Eukaryotic Translation Initiation Factor eIF2α Research Articles

The integrated stress response remodels the microtubule-organizing center to clear unfolded proteins following proteotoxic stress.

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically.

Cell competition is driven by Xrp1-mediated phosphorylation of eukaryotic initiation factor 2α.

Cell competition is a context-dependent cell elimination via cell-cell interaction whereby unfit cells ('losers') are eliminated from the tissue when confronted with fitter cells ('winners'). Despite extensive studies, the mechanism that drives loser's death and its physiological triggers remained elusive. Here, through a genetic screen in Drosophila, we find that endoplasmic reticulum (ER) stress causes cell competition. Mechanistically, ER stress upregulates the bZIP transcription factor Xrp1, which promotes phosphorylation of the eukaryotic translation initiation factor eIF2α via the kinase PERK, leading to cell elimination. Surprisingly, our genetic data show that different cell competition triggers such as ribosomal protein mutations or RNA helicase Hel25E mutations converge on upregulation of Xrp1, which leads to phosphorylation of eIF2α and thus causes reduction in global protein synthesis and apoptosis when confronted with wild-type cells. These findings not only uncover a core pathway of cell competition but also open the way to understanding the physiological triggers of cell competition.

Asp56 in actin is critical for the full activity of the amino acid starvation-responsive kinase Gcn2.

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.

Focus on Translation Initiation of the HIV-1 mRNAs.

To replicate and disseminate, viruses need to manipulate and modify the cellular machinery for their own benefit. We are interested in translation, which is one of the key steps of gene expression and viruses that have developed several strategies to hijack the ribosomal complex. The type 1 human immunodeficiency virus is a good paradigm to understand the great diversity of translational control. Indeed, scanning, leaky scanning, internal ribosome entry sites, and adenosine methylation are used by ribosomes to translate spliced and unspliced HIV-1 mRNAs, and some require specific cellular factors, such as the DDX3 helicase, that mediate mRNA export and translation. In addition, some viral and cellular proteins, including the HIV-1 Tat protein, also regulate protein synthesis through targeting the protein kinase PKR, which once activated, is able to phosphorylate the eukaryotic translation initiation factor eIF2α, which results in the inhibition of cellular mRNAs translation. Finally, the infection alters the integrity of several cellular proteins, including initiation factors, that directly or indirectly regulates translation events. In this review, we will provide a global overview of the current situation of how the HIV-1 mRNAs interact with the host cellular environment to produce viral proteins.

Amyloid-beta Degradation and Neuroprotection of Dauricine Mediated by Unfolded Protein Response in a Caenorhabditis elegans Model of Alzheimer’s disease

Amyloid plaque is a prominent pathologic hallmark in the brains of patients with Alzheimer’s disease (AD), and it has been shown to be associated with endoplasmic reticulum (ER) stress response. However the precise regulation mechanism of amyloid-beta (Aβ) toxicity remains unclear. Here, we found that dauricine could activate X-box binding protein 1 (XBP-1; active form XBP-1S) and eukaryotic translation initiation factor eIF2α and thus delay the progression of AD in the Aβ1-42-transgenic Caenorhabditis elegans CL2120. The ER stress response factor XBP-1 can be activated and shows neuroprotective activity through acceleration of Aβ clearance. Our study reveals that dauricine activates the ire-1/xbp-1 and perk/eIF2α pathways of the unfolded protein response, attenuates translation, and enhances ER-associated degradation, which reduces Aβ expression and attenuates Aβ-associated toxicity. On the contrary, xbp-1 depletion counteracts the effects of dauricine on Aβ-associated toxicity. These results underscore the functional relevance of XBP-1 in Aβ toxicity and degradation, and highlight the potentially pharmacodynamic value of dauricine in preventing the progression of AD.

