Eukaryotic Initiation Factor eIF Research Articles

Signaling plasticity in the integrated stress response.

The Integrated Stress Response (ISR) is an essential homeostatic signaling network that controls the cell's biosynthetic capacity. Four ISR sensor kinases detect multiple stressors and relay this information to downstream effectors by phosphorylating a common node: the alpha subunit of the eukaryotic initiation factor eIF2. As a result, general protein synthesis is repressed while select transcripts are preferentially translated, thus remodeling the proteome and transcriptome. Mounting evidence supports a view of the ISR as a dynamic signaling network with multiple modulators and feedback regulatory features that vary across cell and tissue types. Here, we discuss updated views on ISR sensor kinase mechanisms, how the subcellular localization of ISR components impacts signaling, and highlight ISR signaling differences across cells and tissues. Finally, we consider crosstalk between the ISR and other signaling pathways as a determinant of cell health.

Evaluation of a Series of Turnip Mosaic Virus Chimeric Clones Reveals Two Amino Acid Sites Critical for Systemic Infection in Chinese Cabbage.

Two infectious clones of turnip mosaic virus (TuMV), pKBC-1 and pKBC-8, with differential infectivity in Chinese cabbage (Brassica rapa subsp. pekinensis), were obtained. Both infected Nicotiana benthamiana systemically, inducing similar symptoms, whereas only virus KBC-8 infected Chinese cabbage systemically. To identify the determinants affecting infectivity on Chinese cabbage, chimeric clones were constructed by restriction fragment exchange between the parental clones and tested on several Chinese cabbage cultivars. Chimeric clones p1N8C and p8N1C demonstrated that the C-terminal portion of the polyprotein determines systemic infection of Chinese cabbage despite only three amino acid differences in this region, in the cylindrical inclusion (CI), viral protein genome-linked (VPg), and coat protein (CP). A second pair of hybrid constructs, pHindIII-1N8C and pHindIII-8N1C, failed to infect cultivars CR Victory and Jinseonnorang systemically, yet pHindIII-1N8C caused hypersensitive response-like lesions on inoculated leaves of these cultivars, and could systemically infect cultivars CR Chusarang and Jeongsang; this suggests that R genes effective against TuMV may exist in the first two cultivars but not the latter two. Constructs with single amino acid changes in both VPg (K2045E) and CP (Y3095H) failed to infect Chinese cabbage, implying that at least one of these two amino acid substitutions is essential for successful infection on Chinese cabbage. Successful infection by mutant KBC-8-CP-H and delayed infection with mutant HJY1-VPg-E following mutation or reversion suggested that VPg (2045K) is the residue required for infection of Chinese cabbage and involved in the interaction between VPg and eukaryotic initiation factor eIF(iso)4E, confirmed by yeast two-hybrid assay.

Abstract 3040: The eIF2 kinase GCN2 controls expression of key amino acid transporters and is critical for prostate cancer growth and progression

