eIF2α Kinase Research Articles

Catalytic Activity of Heme-Regulated eIF2 Alpha Kinase (HRI) Regulates Fetal Hemoglobin

Sickle cell disease (SCD) is a hereditary disorder occurring due to a mutation in the β- globin gene resulting in hemoglobin polymerization and sickling of red blood cells that drives an array of severe pathophysiologies. SCD patients with hereditary persistence of fetal hemoglobin mutations show amelioration of disease symptoms. HRI is a heme sensing eIF2α kinase belonging to the integrated stress response pathway, primarily regulating the hemoglobin synthesis in RBCs. Under low levels of heme, HRI undergoes auto-phosphorylation and subsequently phosphorylates its substrate eIF2α. This impedes the global protein translation and selectively activates ATF4 translation thereby initiating a transcriptional stress response. HRI protein deletion has been shown to induce fetal hemoglobin (HbF) in vitro by reducing BCL11A levels (Grevet et al., 2018). It has also been shown that ATF4 binds to enhancer region of BCL11A promoting its expression, revealing a direct regulation of HRI from eIF2α to ATF4 to BCL11A to γ-globin (Huang P, 2020). As it is well-established that protein kinases can exert dual kinase and scaffold functions, we explored whether the regulation of HRI on HbF is driven by its catalytic or non-catalytic activity. We generated HRI kinase dead (KD) mutant HUDEP-2 cells, with a mutation (K196R) in HRI ATP binding site that is predicted to result in loss of its auto-phosphorylation (Bauer B. N., 2001). HRI KD mutant HUDEP-2 was created using CRISPR Cas9 and homologous recombination approach, followed by clonal selection that yielded two homozygous clones, referred as KD clones. To further compare the scaffold and kinase function of HRI, we created HRI knockout (KO) in HUDEP-2 cell line using CRISPR Cas9 approach. We isolated clonal populations with 3 clones each with heterozygous and homozygous editing. Western analysis of KD clones showed that HRI protein levels remain unchanged. About 50% and a 100% loss in HRI protein level was observed in heterozygous and homozygous KO clones respectively. To confirm the loss in kinase activity of HRI, we differentiated KD clones for 7 days in vitro and subjected them to iron depleted conditions. As expected, HRI WT cells under iron depleted conditions showed robust activation in the p-eIF2α levels indicative of increase in HRI activity. KD clones lacked increase in p-eIF2α levels under these conditions confirming the loss of kinase activity of HRI in KD clones. HRI KD and KO clones were characterized to evaluate the HbF regulation by differentiating in vitro for 7-8 days. KD clones showed 4-5-fold induction in HbF levels measured by flow cytometry. Heterozygous and homozygous KO clones showed an equally strong activation in HbF levels. KD and KO clones showed downregulation in BCL11A and ATF4 protein levels. These results indicate that HRI regulates HbF as a function of its catalytic activity. We further employed RNA-seq to study the global transcriptomics in HRI WT and KD mutant clones at day 0 and day 8, under steady state and iron depleted conditions. RNA-seq confirmed the strong activation of HBG1/2 levels and robust silencing of BCL11A levels in KD clones at day 8. A weaker induction in HBG1/2 levels and minor reduction on BCL11A levels were observed at day 0. These results indicate that HRI may play a role in HbF silencing in a stage specific manner. HRI WT cells under iron depleted condition showed activation of ATF4 pathway, indicative of activation of HRI stress pathway. As expected, HRI KD population showed no regulation of ATF4 pathway under iron depleted conditions. Interestingly, we did not observe any changes in the BCL11A gene expression in WT cells under iron depleted conditions. Further evaluation needs to be carried out in order to understand the regulation of BCL11A by ATF4 under low iron mediated stress. Fewer gene changes were observed in KD populations under iron depleted conditions indicative of HRI playing a vital role as a heme sensor. Overall, our results provide evidence to support that HRI regulates HbF though its catalytic activity and demonstrates its role as heme sensor in HUDEP-2 cells. Disclosures Gupta: Sanofi: Current Employment. Hara:Sanofi: Current Employment. Lessard:Sanofi: Current Employment. Sturtevant:Sanofi: Current Employment. Krishnamoorthy:Sanofi: Current Employment. Demers:Sanofi: Current Employment. Hicks:Sanofi: Current Employment.