Downregulation of protein kinase PKR activation by porcine reproductive and respiratory syndrome virus at its early stage infection

The interferon-induced double-strand RNA activated protein kinase (PKR) plays an important role in antiviral response. The objective of this study was to assess the effect of porcine reproductive and respiratory syndrome virus (PRRSV) on PKR activation. Here we report that PRRSV inhibited PKR activation during its early stage infection of primary pulmonary alveolar macrophages (PAMs). PRRSV infection led to lower level of phosphorylated PKR in comparison with mock-infected cells. The PKR inhibition was sustained until 10h post infection in the presence of polyI:C, a synthetic analog of double-stranded RNA activating PKR. PKR-mediated phosphorylation of the eukaryotic translation initiation factor eIF2α was also lower in the PRRSV-infected PAMs during the early stage infection. Interestingly, inactivated PRRSV was capable to inhibit the PKR activation until 6h post infection. This suggests that structural components of PRRSV virions were responsible for the inhibition, although PRRSV replication was needed for longer inhibition. These results indicate that the downregulation of PKR activation during early infection stage should be essential for PRRSV to avoid the antiviral response to initiate replication. This finding contributes to our understanding on PRRSV interaction with host innate immune response and reveal a target for control of PRRSV infection.

Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress.

Multiple transcripts encode for the cell cycle inhibitor p21Cip1. These transcripts produce identical proteins but differ in their 5’ untranslated regions (UTRs). Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through 35S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR) kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5’ upstream open reading frames (uORFs) through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress.

Andes virus nucleocapsid protein interrupts protein kinase R dimerization to counteract host interference in viral protein synthesis.

Pathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2α and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense. Protein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported here reveal that the hantavirus nucleocapsid protein counteracts the PKR antiviral response by inhibiting PKR dimerization, which is required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus-infected cells prevents the PKR-induced host translational shutdown to ensure the continuous synthesis of viral proteins required for efficient virus replication.

The discovery of the BABA receptor: scientific implications and application potential.

The discovery of the BABA receptor: scientific implications and application potential.

Abstract 2934: Activation of the Integrated Stress Response target ATF4 provides resistance towards anoikis- mediated cell death and promotes metastasis.

Abstract In response to diverse environmental and physiological stresses, eukaryotic cells activate a homeostatic program of gene expression known as Integrated Stress Response (ISR). Activation of ISR involves phosphorylation of eukaryotic translation initiation factor eIF2α (eIF2α-P) repressing global translation along with preferential translation of activating transcription factor 4(ATF4), a transcriptional regulator for an array of genes involved in metabolism, nutrient uptake, and anti-oxidant responses. We previously demonstrated that ATF4 expression is significantly increased in tumors as compared to the normal tissue and ablation of ATF4 expression inhibits tumor growth in mice. Recent results from our group also suggest a role of ATF4 in metastasis. Extracellular matrix detachment causes cell death in normal cells by anoikis- an apoptosis mechanism that is known to be deregulated in highly aggressive metastatic tumor cells. Here we report a possible role of ATF4 in rendering resistance towards anoikis mediated cell death. Human adenocarcinoma HT1080 cells harboring ATF4 shRNA injected through tail vein of nude mice fails to establish metastatic lung colonization after 4 weeks as compared to the wild type counterparts. Using suspension cultures we show that detachment of HT1080 cells from extracellular matrix induces ISR via phosphorylation of eIF2α phosphorylation and activation of ATF4. Down regulating ATF4 expression either through shRNA (shATF4) mediated knock down or blocking eIF2α kinase PERK significantly reduced cell survival with increased apoptosis. Inducible knockdown of ATF4 with doxycycline blocked ATF4 expression in suspension and rendered cells sensitive to anoikis induced cell death. Survival following matrix detachment has been shown to be induced through activation of autophagy - a tightly regulated cellular self-digestive process. We observed a similar induction of autophagy in HT1080 fibrosarcoma cells when subjected to anoikis characterized by increased expression of LC3II and degradation of p62. Blocking the induction of autophagy by spautin-1 renders cells sensitive to anoikis suggesting a pro-survival function. Interestingly, ablation of ATF4 expression fails to sustain the autophagic response which explains the sensitivity of these cells towards anoikis mediated cellular apoptosis. Systemic knockdown of several key factors of autophagic process revealed that Atg5 and ULK1 plays in important role in anoikis mediated autophagy. Loss of both Atg5 and ULK1- both transcriptional targets of ATF4, blocked autophagy and significantly increased apoptosis of HT1080 cells. Collectively, our findings divulge a novel role of ATF4 in providing cancer cells protection against physiologically induced autophagy and thus can be exploited as novel target for aggressive metastatic tumors. Citation Format: Souvik Dey, Carly M. Sayers, Constantinos Koumenis. Activation of the Integrated Stress Response target ATF4 provides resistance towards anoikis- mediated cell death and promotes metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2934. doi:10.1158/1538-7445.AM2013-2934