Abstract The Integrated Stress Response (ISR) is central for adaptation and survival of tumor cells in response to exogenous and endogenous stresses. The ISR features a family of protein kinases that phosphorylate the eukaryotic translation initiation factor eIF2 in response to distinct stresses. While phosphoryation of eIF2 reduces global protein synthesis, there is paradoxical induced translation of select stress adapative gene transcripts, including ATF4, which is central for ISR-directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer adaptation to stress. Recently, eIF2 phosphorylation and ATF4 were suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinase(s), its mode of activation, and mechanisms by which the ISR facilitate PCa progression are not known.We discovered that the eIF2 kinase GCN2 (EIF2AK4) is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduced growth in androgen-sensitive and castration-resistant PCa cell lines in culture and cell line-derived and patient-derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2. A transcriptome analysis of PCa cells treated with a specific small molecular inhibitor of GCN2 indicates that this eIF2 kinase was critical for expression of multiple SLC genes, including amino acid transporters previously implicated in different cancers. We determined that GCN2 inhibition decreases intracellular amino acid levels, and reduced aminoacylation of select tRNAs, accounting for reduced growth in PCa cells. Using CRISPR-based phenotypic screens and genome-wide gene expression analyses of control and GCN2-depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is a key SLC gene induced by GCN2 and is essential for PCa proliferation. SLC3A2 has been reported to associate with many SLC7 family transporters, allowing for the localization of these transporters to the plasma membrane for amino acid uptake. Of importance, expression of SLC3A2 partially restored amino acid levels and growth due to loss of GCN2. Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including SLC3A2, which provides for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising new therapeutic targets for the treatment of PCa. Citation Format: Ricardo Cordova, Jagannath Misra, Nur P. Damayanti, Parth H. Amin, Kenneth R. Carlson, Angela J. Klunk, Emily T. Mirek, Marcus J. Miller, Tracy G. Anthony, Roberto Pili, Ronald C. Wek, Kirk A. Staschke. The eIF2 kinase GCN2 controls expression of key amino acid transporters and is critical for prostate cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3040.

Targeting GCN2 Regulation of Amino Acid Homeostasis in Prostate Cancer

The Integrated Stress Response (ISR) plays a critical role in the adaptation and survival of tumor cells to exogenous and endogenous stresses. The ISR features four protein kinases (PERK, GCN2, PKR, and HRI), each activated by different stresses, that phosphorylate the eukaryotic translation initiation factor eIF2, resulting in repression of global protein synthesis. Paradoxically, eIF2 phosphorylation also enhances translation of select gene transcripts, including the transcription factor ATF4, which is central for ISR‐directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer stress adaptation. Moreover, eIF2 phosphorylation and ATF4 have recently been suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinases, their mode of activation, and the mechanisms by which the ISR facilitate PCa progression are unknown.We discovered that GCN2 is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduces growth in androgen‐sensitive and castration‐resistant PCa cell lines in culture and cell line‐derived and patient‐derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2.A transcriptome analysis of PCa cells treated with a specific GCN2 small molecular inhibitor indicates that GCN2 is critical for expression of SLCgenes involved in metabolite transport. We found that GCN2 inhibition decreases intracellular amino acid levels accounting for reduced growth in PCa cells. Using CRISPR‐based phenotypic screens and genome‐wide gene expression analyses of wild‐type and GCN2‐depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is induced by GCN2 and is essential for PCa proliferation. SLC3A2 engages with many nutrient transporters, allowing for their localization to the plasma membrane. Importantly, expression of SLC3A2 reduced GCN2 activation and rescued decreased amino acid levels and growth inhibition due to loss of GCN2.Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including 4F2 (SLC3A2), which provide for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising therapeutic targets for both androgen‐sensitive and castration‐resistant prostate cancer.

A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation.

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.

Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions.

Thermofluor is a fluorescence-based thermal shift assay, which measures temperature-induced protein unfolding and thereby yields valuable information about the integrity of a purified recombinant protein. Analysis of ligand binding to a protein is another popular application of this assay. Thermofluor requires neither protein labeling nor highly specialized equipment, and can be performed in a regular real-time PCR instrument. Thus, for a typical molecular biology laboratory, Thermofluor is a convenient method for the routine assessment of protein quality. Here, we provide Thermofluor protocols using the example of Cdc123. This ATP-grasp protein is an essential assembly chaperone of the eukaryotic translation initiation factor eIF2. We also report on a destabilized mutant protein version and on the ATP-mediated thermal stabilization of wild-type Cdc123 illustrating protein integrity assessment and ligand binding analysis as two major applications of the Thermofluor assay.

Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity.

The translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of the C. elegans RNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron-type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5'UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5'UTR-dependent manner. Our study reveals an in vivo mechanism for eIF3 in governing neuronal protein levels to control neuronal activity states and offers insights into how eIF3 dysregulation contributes to neurological disorders.

