General Control Nonrepressed 2 Research Articles

Involvement of the eIF2α Kinase GCN2 in UV-B Responses.

GCN2 (general control nonrepressed 2) is a serine/threonine-protein kinase that regulates translation in response to stressors such as amino acid and purin deprivation, cold shock, wounding, cadmium, and UV-C exposure. Activated GCN2 phosphorylates the α-subunit of the eukaryotic initiation factor 2 (eIF2) leading to a drastic inhibition of protein synthesis and shifting translation to specific mRNAs. To investigate the role of GCN2 in responses to UV-B radiation its activity was analyzed through eIF2α phosphorylation assays in mutants of the specific UV-B and stress signaling pathways of Arabidopsis thaliana. EIF2α phosphorylation was detectable 30 min after UV-B exposure, independent of the UV-B photoreceptor UV RESISTANCE LOCUS8 and its downstream signaling components. GCN2 dependent phosphorylation of eIF2α was also detectable in mutants of the stress related MAP kinases, MPK3 and MPK6 and their negative regulator map kinase phosphatase1 (MKP1). Transcription of downstream components of the UV-B signaling pathway, the Chalcone synthase (CHS) was constitutively higher in gcn2-1 compared to wildtype and further increased upon UV-B while GLUTATHIONE PEROXIDASE7 (GPX7) behaved similarly to wildtype. The UVR8 independent FAD-LINKED OXIDOREDUCTASE (FADox) had a lower basal expression in gcn2-1 which was increased upon UV-B. Since high fluence rates of UV-B induce DNA damage the expression of the RAS ASSOCIATED WITH DIABETES PROTEIN51 (RAD51) was quantified before and after UV-B. While the basal expression was similar to wildtype it was significantly less induced upon UV-B in the gcn2-1 mutant. This expression pattern correlates with the finding that gcn2 mutants develop less cyclobutane pyrimidine dimers after UV-B exposure. Quantification of translation with the puromycination assay revealed that gcn2 mutants have an increased rate of translation which was also higher upon UV-B. Growth of gcn2 mutants to chronic UV-B exposure supports GCN2’s role as a negative regulator of UV-B responses. The elevated resistance of gcn2 mutants towards repeated UV-B exposure points to a critical role of GCN2 in the regulation of translation upon UV-B.

Defining meal requirements for protein to optimize metabolic roles of amino acids

Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signals that influence the rate of protein synthesis, inflammation responses, mitochondrial activity, and satiety, exerting their influence through signaling systems including mammalian/mechanistic target of rapamycin complex 1 (mTORC1), general control nonrepressed 2 (GCN2), glucagon-like peptide 1 (GLP-1), peptide YY (PYY), serotonin, and insulin. These signals represent meal-based responses to dietary protein. The best characterized of these signals is the leucine-induced activation of mTORC1, which leads to the stimulation of skeletal muscle protein synthesis after ingestion of a meal that contains protein. The response of this metabolic pathway to dietary protein (i.e., meal threshold) declines with advancing age or reduced physical activity. Current dietary recommendations for protein are focused on total daily intake of 0.8 g/kg body weight, but new research suggests daily needs for older adults of ≥1.0 g/kg and identifies anabolic and metabolic benefits to consuming at least 20–30 g protein at a given meal. Resistance exercise appears to increase the efficiency of EAA use for muscle anabolism and to lower the meal threshold for stimulation of protein synthesis. Applying this information to a typical 3-meal-a-day dietary plan results in protein intakes that are well within the guidelines of the Dietary Reference Intakes for acceptable macronutrient intakes. The meal threshold concept for dietary protein emphasizes a need for redistribution of dietary protein for optimum metabolic health.

Control of the translational machinery by amino acids

Amino acids are the precursors for the synthesis of proteins. In humans, approximately half of the 20 different amino acids are essential, ie, must be obtained from the diet. Cells must therefore take account of amino acid availability to achieve sustainable rates of protein synthesis. One of the major mechanisms involved in this is signaling through a complex of proteins termed mammalian target of rapamycin complex (mTORC) 1, which is activated by amino acids. In turn, mTORC1 regulates the production of ribosomes, the molecular machines that make proteins, and the activity of other cellular components required for protein synthesis. mTORC1 signaling promotes the transcription of the genes for ribosomal RNAs and many other components involved in ribosome production. It also positively regulates the translation of the messenger RNAs (mRNAs) for ribosomal proteins. Indeed, recent studies have shown that mammalian target of rapamycin signaling drives the translation of mRNAs for many anabolic enzymes and other proteins involved in diverse cellular functions. The translational machinery is also regulated by the absence of amino acids through the protein kinase GCN2 (general control nonrepressed 2), which phosphorylates and in end-effect inhibits the translation initiation factor eIF2 (eukaryotic initiation factor 2). This process shuts down general protein synthesis to conserve amino acids.