The IE180 Protein of Pseudorabies Virus Suppresses Phosphorylation of Translation Initiation Factor eIF2α

We have previously shown that the porcine alphaherpesvirus pseudorabies virus (PRV) efficiently interferes with phosphorylation of the eukaryotic translation initiation factor eIF2α. Inhibition of phosphorylation of eIF2α has been reported earlier for the closely related alphaherpesvirus herpes simplex virus 1 (HSV-1) through its ICP34.5 and US11 proteins. PRV, however, does not encode an ICP34.5 or US11 orthologue. Assays using cycloheximide, UV-inactivated PRV, or phosphonoacetic acid (PAA) showed that de novo expression of one or more (immediate) early viral protein(s) is required for interference with eIF2α phosphorylation. In line with this, a time course assay showed that eIF2α phosphorylation was abolished within 2 h after PRV inoculation. PRV encodes only one immediate-early protein, IE180, the orthologue of HSV-1 ICP4. As reported earlier, a combinational treatment of cells with cycloheximide and actinomycin D allowed expression of IE180 without detectable expression of the US3 early protein in PRV-infected cells. This led to a substantial reduction in eIF2α phosphorylation levels, indicative for an involvement of IE180. In support of this, transfection of IE180 also potently reduced eIF2α phosphorylation. IE180-mediated interference with eIF2α phosphorylation was not cell type dependent, as it occurred both in rat neuronal 50B11 cells and in swine testicle cells. Inhibition of the cellular phosphatase PP1 impaired PRV-mediated interference with eIF2α phosphorylation, indicating that PP1 is involved in this process. In conclusion, the immediate-early IE180 protein of PRV has the previously uncharacterized ability to suppress phosphorylation levels of the eukaryotic translation initiation factor eIF2α.

Immunogenic Tumor Cell Death for Optimal Anticancer Therapy: The Calreticulin Exposure Pathway

In response to some chemotherapeutic agents such as anthracyclines and oxaliplatin, cancer cells undergo immunogenic apoptosis, meaning that their corpses are engulfed by dendritic cells and that tumor cell antigens are presented to tumor-specific CD8(+) T cells, which then control residual tumor cells. One of the peculiarities of immunogenic apoptosis is the early cell surface exposure of calreticulin (CRT), a protein that usually resides in the lumen of the endoplasmic reticulum (ER). When elicited by anthracyclines or oxaliplatin, the CRT exposure pathway is activated by pre-apoptotic ER stress and the phosphorylation of the eukaryotic translation initiation factor eIF2alpha by the kinase PERK, followed by caspase-8-mediated proteolysis of the ER-sessile protein BAP31, activation of the pro-apoptotic proteins Bax and Bak, anterograde transport of CRT from the ER to the Golgi apparatus and exocytosis of CRT-containing vesicles, finally resulting in CRT translocation onto the plasma membrane surface. Interruption of this complex pathway abolishes CRT exposure, annihilates the immunogenicity of apoptosis, and reduces the immune response elicited by anticancer chemotherapies. We speculate that human cancers that are incapable of activating the CRT exposure pathway are refractory to the immune-mediated component of anticancer therapies.