Abstract PO-47: BTM-3566 induces complete tumor regression in diffuse large B-cell lymphoma: Regulation by the eIF2α kinase “heme-regulated inhibitor” and the mitochondrial protein FAM210B

Abstract BTM-3566 is a novel small-molecule compound with broad anticancer activity in hematologic and solid cancers, with exceptional activity against diffuse large B-cell lymphoma (DLBCL). The compound's mechanism of action (MoA) is dependent on activation of the ATF4 integrated stress response (ISR) by the eIF2 kinase heme-regulated inhibitor (EIF2AK1, HRI) resulting in translational repression, increased expression of ATF4, and apoptosis. To evaluate HRI's role in mediating the ISR and apoptosis by BTM-3566 in DLBCL, homozygous HRI knockout BJAB cells were generated. HRI gene deletion resulted in inhibition of BTM-3566 induction of ATF4 expression, reduction of cell growth inhibition, and abrogation of apoptosis. Expression of the antiapoptotic proteins Mcl1 and Bcl6 was reduced following treatment with BTM-3566 in wild-type BJAB cells but not in HRI KO BJAB cells. To further elucidate BTM-3566 MoA, an activity screen was performed across 407 hematologic and solid cancer cell lines. Expression of FAM210B, a mitochondrial membrane protein, emerged as a key predictor of response to BTM-3566. FAM210B expression was negatively correlated with response to compound; the cell lines most responsive to BTM-3566 were the B-cell malignancies, notably DLBCL, which have the lowest expression of FAM210B among all cancers. The relevance of FAM210B to BTM-3566 activity was confirmed by overexpression of FAM210B in the DLBCL cell line BJAB and the Burkitt lymphoma cell line RAMOS. Overexpression of FAM210B abrogated BTM-3566 antiproliferative activity in these cell lines. The specificity of BTM-3566 activity regulation by FAM210B was demonstrated by comparison to treatments known to activate the ATF4 ISR through eIF2α kinases; tunicamycin (PERK), amino acid deprivation (GCN2), ONC201, and bortezomib (HRI) were tested in FAM210B-overexpressing BJAB cells. FAM210B overexpression inhibited BTM-3566-mediated induction of ATF4 protein expression but had no effect on ATF4 induction by the other treatments. The antitumor activity of BTM-3566 in DLBCL was evaluated in patient-derived DLBCL xenograft (PDX) mouse models. Eight models (n=3/model) were tested, which included GCB and ABC subtypes, and as typical for DLBCL, all had low FAM210B expression. Treatment was initiated with BTM-3566 at 20 mg/kg po daily after tumor volume reached 200 mm3. Complete tumor regression was observed in 79% of the mice with no evidence of tumor progression during the 21-day treatment period. Treatment was well tolerated with only mild weight loss noted. In summary, BTM-3566 induces robust complete regressions in DLBCL PDX models via a unique MoA characterized by apoptosis mediated by HRI induction of the ATF4 ISR in an FAM210B-regulated manner. BTM-3566 has the potential to meaningfully address unmet clinical needs in DLBCL and other B-cell malignancies with low FAM210B expression. Citation Format: Matthew Kostura, Jedd Levine, Alan Cooper, Andy Anantha, Michael Stocum, Michael Luther. BTM-3566 induces complete tumor regression in diffuse large B-cell lymphoma: Regulation by the eIF2α kinase “heme-regulated inhibitor” and the mitochondrial protein FAM210B [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-47.

GZD824 Inhibits GCN2 and Sensitizes Cancer Cells to Amino Acid Starvation Stress.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

The eIF2α kinase HRI in innate immunity, proteostasis, and mitochondrial stress.

