The eukaryotic initiation factor 2 kinase GCN2 protects against hepatotoxicity during asparaginase treatment

Asparaginase is an important drug in the treatment regimen for acute lymphoblastic leukemia. Asparaginase depletes circulating asparagine and glutamine, activating an amino acid stress response (AAR) involving phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible kinase 2 (GCN2). We hypothesized that GCN2 functions to mitigate hepatic stress during asparaginase therapy by activating the AAR. To test this idea, C57BL/6J wild-type mice (Gcn2(+/+)) and those deleted for Gcn2 (Gcn2(-/-)) were injected with asparaginase or saline excipient one time daily for 1 or 6 days. In liver, increased phosphorylation of eIF2 and mRNA expression of AAR target genes activating transcription factor 4, asparagine synthetase, eIF4E-binding protein 1, and CAAT enhancer-binding protein homologous protein were significantly blunted or blocked in the liver of Gcn2(-/-) mice. Loss of AAR during asparaginase coincided with increases in mammalian target of rapamycin signaling, hepatic triglyceride accumulation, and DNA damage in association with genetic markers of oxidative stress (glutathione peroxidase) and inflammation (tumor necrosis factor alpha-α). Although asparaginase depleted circulating asparagine in both Gcn2(+/+) and Gcn2(-/-) mice, all other amino acids, including plasma glutamine, were elevated in the plasma of Gcn2(-/-) mice. This study shows that loss of GCN2 promotes oxidative stress and inflammatory-mediated DNA damage during asparaginase therapy, suggesting that patients with reduced or dysfunctional AAR may be at risk of developing hepatic complications during asparaginase treatment

Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice

Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted

Phosphorylation of Eukaryotic Initiation Factor 2 Alpha Regulates Stress in the Human Protozoan Parasite Entamoeba Histolytica

Entamoeba histolytica is a food- and water-borne intestinal parasite responsible for amoebic dysentery and amoebic liver abscess. The life cycle of E. histolytica alternates between the host-restricted trophozoite form and the highly infective latent cyst stage that is able to persist in the environment. Throughout its life cycle, which may include invasion of tissues in the human host, the parasite is subjected to a variety of stressful conditions. In other systems, stress can trigger the activation of kinases that phosphorylate a serine residue on eukaryotic translation initiation factor-2α (eIF2α). This modification inhibits the activity of eIF2 resulting in a general decline in protein synthesis, and, paradoxically, an up-regulation of the expression of certain genes that permit the cell to counter the stress. Genomic data reveal that E. histolytica possesses eIF2α with a conserved phosphorylatable serine at position 59. Thus, this pathogen may have the machinery for stress-induced translational control. To test this, we exposed E. histolytica trophozoites to six different stress conditions and assessed viability, as well as the level of total and phospho-EheIF2α via Western blot of cell lysates. Long term serum starvation induced an increase in the level of phospho-EheIF2α, but no other stress condition caused a significant change. Long term serum starvation also showed a decrease in polyribosome abundance as observed through sucrose gradient ultracentrifugation; this is consistent with the observation that this condition also induces phosphorylation of EheIF2α. This suggests that the eIF2α-dependent stress response system is operational in E. histolytica and that the system may be activated only by certain stresses. To further examine the role of phosphorylation of EheIF2α during stress, three transgenic cell lines were created. EheIF2α-S59 over-expresses wild type eIF2α protein. EheIF2α-S59A expresses eIF2α with the serine-59 residue mutated to an alanine, creating a non-phosphorylatable subunit. EheIF2α-S59D expresses eIF2α with the serine-59 residue mutated to an aspartic acid to mimic a phosphorylated residue. EheIF2α-S59 exhibited a high level of phosphorylation of the exogenous protein, leading to a decreased growth and polyribosome abundance when compared to the control cell line. EheIF2α-S59A had the highest growth rate and retained a high abundance of polyribosome. EheIF2α-S59D exhibited the slowest growth rate and had a decrease in polyribosome when compared to control; however, EheIF2α-S59D did exhibit the highest survival rate in over half the stress conditions tested. This may indicate the protective nature of phosphorylation of EheIF2α during times of stress

