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Although the physiological role of tissue-specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme-regulated eIF2alpha kinase (HRI) in mice. We establish that HRI, which is expre...
Citations
... As expected for an iron-dependent process, we observed a significant decrease in bulk translation in response to iron deficiency. Similar to previous studies in yeast and erythroid cells 20,23,24,42,43 , we show that U-2 OS cells limit global translation by phosphorylating eIF2α and inhibiting mTORC1 signaling when iron is scarce. In yeast, Gcn2 kinase limits protein synthesis during iron deficiency by phosphorylating eIF2α through a mechanism dependent on the uncharged tRNA-sensing Gcn1-Gcn20 complex 21 . ...
... In yeast, Gcn2 kinase limits protein synthesis during iron deficiency by phosphorylating eIF2α through a mechanism dependent on the uncharged tRNA-sensing Gcn1-Gcn20 complex 21 . In red blood cells, HRI kinase senses low heme levels and phosphorylates eIF2α, thereby inhibiting globin synthesis 23 . Although mammalian cells possess four eIF2α kinases, little is known about the kinases responsible for eIF2α phosphorylation upon iron deficiency in other tissues. ...
Protein synthesis is a highly energy-consuming process that is downregulated in response to many environmental stresses or adverse conditions. Studies in the yeast Saccharomyces cerevisiae have shown that bulk translation is inhibited during adaptation to iron deficiency, which is consistent with its requirement for ribosome biogenesis and recycling. Although iron deficiency anemia is the most common human nutritional disorder, how iron modulates translation in mammals is poorly understood. Studies during erythropoiesis have shown that iron bioavailability is coordinated with globin synthesis via bulk translation regulation. However, little is known about the control of translation during iron limitation in other tissues. Here, we investigated how iron depletion affects protein synthesis in human osteosarcoma U-2 OS cells. By adding an extracellular iron chelator, we observed that iron deficiency limits cell proliferation, induces autophagy, and decreases the global rate of protein synthesis. Analysis of specific molecular markers indicates that the inhibition of bulk translation upon iron limitation occurs through the eukaryotic initiation factor eIF2α and mechanistic target of rapamycin (mTOR) pathways. In contrast to other environmental and nutritional stresses, iron depletion does not trigger the assembly of messenger ribonucleoprotein stress granules, which typically form upon polysome disassembly.
... HRI suppresses HbF by elevating ATF4 levels (Huang et al., 2020). In vitro studies have demonstrated that the loss of HRI is well-tolerated in human erythroid cells (Han et al., 2001;Huang et al., 2020), suggesting it as a potential drug target for increasing HbF levels. Therefore, HRI stands out as a molecular target in the pathophysiology of SCD. ...
... HRI suppresses HbF by elevating ATF4 levels [14]. In vitro studies have demonstrated that the loss of HRI is well-tolerated in human erythroid cells [14,15], suggesting it as a potential drug target for increasing HbF levels. Therefore, HRI stands out as a molecular target in the pathophysiology of SCD. ...
Background
Sickle cell disease (SCD) poses a significant health challenge and therapeutic approaches often target fetal hemoglobin (HbF) to ameliorate symptoms. Hydroxyurea, a current therapeutic option for SCD, has shown efficacy in increasing HbF levels. However, concerns about myelosuppression and thrombocytopenia necessitate the exploration of alternative compounds. Heme-regulated inhibitor (HRI) presents a promising target for pharmacological intervention in SCD due to its association with HbF modulation. This study systematically screened compounds for their potential inhibitory functions against HRI.
Methods
Small-molecule compounds from 17 plants commonly utilized in traditional SCD management were subjected to in silico screening against HRI. Molecular docking was performed, and free binding energy calculations were determined using molecular mechanics with generalized born and surface area (MMGBSA). The lead compounds were subjected to molecular dynamics simulation at 100 ns. Computational quantum mechanical modelling of the lead compounds was subsequently performed. We further examined the pharmacodynamics, pharmacokinetic and physiological properties of the identified compounds.
Results
Five potential HRI inhibitors, including kaempferol-3-(2G-glucosyrutinoside), epigallocatechin gallate, tiliroside, myricetin-3-O-glucoside, and cannabiscitrin, with respective docking scores of -16.0, -12.17, -11.37, -11.56 and 11.07 kcal/mol, were identified. The MMGBSA analysis of the complexes yielded free-binding energies of -69.76, -71.17, -60.44, 53.55, and − 55 kcal/mol, respectively. The identified leads were stable within HRI binding pocket for the duration of 100 ns simulation.
Conclusions
The study successfully identified five phytoligands with potential inhibitory effects on HRI, opening avenues for their use as modulators of HbF in SCD patients. This finding holds promise for advancing treatment strategies in SCD. However, additional preclinical analyses are warranted to validate the chemotherapeutic properties of the lead compounds.
... In the past decade, the repertoire of stress inputs detected by the ISR sensor kinases-directly, or indirectly through relay proteins-has expanded considerably. For HRI, it is wellestablished that its kinase domain can directly detect heme to coordinate the translation of globin mRNAs with heme and iron availability (Rafie-Kolpin et al., 2000;Han et al., 2001;Ricketts et al., 2022). However, it can function independently of heme to relay information about mitochondrial stress, ranging from redox imbalances to loss of mitochondrial proteostasis. ...