Inhibition of indoleamine 2,3-dioxygenase in mixed lymphocyte reaction affects glucose influx and enzymes involved in aerobic glycolysis and glutaminolysis in alloreactive T-cells

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. T-cell proliferation and differentiation to effector cells require increased glucose consumption, aerobic glycolysis and glutaminolysis. The effect of IDO on the above metabolic pathways was evaluated in alloreactive T-cells. Mixed lymphocyte reaction (MLR) in the presence or not of the IDO inhibitor, 1-methyl-dl-tryptophan (1-MT), was used. In MLRs, 1-MT decreased tryptophan consumption, increased cell proliferation, glucose influx and lactate production, whereas it decreased tricarboxylic acid cycle activity. In T-cells, from the two pathways that could sense tryptophan depletion, i.e. general control nonrepressed 2 (GCN2) kinase and mammalian target of rapamycin complex 1, 1-MT reduced only the activity of the GCN2 kinase. Additionally 1-MT treatment of MLRs altered the expression and/or the phosphorylation state of glucose transporter-1 and of key enzymes involved in glucose metabolism and glutaminolysis in alloreactive T-cells in a way that favors glucose influx, aerobic glycolysis and glutaminolysis. Thus in alloreactive T-cells, IDO through activation of the GCN2 kinase, decreases glucose influx and alters key enzymes involved in metabolism, decreasing aerobic glycolysis and glutaminolysis. Acting in such a way, IDO could be considered as a constraining factor for alloreactive T-cell proliferation and differentiation to effector T-cell subtypes.

General control nonrepressed 2 (GCN2) kinase participation in the remission phase of experimental autoimmune encephalomyelitis (EAE) in mice

General control nonrepressed 2 (GCN2) kinase participation in the remission phase of experimental autoimmune encephalomyelitis (EAE) in mice

Anti-tumorigenic effects of Type 1 interferon are subdued by integrated stress responses

Viral and pharmacological inducers of protein kinase RNA-activated (PKR)-like ER kinase (PERK) were shown to accelerate the phosphorylation-dependent degradation of the IFNAR1 chain of the Type 1 interferon (IFN) receptor and to limit cell sensitivity to IFN. Here we report that hypoxia can elicit these effects in a PERK-dependent manner. The altered fate of IFNAR1 affected by signaling downstream of PERK depends on phosphorylation of eIF2α (eukaryotic translational initiation factor 2-α) and ensuing activation of p38α kinase. Activators of other eIF2α kinases such as PKR or GCN2 (general control nonrepressed-2) are also capable of eliminating IFNAR1 and blunting IFN responses. Modulation of constitutive PKR activity in human breast cancer cells stabilizes IFNAR1 and sensitizes these cells to IFNAR1-dependent anti-tumorigenic effects. Although downregulation of IFNAR1 and impaired IFNAR1 signaling can be elicited in response to amino-acid deficit, the knockdown of GCN2 in melanoma cells reverses these phenotypes. We propose that, in cancer cells and the tumor microenvironment, activation of diverse eIF2α kinases followed by IFNAR1 downregulation enables multiple cellular components of tumor tissue to evade the direct and indirect anti-tumorigenic effects of Type 1 IFN.

Dual role of methionyl-tRNA synthetase in the regulation of translation and tumor suppressor activity of aminoacyl-tRNA synthetase-interacting multifunctional protein-3

Mammalian methionyl-tRNA synthetase (MRS) plays an essential role in initiating translation by transferring Met to initiator tRNA (tRNA(i)(Met)). MRS also provides a cytosolic anchoring site for aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3)/p18, a potent tumor suppressor that is translocated to the nucleus for DNA repair upon DNA damage. However, the mechanism by which this enzyme mediates these two seemingly unrelated functions is unknown. Here we demonstrate that AIMP3 is released from MRS by UV irradiation-induced stress. Dissociation was induced by phosphorylation of MRS at Ser662 by general control nonrepressed-2 (GCN2) following UV irradiation. Substitution of Ser662 to Asp (S662D) induced a conformational change in MRS and significantly reduced its interaction with AIMP3. This mutant possessed significantly reduced MRS catalytic activity because of loss of tRNA(Met) binding, resulting in down-regulation of global translation. According to the Met incorporation assay using stable HeLa cells expressing MRS S662A or eukaryotic initiation factor-2 subunit-α (eIF2α) S51A, inactivation of GCN2-induced phosphorylation at eIF2α or MRS augmented the role of the other, suggesting a cross-talk between MRS and eIF2α for efficient translational inhibition. This work reveals a unique mode of regulation of global translation as mediated by aminoacyl-tRNA synthetase, specifically MRS, which we herein identified as a previously unidentified GCN2 substrate. In addition, our research suggests a dual role for MRS: (i) as a coregulator with eIF2α for GCN2-mediated translational inhibition; and (ii) as a coupler of translational inhibition and DNA repair following DNA damage by releasing bound tumor suppressor AIMP3 for its nuclear translocation.

IDO Upregulates Regulatory T Cells via Tryptophan Catabolite and Suppresses Encephalitogenic T Cell Responses in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA-induced Tregs contribute to inhibition of Th17 cells. By using a DC-T cell coculture, we found that 3-HAA-treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28-stimulated naive CD4(+) T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.

Regulation of protein synthesis initiation in eucaryotes

Eukaryotic initiation factor (eIF)2B is a guanine nucleotide exchange factor (GEF) for a second initiation factor, eIF2. During the initiation of messenger RNA (mRNA) translation, eIF2 binds Guanosine-5'-triphosphate (GTP) and initiator methionyl- transfer RNA (tRNAi) (met-tRNAi) and the resulting ternary complex subsequently binds to the 40S ribosomal subunit. Alterations in ternary complex formation due to changes in eIF2B GEF activity not only produce alterations in global rates of protein synthesis but also cause specific changes in the translation of mRNAs encoding certain proteins, such as the transcription factors general control nonrepressed 2 (GCN2) and activating transcription factor 4 (ATF4). Studies have implicated changes in eIF2B GEF activity in alterations in protein synthesis in a number of pathophysiological and physiological conditions, such as diabetes, cancer, sepsis, vanishing white matter disease, and resistance exercise. The chapter discusses the state of knowledge concerning the function of eIF2B and its role in regulating mRNA translation.