The integrated stress response (ISR) is an evolutionary conserved stress response pathway that leads to a global arrest in translation as well as to the expression of specific genes, such as the transcription factor ATF4, to promote cellular recovery. The central nexus of this pathway is the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) by one of the four eIF2α kinases that sense specific cellular stressors. The heme-regulated inhibitor (HRI) is one of these kinases, and it was initially reported to be activated in response to heme deprivation. Nevertheless, further studies have established that cytosolic proteotoxicity, resulting from oxidative or osmotic stress, heat shock, and proteasome inhibition, is the predominant trigger for HRI to induce the ISR. In this review, we present newly identified functions of HRI in innate immunity, proteostasis, and mitochondrial stress. Indeed, HRI-mediated signaling defines a novel cytosolic unfolded protein response (cUPR) required for the proper formation of some innate immune signalosomes and the control of toxic protein aggregates, and this eIF2α kinase also serves as a relay for mitonuclear communication after a mitochondrial stress.

New molecular marker for phylogenetic reconstruction of black yeast-like fungi (Chaetothyriales) with hypothetical EIF2AK2 kinase gene

In eukaryotes, phosphorylation of the α-subunit of eIF2 is a mechanism to adjust cellular gene expression profiles in response to specific signals. The eIF2α kinases are a group of serine–threonine kinases that perform important functions in response to infection, proteotoxicity, and nutrient scavenging. The conserved nature of eIF2α kinases among fungi makes them potential evolutionary markers, which may contribute to deeper understanding of taxonomy and evolution. To date, only few studies are available of eIF2α kinases in black yeasts, which are members of Chaetothyriales containing potential agents of a gamut of major human diseases, such as chromoblastomycosis, phaeohyphomycosis and mycetoma. To establish the phylogenetic validity of sequences of eIF2α kinases hypothetical genes, we compared these genes between members of different classes of fungi, including black yeasts and allies, aiming at evaluation of the phylogeny of this group using an alternative molecular marker, compared to standard ribosomal genes. Trees generated with eIF2α kinase sequences of fungi were compared with those generated by ribosomal internal transcribed spacers (ITS rDNA) sequences from the same species. Sequences used were obtained from the protein Non-redundant database of NCBI, were aligned using CLUSTALX v1.8 and alignments were analyzed with RAxML v8.2.9 on the CIPRES Science Gateway portal. The trees generated had similar topologies, demonstrating that eIF2α kinases hypothetical gene sequences present a coherent reflection of evolution among fungi, compared to trees reconstructed by the use of ribosomal sequences. Our preliminary findings with a limited dataset strongly suggest that the evolution of kinases among black yeasts follows a similar path as revealed by ribosomal data, which underlines the validity of current taxonomy of black yeasts and relatives.

Abstract 5837: Exploring integrated stress response pathway to target MYC-amplified medulloblastoma

Abstract Medulloblastoma (MB) accounts for 20% of diagnosed brain tumors in children. The Group 3 (G3) MB subtype is found to be especially malignant. Current G3 MB treatment regimens provide for 50% 5-year survival rates and are associated with severe side effects, including hearing loss and neurocognitive deficits. G3 MB is characterized by MYC overexpression, which drives elevated protein translation in tumor cells. High expression levels of PERK correlate with poor survival outcome in G3 MB, and knockdown of PERK combined with hypoxic stress induces apoptosis in MB cells. PERK is an eIF2α kinase that responds to ER stress and is one of the key mediators of unfolded protein response. When ER stress is detected, PERK phosphorylates eIF2α to shut down global translation and to initiate integrated stress response. However, there are 3 other eIF2α kinases that work alongside PERK to help cells respond to various stresses. ISRIB is in an integrated stress response inhibitor that blocks P-eIF2α attenuation by eIF2B, thus maintaining high translation levels under stress. Combining ISRIB with stress such as hypoxia induces apoptosis in MB cells. In addition, combination of ISRIB and hypoxia is correlated with increased levels of oxidative stress. Apart from restoring global translation levels, ISRIB can prevent stress granules (SGs) from being formed. High expression of SG proteins such as G3BP1 and YB1 also correlates with poor survival outcome in G3 MB patients. When SG formation is inhibited by knocking down expression of G3BP1 and G3BP2, MB cells undergo higher levels of apoptosis under vincristine induced stress. We show that preventing G3 MB cells from undergoing integrated stress response and inhibiting formation of stress granules induces higher levels of cancer cell death. Overall, combining ISRIB with conventional chemotherapy may enhance the effects of the latter, which is increasingly relevant for patients suffering from G3 MB. Citation Format: Sofya Langman, Poul H. Sorensen. Exploring integrated stress response pathway to target MYC-amplified medulloblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5837.