Translational Control in the Latency of Apicomplexan Parasites

Apicomplexan parasites Toxoplasma gondii and Plasmodium spp. use latent stages to persist in the host, facilitate transmission, and thwart treatment of infected patients. Therefore, it is important to understand the processes driving parasite differentiation to and from quiescent stages. Here, we discuss how a family of protein kinases that phosphorylate the eukaryotic initiation factor-2 (eIF2) function in translational control and drive differentiation. This translational control culminates in reprogramming of the transcriptome to facilitate parasite transition towards latency. We also discuss how eIF2 phosphorylation contributes to the maintenance of latency and provides a crucial role in the timing of reactivation of latent parasites towards proliferative stages

ESTABLISHING THE FIDELITY OF START CODON RECOGNITION: ROLE OF EUKARYOTIC INITIATION FACTOR 2

In eukaryotes, start codon selection is governed by base pairing between the start codon and the anticodon of the initiator tRNA (Met-tRNAi) and is achieved via a complex machinery involving at least twelve initiation factors. eIF2 is a heterotrimeric G-protein that, in its active GTP-bound state, binds and delivers the Met-tRNAi to the P-site of the small (40S) subunit of the ribosome, in a process facilitated by multiple other initiation factors forming a pre-initiation complex (PIC). The process of initiation is composed of two phases with distinct conformations of the PIC. First, the PIC in an open conformation scans the mRNA 5’ UTR in a manner allowing for the Met-tRNAi to sample the nucleotides for complementarity. Upon a cognate codon:anticodon interaction, the PIC then adopts a closed conformation accompanied by the irreversible hydrolysis of eIF2-bound GTP and Pi release. The molecular mechanisms by which cognate codon:anticodon base pairing is linked to the hydrolysis of eIF2-bound GTP and Pi release, formation of the closed conformation, and start codon selection are not well understood. In this study, we provide genetic and biochemical evidence that suggests a new function for domain-III of eIF2γ in coordinating these processes and maintaining the equilibrium between the two conformations of the PIC. In order to identify the structural elements in eIF2 essential for start codon selection, we isolated novel mutations in the yeast γ subunit that alter the accuracy of this process. We identified two classes of mutations with opposing effects: mutations that reduce the stringency of start codon recognition and those that, conversely, restore initiation fidelity. The isolated mutations localize to distinct regions on the surface of domain-III in close proximity of the proposed binding interface between eIF2 and the 40S subunit. We propose a model in which eIF2(γ) maintains a dynamic interaction with the 40S subunit during the scanning of the mRNA 5’ UTR. Upon cognate codon:anticodon base pairing, however, new contact points between eIF2γ domain-III and the 40S subunit are created that stabilize the closed conformation, hence stabilizing the accommodation of the Met-tRNAi in the P-site and allowing for the progress of translation initiation

The <i>Plasmodium</i> eukaryotic initiation factor-2α kinase IK2 controls the latency of sporozoites in the mosquito salivary glands

Sporozoites, the invasive form of malaria parasites transmitted by mosquitoes, are quiescent while in the insect salivary glands. Sporozoites only differentiate inside of the hepatocytes of the mammalian host. We show that sporozoite latency is an active process controlled by a eukaryotic initiation factor-2α (eIF2α) kinase (IK2) and a phosphatase. IK2 activity is dominant in salivary gland sporozoites, leading to an inhibition of translation and accumulation of stalled mRNAs into granules. When sporozoites are injected into the mammalian host, an eIF2α phosphatase removes the PO4 from eIF2α-P, and the repression of translation is alleviated to permit their transformation into liver stages. In IK2 knockout sporozoites, eIF2α is not phosphorylated and the parasites transform prematurely into liver stages and lose their infectivity. Thus, to complete their life cycle, Plasmodium sporozoites exploit the mechanism that regulates stress responses in eukaryotic cells