Richard Jackson (1940–2020) ‐ A towering presence in translation

Richard Jackson (1940–2020) ‐ A towering presence in translation

Relevance of oxidative stress in inhibition of eIF2 alpha phosphorylation and stress granules formation during Usutu virus infection.

Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile, Japanese encephalitis, Zika, and dengue viruses. USUV emerged in 1996 in Europe, where quickly spread across the continent causing a considerable number of bird deaths and varied neurological disorders in humans, including encephalitis, meningoencephalitis, or facial paralysis, thus warning about USUV as a potential health threat. USUV replication takes place on the endoplasmic reticulum (ER) of infected cells, inducing ER stress and resulting in the activation of stress-related cellular pathways collectively known as the integrated stress response (ISR). The alpha subunit of the eukaryotic initiation factor eIF2 (eIF2α), the core factor in this pathway, is phosphorylated by stress activated kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), heme-regulated inhibitor kinase (HRI), and general control non-repressed 2 kinase (GCN2). Its phosphorylation results, among others, in the downstream inhibition of translation with accumulation of discrete foci in the cytoplasm termed stress granules (SGs). Our results indicated that USUV infection evades cellular stress response impairing eIF2α phosphorylation and SGs assembly induced by treatment with the HRI activator ArsNa. This protective effect was related with oxidative stress responses in USUV-infected cells. Overall, these results provide new insights into the complex connections between the stress response and flavivirus infection in order to maintain an adequate cellular environment for viral replication.

Small molecule cognitive enhancer reverses age-related memory decline in mice.

With increased life expectancy, age-associated cognitive decline becomes a growing concern, even in the absence of recognizable neurodegenerative disease. The integrated stress response (ISR) is activated during aging and contributes to age-related brain phenotypes. We demonstrate that treatment with the drug-like small-molecule ISR inhibitor ISRIB reverses ISR activation in the brain, as indicated by decreased levels of activating transcription factor 4 (ATF4) and phosphorylated eukaryotic translation initiation factor eIF2. Furthermore, ISRIB treatment reverses spatial memory deficits and ameliorates working memory in old mice. At the cellular level in the hippocampus, ISR inhibition (i) rescues intrinsic neuronal electrophysiological properties, (ii) restores spine density and (iii) reduces immune profiles, specifically interferon and T cell-mediated responses. Thus, pharmacological interference with the ISR emerges as a promising intervention strategy for combating age-related cognitive decline in otherwise healthy individuals.

Light-Dependent Activation of the GCN2 Kinase Under Cold and Salt Stress Is Mediated by the Photosynthetic Status of the Chloroplast.

Regulation of cytosolic mRNA translation is a key node for rapid adaptation to environmental stress conditions. In yeast and animals, phosphorylation of the α-subunit of eukaryotic translation initiation factor eIF2 is the most thoroughly characterized event for regulating global translation under stress. In plants, the GCN2 kinase (General Control Nonderepressible-2) is the only known kinase for eIF2α. GCN2 is activated under a variety of stresses including reactive oxygen species (ROS). Here, we provide new evidence that the GCN2 kinase in Arabidopsis is also activated rapidly and in a light-dependent manner by cold and salt treatments. These treatments alone did not repress global mRNA ribosome loading in a major way. The activation of GCN2 was accompanied by a more oxidative environment and was attenuated by inhibitors of photosynthetic electron transport, suggesting that it is gated by the redox poise or the reactive oxygen status of the chloroplast. In keeping with these results, gcn2 mutant seedlings were more sensitive than wild type to both cold and salt in a root elongation assay. These data suggest that cold and salt stress may both affect the status of the cytosolic translation apparatus via the conserved GCN2-eIF2α module. The potential role of the GCN2 kinase pathway in the global repression of translation under abiotic stress is discussed.

EIF2B and the Integrated Stress Response: A Structural and Mechanistic View.

