-4 can bind to the promoter sequences involved in the response to amino acid starvation.

ATF-2 and amino acid signaling pathways.

The transactivating capacity of ATF-2 is activated via phosphorylation of N-terminal residues Thr-69, Thr-71 and Ser-90 (66,67). There are two lines of evidence suggesting that ATF-2 phosphorylation belongs to the amino acid-response pathway leading to the transcriptional activation of CHOP by amino acids: 1) leucine starvation induces ATF-2 phosphorylation in human cell lines (J. Averous, personal communication); and 2) an ATF-2 dominant-negative mutant (68) in which the three residues cannot be phosphorylated inhibits the CHOP promoter activity enhanced by leucine starvation. These data suggest that a specific amino acid-regulated pathway that leads to the transcriptional activation of CHOP may involve a phosphorylation of prebound ATF-2 rather than an increase in ATF-2 binding. However, the identity of the kinases involved in ATF-2 phosphorylation by amino acid starvation remains to be discovered (see Fig. 3).

It appears that at least two different pathways that lead to ATF-2 phosphorylation and ATF-4 expression are necessary to induce CHOP expression in response to one stimulus (amino acid starvation). In addition, ATF-2 and -4 belong to the basic leucine zipper transcription-factor family. These proteins have the ability to interact with several transcription factors to bind the target DNA sequence. In the case of amino acid regulation of CHOP expression, we have no evidence that ATF-2 and -4 form a dimer that binds the AARE sequence, but they could be included in a larger regulatory protein complex. For example, it has been shown (69) that ATF-2 interacts with at least two transcriptions factors (CP1 and NF1) in a large protein complex to regulate transcription of the fibronectin gene.

Defining the precise cascade of molecular events by which the cellular concentration of an individual amino acid regulates gene expression will be an important contribution to our understanding of metabolite control in mammalian cells. These studies will provide insight into the role of amino acids in the regulation of cellular functions such as cell division, protein synthesis or proteolysis.

FOOTNOTES

Presented at the conference "The Second Workshop on the Assessment of Adequate Intake of Dietary Amino Acids" held October 31-November 1, 2002, in Honolulu, Hawaii. The conference was sponsored by the International Council on Amino Acid Science. The Workshop Organizing Committee included Vernon R. Young, Yuzo Hayashi, Luc Cynober and Motoni Kadowaki. Conference proceedings were published in a supplement to The Journal of Nutrition. Guest editors for the supplement publication were Dennis M. Bier, Luc Cynober, Yuzo Hayashi and Motoni Kadowaki.

Abbreviations used: AARE, amino acid regulatory element; AS, asparagine synthetase; ATF, activating transcription factor; Cat-1, cationic amino acid transporter-1; C/EBP, CCAAT/enhancer binding protein; CHOP, C/EBP homologous protein; eIF2, eukaryotic initiation factor-2; ER, endoplasmic reticulum; ERSE, ER stress response element; GCN, general control nondepressible protein kinase; IGFBP-1, insulin-like growth factor binding protein-1; IRES, internal ribosome entry site; mTOR, mammalian target of rapamycin; NSRE-1 and NSRE-2, nutrient-sensing response elements-1 and -2; SPS, Ssy1p-Ptr3p-Ssy5p complex; UTR, untranslated region.

The following abbreviations are related to genes expressed in yeast: Ssy1p, Ssy5p, Ptr3p, BAP3, TAT2, CHA1, DAL4, MET3 and MMP1. The meaning of these abbreviations is not related to the function of the encoded protein.

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