Abstract 688: Targeting tumor associated host cells by modifiers of integrated endoplasmic reticulum stress response

Abstract Solid tumors are composed of cancer cells, tumor associated host cells (TAHCs) and extracellular matrix components. TAHCs form tumor vasculature, secrete extracellular matrix components, provide protection from host immune-defense, and contribute to therapy resistance. Agents that can abrogate the survival and/or proliferation of both cancer cells and TAHCs would be more potent and less vulnerable to acquired resistance. Solid tumors are under stress due to host immune surveillance, lagging vascularization, and an unstable nascent vasculature that results in nutrients and oxygen restriction. Tumors utilize adaptive pathways including hypoxia activated signaling, integrated endoplasmic reticulum (ER) stress response (IERSR) signaling, and immune evasion for survival and progression. The IERSR is a double-edged sword that is pro-survival if activated transiently but cause cell death if activated in a sustained manner. In particular, sustained activation by eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation (eIF2α-P) arm of the IERSR causes cell death. eIF2α-P reduces overall protein synthesis while inducing expression of some pro-apoptotic and tumor suppressor genes. Because TAHCs actively synthesize proteins in supporting tumor cells, we hypothesized that specific activators of eIF2α kinases will abrogate tumor survival and/or progression by dual effect on TACHs and cancer cells. We tested this hypothesis by evaluating chemical activators of eIF2α kinase heme regulated inhibitor (HRI) in ex-vivo aortic ring angiogenesis assay and against tumor organoids formed by co-culture of macrophages, fibroblasts, endothelial cells, and human breast or melanoma cancer cells. All HRI activators inhibited angiogenesis in the aortic ring assay (ARA) and had profound effects on tumor angiogenesis and macrophages. At sublethal concentration, HRI activators disrupted tumor vasculature and significantly reduced macrophages in the tumor organoids. These agents also significantly inhibited cell migration as measured by wound healing assay and by cell-migration assay of xCELLigence® RTCA DP instrument. These data indicate that HRI activators can target both tumor and TAHCs in a dose dependent manner and support further development of these agents for cancer therapy. Citation Format: Berin Upcin, Daniel Szi-Marton, Erik Henke, Süleyman Ergün, Bertal Huseyin Aktas. Targeting tumor associated host cells by modifiers of integrated endoplasmic reticulum stress response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 688.

Gcn2 eIF2α kinase mediates combinatorial translational regulation through nucleotide motifs and uORFs in target mRNAs.

The protein kinase Gcn2 is a central transducer of nutritional stress signaling important for stress adaptation by normal cells and the survival of cancer cells. In response to nutrient deprivation, Gcn2 phosphorylates eIF2α, thereby repressing general translation while enhancing translation of specific mRNAs with upstream ORFs (uORFs) situated in their 5′-leader regions. Here we performed genome-wide measurements of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysome analyses were combined with microarray measurements to identify gene transcripts whose translation was up-regulated in response to the stress in a Gcn2-dependent manner. We determined that translation is reprogrammed to enhance RNA metabolism and chromatin regulation and repress ribosome synthesis. Interestingly, translation of intron-containing mRNAs was up-regulated. The products of the regulated genes include additional eIF2α kinase Hri2 amplifying the stress signaling and Gcn5 histone acetyl transferase and transcription factors, together altering genome-wide transcription. Unique dipeptide-coding uORFs and nucleotide motifs, such as ‘5′-UGA(C/G)GG-3′, are found in 5′ leader regions of regulated genes and shown to be responsible for translational control.