The eukaryotic translation initiation factor eIF2 is a GTPase, which brings the initiator Met-tRNAi to the ribosome as the eIF2-GTP·Met-tRNAi ternary complex (TC). TC regeneration is catalyzed by the guanine nucleotide exchange factor (GEF) eIF2B. eIF2 phosphorylation by several stress-induced kinases converts it into a competitive inhibitor of eIF2B. Inhibition of eIF2B activity lowers cellular TC concentrations, which in turn triggers the integrated stress response (ISR). Depending on its degree of activation and duration, the ISR protects the cell from the stress or can itself induce apoptosis. ISR dysregulation is a causative factor in the pathology of multiple neurodegenerative disorders, while ISR inhibitors are neuroprotective. The realization that eIF2B is a promising therapeutic target has triggered significant interest in its structure and its mechanisms of action and regulation. Recently, four groups published the cryo-electron microscopy structures of eIF2B with its substrate eIF2 and/or its inhibitor, phosphorylated eIF2 [eIF2(α-P)]. While all three structures of the nonproductive eIF2B·eIF2(α-P) complex are similar to each other, there is a sharp disagreement between the published structures of the productive eIF2B·eIF2 complex. One group reports a structure similar to that of the nonproductive complex, whereas two others observe a vastly different eIF2B·eIF2 complex. Here, we discuss the recent reports on the structure, function, and regulation of eIF2B; the preclinical data on the use of ISR inhibitors for the treatment of neurodegenerative disorders; and how the new structural and biochemical information can inform and influence the use of eIF2B as a therapeutic target.

Suppression of MEHMO Syndrome Mutation in eIF2 by Small Molecule ISRIB.

Dysregulation of cellular protein synthesis is linked toa variety of diseases. Mutations in EIF2S3, encoding the γ subunit of the heterotrimeric eukaryotic translation initiation factor eIF2, cause MEHMO syndrome, an X-linked intellectual disability disorder. Here, using patient-derived induced pluripotent stem cells, we show that a mutation at the C terminus of eIF2γ impairs CDC123 promotion of eIF2 complex formation and decreases the level of eIF2-GTP-Met-tRNAiMet ternary complexes. This reduction in eIF2 activity results in dysregulation of global and gene-specific protein synthesis and enhances cell death upon stress induction. Addition of the drug ISRIB, an activator of the eIF2 guanine nucleotide exchange factor, rescues the cell growth, translation, and neuronal differentiation defects associated with the EIF2S3 mutation, offering the possibility of therapeutic intervention for MEHMO syndrome.

Expression, purification, and in vitro characterization of kinase domain of NtGCN2 from tobacco

General control nonderepressible 2 (GCN2) can phosphorylate the α subunit of eukaryotic initiation factor eIF2 (eukaryotic translation initiation factor 2) to down-regulateprotein synthesis in response to various biotic and abiotic stresses. However, the kinase activity of plant GCN2 has not been well-characterized in vitro. In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expressionin Escherichia coli BL21-CodonPlus-(DE3)-RIPL after induction by 0.5 mmol L−1 IPTG for 13 h at 16 °C. The soluble protein was collected and purified by Ni2+-NTA agarose column, anion exchange, and molecular sieve, and the purified proteinwas used for kinase assays and the preparation of a polyclonal antibody. Enzyme-linked immunosorbent assay results showed that the titer of the antiserum was 1:520K. Western blot analysis showed that the prepared antibody reacted with GCN2 in tobacco. Additionally, the kinase activity of NtGCN2 was characterized by using recombinant NteIF2α protein as a substrate in vitro. The results showed that NtGCN2 phosphorylated NteIF2α in vitro, with the level of phosphorylation positively correlated with the NtGCN2 concentration and reaction time. Our study has prepared a specific antibody, and proves NtGCN2 can phosphorylate NteIF2α in vitro, which lays a foundation for further study of the function and interaction network of NtGCN2.

MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2.