Regulation of arginine transport by GCN2 eIF2 kinase is important for replication of the intracellular parasite Toxoplasma gondii

Toxoplasma gondii is a prevalent protozoan parasite that can infect any nucleated cell but cannot replicate outside of its host cell. Toxoplasma is auxotrophic for several nutrients including arginine, tryptophan, and purines, which it must acquire from its host cell. The demands of parasite replication rapidly deplete the host cell of these essential nutrients, yet Toxoplasma successfully manages to proliferate until it lyses the host cell. In eukaryotic cells, nutrient starvation can induce the integrated stress response (ISR) through phosphorylation of an essential translation factor eIF2. Phosphorylation of eIF2 lowers global protein synthesis coincident with preferential translation of gene transcripts involved in stress adaptation, such as that encoding the transcription factor ATF4 (CREB2), which activates genes that modulate amino acid metabolism and uptake. Here, we discovered that the ISR is induced in host cells infected with Toxoplasma. Our results show that as Toxoplasma depletes host cell arginine, the host cell phosphorylates eIF2 via protein kinase GCN2 (EIF2AK4), leading to induced ATF4. Increased ATF4 then enhances expression of the cationic amino acid transporter CAT1 (SLC7A1), resulting in increased uptake of arginine in Toxoplasma-infected cells. Deletion of host GCN2, or its downstream effectors ATF4 and CAT1, lowers arginine levels in the host, impairing proliferation of the parasite. Our findings establish that Toxoplasma usurps the host cell ISR to help secure nutrients that it needs for parasite replication

Eukaryotic translation initiation machinery can operate in a prokaryotic-like mode without eIF2

Unlike prokaryotes, a specialized eukaryotic initiation factor 2 (eIF2), in the form of the ternary complex eIF2*GTP*Met-tRNAiMet is utilized to deliver the initiator tRNA to the ribosome within all eukaryotic cells1. Phosphorylation of eIF2 is known to be central to the global regulation of protein synthesis under stress conditions and infection2. Another distinctive feature of eukaryotic translation is scanning of mRNA 5&#x27;-leaders, whose origin in evolution may be relevant to the appearance of eIF2 in eukaryotes. Translation initiation on the hepatitis C virus (HCV) internal ribosome entry site (IRES) occurs without scanning3,4. Whether these unique features of the HCV IRES account for the formation of the final 80S initiation complex is unknown. Here we show that the HCV IRES-directed translation can occur without either eIF2 or its GTPase activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a prokaryotic-like pathway which involves eIF5B, the analogue of bacterial IF25,6, instead of eIF2. This switch from a eukaryotic-like mode of AUG selection to a &#x22;bacterial&#x22; one occurs when eIF2 is inactivated by phosphorylation, a way with which host cells counteract infection. The relative resistance of HCV IRES-directed translation to eIF2 phosphorylation may represent one more line of defense used by this virus against host antiviral responses and can contribute to the well known resistance of HCV to interferon based therapy

Flightless-I Controls Fat Storage in Drosophila

Triglyceride homeostasis is a key process of normal development and is essential for the maintenance of energy metabolism. Dysregulation of this process leads to metabolic disorders such as obesity and hyperlipidemia. Here, we report a novel function of the Drosophila flightless-I (fliI) gene in lipid metabolism. Drosophila fliI mutants were resistant to starvation and showed increased levels of triglycerides in the fat body and intestine, whereas fliI overexpression decreased triglyceride levels. These flies suffered from metabolic stress indicated by increased levels of trehalose in hemolymph and enhanced phosphorylation of eukaryotic initiation factor 2 alpha (eIF2??). Moreover, upregulation of triglycerides via a knockdown of fliI was reversed by a knockdown of desat1 in the fat body of flies. These results indicate that fliI suppresses the expression of desat1, thereby inhibiting the development of obesity; fliI may, thus, serve as a novel therapeutic target in obesity and metabolic diseases