Pseudorabies virus infection inhibits stress granules formation via dephosphorylating eIF2α

Pseudorabies virus (PRV) is one of the most notorious pathogens in the global pig industry. During infection, viruses may evolve various strategies, such as modulating stress granules (SGs) formation, to create an optimal surroundings for viral replication. However, the interplay between PRV infection and SGs formation remains largely unknown. Here we showed that PRV infection markedly blocked SGs formation induced by sodium arsenate (AS) and DL-Dithiothreitol (DTT). Accordantly, the phosphorylation of eIF2α was markedly inhibited in PRV-infected cells, although two eIF2α kinases double-stranded RNA-activated protein kinase (PKR) and PKR-like ER kinase (PERK) were activated during PRV infection. Furthermore, we also found that the dephosphorylation of eIF2α occurred at the early stage of virus infection but without the elevated production of GADD34 and PP1. Moreover, inhibition of PP1 activity by salubrinal could counteract PRV-mediated eIF2α dephosphorylation partially and inhibit virus replication. Our results revealed that, on the one hand, PRV infection activated eIF2α kinases PKR (latter inhibited) and PERK, and on the other hand, PRV encoded-functions dephosphorylated eIF2α and inhibited SGs formation to facilitate virus replication.

The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression

AbstractReactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and β-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9–guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.

Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell.

Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR) but the underlying triggers remain unclear. We systematically combined acute mitochondrial inhibitors with genetic tools for compartment-specific NADH oxidation to trace mechanisms linking different forms of mitochondrial dysfunction to the ISR in proliferating mouse myoblasts and in differentiated myotubes. In myoblasts, we find that impaired NADH oxidation upon electron transport chain (ETC) inhibition depletes asparagine, activating the ISR via the eIF2α kinase GCN2. In myotubes, however, impaired NADH oxidation following ETC inhibition neither depletes asparagine nor activates the ISR, reflecting an altered metabolic state. ATP synthase inhibition in myotubes triggers the ISR via a distinct mechanism related to mitochondrial inner-membrane hyperpolarization. Our work dispels the notion of a universal path linking mitochondrial dysfunction to the ISR, instead revealing multiple paths that depend both on the nature of the mitochondrial defect and on the metabolic state of the cell.

Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition.

We examined the feedback between the major protein degradation pathway, the ubiquitin-proteasome system (UPS), and protein synthesis in rat and mouse neurons. When protein degradation was inhibited, we observed a coordinate dramatic reduction in nascent protein synthesis in neuronal cell bodies and dendrites. The mechanism for translation inhibition involved the phosphorylation of eIF2α, surprisingly mediated by eIF2α kinase 1, or heme-regulated kinase inhibitor (HRI). Under basal conditions, neuronal expression of HRI is barely detectable. Following proteasome inhibition, HRI protein levels increase owing to stabilization of HRI and enhanced translation, likely via the increased availability of tRNAs for its rare codons. Once expressed, HRI is constitutively active in neurons because endogenous heme levels are so low; HRI activity results in eIF2α phosphorylation and the resulting inhibition of translation. These data demonstrate a novel role for neuronal HRI that senses and responds to compromised function of the proteasome to restore proteostasis.

Dynamic Malignant Wave of Ribosome-Insulted Gut Niche via the Wnt-CTGF/CCN2 Circuit.

SummaryStress-driven ribosome dysfunction triggers an eIF2α-mediated integrated stress response to maintain cellular homeostasis. Among four key eIF2α kinases, protein kinase R (PKR) expression positively associates with poor prognoses for colorectal cancer (CRC) patients. We identified PKR-linked Wnt signaling networks that facilitate early inflammatory niche and epithelial-mesenchymal transitions of tumor tissues in response to ribosomal insults. However, the downstream Wnt signaling target fibrogenic connective tissue growth factor (CTGF/CCN2) regulates the nuclear translocation of β-catenin in a negative feedback manner. Moreover, dwindling expression of the Wnt/β-catenin pathway-regulator CTGF triggers noncanonical Wnt pathway-mediated exacerbation of intestinal cancer progression such as an increase in cancer stemness and acquisition of chemoresistance in the presence of ribosomal insults. The Wnt-CTGF-circuit-associated landscape of oncogenic signaling events was verified with clinical genomic profiling. This ribosome-associated wave of crosstalk between stress and oncogenes provides valuable insight into potential molecular interventions against intestinal malignancies.

GCN2-Like Kinase Modulates Stress Granule Formation During Nutritional Stress in Trypanosoma cruzi.