The heterotrimeric eukaryotic translation initiation factor (eIF) 2 plays critical roles in delivering initiator Met-tRNAiMet to the 40S ribosomal subunit and in selecting the translation initiation site. Genetic analyses of patients with MEHMO syndrome, an X-linked intellectual disability syndrome, have identified several unique mutations in the EIF2S3 gene that encodes the γ subunit of eIF2. To gain insights into the molecular consequences of MEHMO syndrome mutations on eIF2 function, we generated a yeast model of the human eIF2γ-I259M mutant, previously identified in a patient with MEHMO syndrome. The corresponding eIF2γ-I318M mutation impaired yeast cell growth and derepressed GCN4 expression, an indicator of defective eIF2–GTP–Met-tRNAiMet complex formation, and, likewise, overexpression of human eIF2γ-I259M derepressed ATF4 messenger RNA translation in human cells. The yeast eIF2γ-I318M mutation also increased initiation from near-cognate start codons. Biochemical analyses revealed a defect in Met-tRNAiMet binding to the mutant yeast eIF2 complexes in vivo and in vitro. Overexpression of tRNAiMet restored Met-tRNAiMet binding to eIF2 in vivo and rescued the growth defect in the eIF2γ-I318M strain. Based on these findings and the structure of eIF2, we propose that the I259M mutation impairs Met-tRNAiMet binding, causing altered control of protein synthesis that underlies MEHMO syndrome.

Disrupted sphingolipid metabolism following acute clozapine and olanzapine administration

BackgroundSecond generation antipsychotics (SGAs) induce glucometabolic side-effects, such as hyperglycemia and insulin resistance, which pose a therapeutic challenge for mental illness. Sphingolipids play a role in glycaemic balance and insulin resistance. Endoplasmic reticulum (ER) stress contributes to impaired insulin signalling and whole-body glucose intolerance. Diabetogenic SGA effects on ER stress and sphingolipids, such as ceramide and sphingomyelin, in peripheral metabolic tissues are unknown. This study aimed to investigate the acute effects of clozapine and olanzapine on ceramide and sphingomyelin levels, and protein expression of key enzymes involved in lipid and glucose metabolism, in the liver and skeletal muscle.MethodsFemale rats were administered olanzapine (1 mg/kg), clozapine (12 mg/kg), or vehicle (control) and euthanized 1-h later. Ceramide and sphingomyelin levels were examined using electrospray ionization (ESI) mass spectrometry. Expression of lipid enzymes (ceramide synthase 2 (CerS2), elongation of very long-chain fatty acid 1 (ELOVL1), fatty acid synthase (FAS) and acetyl CoA carboxylase 1 (ACC1)), ER stress markers (inositol-requiring enzyme 1 (IRE1) and eukaryotic initiation factor (eIF2α) were also examined.ResultsClozapine caused robust reductions in hepatic ceramide and sphingolipid levels (p < 0.0001), upregulated CerS2 (p < 0.05) and ELOVL1 (+ 37%) and induced significant hyperglycemia (vs controls). In contrast, olanzapine increased hepatic sphingomyelin levels (p < 0.05 vs controls). SGAs did not alter sphingolipid levels in the muscle. Clozapine increased (+ 52.5%) hepatic eIF2α phosphorylation, demonstrating evidence of activation of the PERK/eIF2α ER stress axis. Hepatic IRE1, FAS and ACC1 were unaltered.ConclusionsThis study provides the first evidence that diabetogenic SGAs disrupt hepatic sphingolipid homeostasis within 1-h of administration. Sphingolipids may be key candidates in the mechanisms underlying the diabetes side-effects of SGAs; however, further research is required.

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle.

Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream—in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3–40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3’s impact on translational control in eukaryotic cells.

Could the eIF2α-Independent Translation Be the Achilles Heel of Cancer?