The RNA-activated Protein Kinase Enhances the Induction of Interferon-β and Apoptosis Mediated by Cytoplasmic RNA Sensors

Detection of foreign RNA by the innate immune system can trigger the induction of type I interferon (IFN) and apoptosis. Important antiviral defense pathways that result in type I IFN production following the recognition of foreign double-stranded RNA (dsRNA) include the RIG-I family helicases and IPS-1 adaptor cytosolic pathway and the Toll-like receptor 3 and TIR domain-containing adaptor-inducing IFN-beta (TRIF) adaptor membrane-associated pathway, both of which activate IFN regulatory factor 3 (IRF3). In addition to triggering an innate immune response, dsRNAs are widely used to mediate gene-selective silencing in mammalian cells by the RNA interference pathway. We investigated the ability of short interfering RNAs, including T7 phage polymerase-synthesized RNA (PRNA), which like some viral RNAs contains a 5'-triphosphate, to selectively silence gene expression and to cause induction of IFN-beta and apoptosis. We found that PRNA-mediated gene silencing and associated nonspecific pro-apoptotic and IFN-inducing effects were dependent on the cell line and RNA length. Double-stranded PRNAs 50 nucleotides long as well as polyinosinic-polycytidylic acid activated the RNA-dependent protein kinase (PKR) and induced significant levels of IFN-beta and apoptosis, whereas shorter PRNAs and chemically synthesized dsRNAs did not. Effector caspase activation and apoptosis following RNA transfection was enhanced by pretreatment with IFN, and removal of the 5'-phosphate from PRNAs decreased induction of both IFN-beta and apoptosis. PKR, in addition to IPS-1 and IRF3 but not TRIF, was required for maximal type I IFN-beta induction and the induction of apoptosis by both transfected PRNAs and polyinosinic-polycytidylic acid.

The Cellular Protein P58 IPK Regulates Influenza Virus mRNA Translation and Replication through a PKR-Mediated Mechanism

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58(IPK), the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2alpha. P58(IPK) also inhibits PERK, an eIF2alpha kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58(IPK) to interact with and inhibit multiple eIF2alpha kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58(IPK) during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58(IPK-/-) mice, we demonstrated that the absence of P58(IPK) led to an increase in eIF2alpha phosphorylation and decreased influenza virus mRNA translation. The absence of P58(IPK) also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58(IPK) target, PKR, the trends were reversed-eIF2alpha phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58(IPK) also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2alpha phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2alpha. Taken together, our results support a model in which P58(IPK) regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

Evasion of cellular antiviral responses by human cytomegalovirus TRS1 and IRS1.

During infection with human cytomegalovirus (HCMV), cellular protein synthesis continues even as viral proteins are being synthesized in abundance. Thus, HCMV may have a mechanism for counteracting host cell antiviral pathways that act by shutting off translation. Consistent with this view, HCMV infection of human fibroblasts rescues the replication of a vaccinia virus mutant lacking the double-stranded RNA-binding protein gene E3L (VVdeltaE3L). HCMV also prevents the phosphorylation of the eukaryotic translation initiation factor eIF-2alpha, the activation of RNase L, and the shutoff of viral and cellular protein synthesis that otherwise result from VVdeltaE3L infection. To identify the HCMV gene(s) responsible for these effects, we prepared a library of VVdeltaE3L recombinants containing HCMV genomic fragments. By infecting nonpermissive cells with this library and screening for VV gene expression and replication, we isolated a virus containing a 2.8-kb HCMV fragment that rescues replication of VVdeltaE3L. The fragment comprises the 3' end of the J1S open reading frame through the entire TRS1 gene. Analyses of additional VVdeltaE3L recombinants revealed that the protein encoded by TRS1, pTRS1, as well as the closely related IRS1 gene, rescues VVdeltaE3L replication and prevent the shutoff of protein synthesis, the phosphorylation of eIF-2alpha, and activation of RNase L. These results demonstrate that TRS1 and IRS1 are able to counteract critical host cell antiviral response pathways.

Translational initiation factors IF-1 and eIF-2α share an RNA-binding motif with prokaryotic ribosomal protein S1 and polynucleotide phosphorylase

Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2α chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2α may bind to RNA.