The integrated stress response in eukaryotic cells is an orchestrated pathway that leads to eukaryotic Initiation Factor 2 alpha subunit (eIF2α) phosphorylation at ser51 and ultimately activates pathways to mitigate cellular damages. Three putative kinases (Tck1, Tck2, and Tck3) are found in the Trypanosoma cruzi genome, the flagellated parasite that causes Chagas disease. These kinases present similarities to other eukaryotic eIF2α kinases, exhibiting a typical insertion loop in the kinase domain of the protein. We found that this insertion loop is conserved among kinase 1 of several T. cruzi strains but differs among various Kinetoplastidae species, suggesting unique roles. Kinase 1 is orthologous of GCN2 of several eukaryotes, which have been implicated in the eIF2α ser51 phosphorylation in situations that mainly affects the nutrients levels. Therefore, we further investigated the responses to nutritional stress of T. cruzi devoid of TcK1 generated by CRISPR/Cas9 gene replacement. In nutrient-rich conditions, replicative T. cruzi epimastigotes depleted of TcK1 proliferate as wild type cells but showed increased levels of polysomes relative to monosomes. Upon nutritional deprivation, the polysomes decreased more than in TcK1 depleted line. However, eIF2α is still phosphorylated in TcK1 depleted line, as in wild type parasites. eIF2α phosphorylation increased at longer incubations times, but KO parasites showed less accumulation of ribonucleoprotein granules containing ATP-dependent RNA helicase involved in mRNA turnover (DHH1) and Poly-A binding protein (PABP1). Additionally, the formation of metacyclic-trypomastigotes is increased in the absence of Tck1 compared to controls. These metacyclics, as well as tissue culture trypomastigotes derived from the TcK1 knockout line, were less infective to mammalian host cells, although replicated faster inside mammalian cells. These results indicate that GCN2-like kinase in T. cruzi affects stress granule formation, independently of eIF2α phosphorylation upon nutrient deprivation. It also modulates the fate of the parasites during differentiation, invasion, and intracellular proliferation.

Proteomic analysis of the ila2 mutant of Arabidopsis links translational regulation with photosynthesis, protein folding and ribosomal proteins

A key component of the conserved GCN (General Control Non-repressive) signalling pathway in plants is the ILITHYIA(ILA) protein, homologous to the yeast GCN1 protein. Similar to yeast and animals, ILA seems to be involved in the activation of the eIF2α kinase GCN2, allowing protein arrest under stress conditions. Recently, it has been reported that, in plants, ILA could be also playing a role in development independent of GCN2. To gain insight into this new function of the ILA protein, we have performed proteomic analysis to identify differentially-expressed proteins in the strong loss-of-function allele of ILA (ila2). ila2 plants present a reduced expression of photosynthetic proteins, and an increased expression of the translational initiation factor eiF5A, ribosomal proteins and heat-shock proteins. These results open new hypothesis to understand the roles of this important translational regulator in plant tissues.

PKZ, a Fish-Unique eIF2α Kinase Involved in Innate Immune Response.

PKZ is a novel and unique eIF2α protein kinase identified in fish. Although PKZ is most homologous to PKR, particularly in the C-terminal catalytic domain, it contains two N-terminal Z-DNA-binding domains (Zα1 and Zα2) instead of the dsRNA binding domains (dsRBDs) in PKR. As a novel member of eIF2α kinase family, the available data suggest that PKZ has some distinct mechanisms for recognition, binding, and B-Z DNA transition. Functionally, PKZ seems to be activated by the binding of Zα to Z-DNA and participates in innate immune responses. In this review, we summarize the recent progress on fish PKZ.

Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation.

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.

Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response (ISR), in which eIF2α phosphorylation induces the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. We found that HRI is the eIF2α kinase necessary and sufficient for this relay. In a genome-wide CRISPRi screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease, and DELE1, a little-characterized protein we found to be associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1, leading to its accumulation in the cytosol, where it interacts with HRI and activates its eIF2α kinase activity. Additionally, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response inducing specific molecular chaperones. Therefore, this pathway is a potential therapeutic target enabling fine-tuning of the ISR for beneficial outcomes in diseases involving mitochondrial dysfunction.