Eukaryotic initiation factor eIF2 is a key component of the ternary complex whose role is to deliver initiator tRNA into the ribosome. A variety of stimuli, both physiologic and pathophysiologic activate eIF2 kinases that phosphorylate the α subunit of eIF2, preventing it from forming the ternary complex, thus attenuating cellular protein synthesis. Paradoxically, in cancer cells, the phosphorylation of eIF2α is associated with activation of survival pathways. This review explores the recently emerged novel mechanism of eIF2α-independent translation initiation. This mechanism, which appears to be shared by some RNA viruses and Internal Ribosome Entry Site-containing cellular mRNAs and utilizes auxiliary proteins, such as eIF5B, eIF2D, and MCT-1, is responsible for the selective translation of cancer-associated genes and could represent a weak point amenable to specific targeting for the treatment of cancer.

Signaling through alternative Integrated Stress Response pathways compensates for GCN2 loss in a mouse model of soft tissue sarcoma.

The tumor microenvironment is characterized by deficiencies in oxygen and nutrients, such as glucose and amino acids. Activation of the GCN2 arm of the Integrated Stress Response (ISR) in response to amino acid deprivation is one mechanism by which tumor cells cope with nutrient stress. GCN2 phosphorylates the alpha subunit of the eukaryotic translation initiation factor eIF2, leading to global downregulation of translation to conserve amino acids and initiation of a transcriptional program through ATF4 to promote recovery from nutrient deprivation. Loss of GCN2 results in decreased tumor cell survival in vitro under amino acid deprivation and attenuated tumor growth in xenograft tumor models. However, it is not known what effects GCN2 loss has on the growth of autochthonous tumors that arise in their native microenvironment. Here, we demonstrate in a genetically engineered mouse model of soft tissue sarcoma that loss of GCN2 has no effect on tumor growth or animal survival. The sarcomas displayed compensatory activation of PERK or phospho-eIF2α independent upregulation of ATF4 in order to maintain ISR signaling, indicating that this pathway is critical for tumorigenesis. These results have important implications for the development and testing of small molecule inhibitors of ISR kinases as cancer therapeutics.

Effects of Nandrolone in the Counteraction of Skeletal Muscle Atrophy in a Mouse Model of Muscle Disuse: Molecular Biology and Functional Evaluation.

Muscle disuse produces severe atrophy and a slow-to-fast phenotype transition in the postural Soleus (Sol) muscle of rodents. Antioxidants, amino-acids and growth factors were ineffective to ameliorate muscle atrophy. Here we evaluate the effects of nandrolone (ND), an anabolic steroid, on mouse skeletal muscle atrophy induced by hindlimb unloading (HU). Mice were pre-treated for 2-weeks before HU and during the 2-weeks of HU. Muscle weight and total protein content were reduced in HU mice and a restoration of these parameters was found in ND-treated HU mice. The analysis of gene expression by real-time PCR demonstrates an increase of MuRF-1 during HU but minor involvement of other catabolic pathways. However, ND did not affect MuRF-1 expression. The evaluation of anabolic pathways showed no change in mTOR and eIF2-kinase mRNA expression, but the protein expression of the eukaryotic initiation factor eIF2 was reduced during HU and restored by ND. Moreover we found an involvement of regenerative pathways, since the increase of MyoD observed after HU suggests the promotion of myogenic stem cell differentiation in response to atrophy. At the same time, Notch-1 expression was down-regulated. Interestingly, the ND treatment prevented changes in MyoD and Notch-1 expression. On the contrary, there was no evidence for an effect of ND on the change of muscle phenotype induced by HU, since no effect of treatment was observed on the resting gCl, restCa and contractile properties in Sol muscle. Accordingly, PGC1α and myosin heavy chain expression, indexes of the phenotype transition, were not restored in ND-treated HU mice. We hypothesize that ND is unable to directly affect the phenotype transition when the specialized motor unit firing pattern of stimulation is lacking. Nevertheless, through stimulation of protein synthesis, ND preserves protein content and muscle weight, which may result advantageous to the affected skeletal muscle for functional recovery.