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.
... Finally, the red cell distribution width (RDW) measures the variability of red blood cell size. There is no information on the correlation between SF and EI in newborns from high-altitude cities, but there is a 21% positive correlation with MCHC and a 25% negative correlation with RDW in pregnant women from high-altitude cities 24 . The main objective of this study was to establish the correlation between SF and EI, as well as with Hb. ...
... The correlation between MCV and MCH as well as the cubic and logarithmic model relating these to SF, in 6-month-old newborns living at 3400 m.a.s.l., are explained by a decrease in the production of glo-Anemia -W. Villamonte-Calanche et al bin chains in the red blood cell due to ID, so there is a lower synthesis of Hb and, consequently, a lower cell volume 24 , therefore, MCH is an early and more sensitive indicator of ID 23 . In the initial phase of anemia due to ID, erythrocytes are of different sizes, between normal and small, consequently, RDW increases 27 and explains the negative correlation observed in our stu- 25 found a lower correlation using Pearson's test between SF and MCV (r = 0.282; p < 0.001) in 2-year-old children living at sea level, which could be explained by a higher frequency of inflammatory pathology in this population, which were not excluded in this study and which would have conditioned a higher ID and increased SF 20 . ...
Objetivo:
Correlacionar la ferritina sérica (FS) con la Hb e IE, y determinar el punto de corte de Hb para predecir DH en lactantes de 6 meses a 3400 m de altitud.
Sujetos y Método:
Se evaluaron 128 lactantes de 6 meses a 3.400 m de altitud. Se consideró variable independiente a la FS. Los IE y la Hb fueron las variables dependientes. La An en el lactante fue definida con una Hb < 13,4 g/dl. La DH se definió por FS < 12 ug/dL. Los datos se procesaron en el programa SPSS® versión 25. Se usó la correlación Spearman para el análisis bivariado. Se construyó la curva ROC para determinar el punto de corte de Hb para DH.
Resultados:
La mayor correlación de FS se observó con la hemoglobina corpuscular media (HCM), rho = 0,449 (p < 0,001) y el volumen corpuscular medio (VCM) rho = 0,423 (p < 0,001). El punto de corte de la Hb, según la FS, que define DH fue de 12,15 g/dL (curva ROC: 0,704; IC 95%: 0,597-0,811; p < 0,001).
Conclusión:
El VCM y la HCM presentaron mejor correlación con la FS. El modelo cúbico y logarítmico son los que representaron mejor estas relaciones respectivamente. La Hb < 12,15 g/dL permite diagnosticar DH en lactantes de 6 meses a 3400 m de altura.
... Показано, что в ходе инфекции EMCV нарушается активация киназы PKR [47], а белок L способен подавлять выработку интерферонов и моделировать каскады MAPK, участвующие в регуляции PKR [8,27,48]. Киназа HRI активируется и фосфорилирует eIF2α при снижении концентрации гема, тем самым регулируя синтез белков в ретикулоцитах [49]. Об активации этой киназы в ходе пикорнавирусной инфекции ничего не известно. ...
Introduction. Infection of cells with encephalomyocarditis virus type 1 (EMCV-1, Cardiovirus A: Picornaviridae) is accompanied by suppression of cellular protein synthesis. The main role in the inhibition of cellular translation is assigned to the L and 2A security proteins. The mechanism of the possible influence of the L protein on cellular translation is unknown. There are hypotheses about the mechanism of influence of 2A protein on the efficiency of cap-dependent translation, which are based on interaction with translation factors and ribosome subunits. However, the available experimental data are contradictory, obtained using different approaches, and do not form a unified model of the interaction between the L and 2A proteins and the cellular translation machinery. Aim. To study the role of L and 2A security proteins in the suppression of translation of cellular proteins and the efficiency of translation and processing of viral proteins in infected cells. Materials and methods. Mutant variants of EMCV-1 were obtained to study the properties of L and 2A viral proteins: Zfmut, which has a defective L; 2A encoding a partially deleted 2A; Zfmut2A containing mutations in both proteins. Translational processes in infected cells were studied by Western-blot and the pulse method of incorporating radioactively labeled amino acids (14C) into newly synthesized proteins, followed by radioautography. Results. The functional inactivation of the 2A protein does not affect the inhibition of cellular protein synthesis. A direct correlation was found between the presence of active L protein and specific inactivation of cellular protein synthesis at an early stage of viral infection. Nonspecific suppression of the translational processes of the infected cell, accompanied by phosphorylation of eIF2, occurs at the late stage of infection. Partial removal of the 2A protein from the EMCV-1 genome does not affect the development of this process, while inactivation of the L protein accelerates the onset of complete inhibition of protein synthesis. Partial deletion of the 2A disrupts the processing of viral capsid proteins. Suppression of L protein functions leads to a decrease in the efficiency of viral translation. Conclusion. A study of the role of EMCV-1 L and 2A proteins during the translational processes of an infected cell, first performed using infectious viral pathogens lacking active L and 2A proteins in one experiment, showed that 2A protein is not implicated in the inhibition of cellular translation in HeLa cells; L protein seems to play an important role not only in the specific inhibition of cellular translation but also in maintaining the efficient synthesis of viral proteins; 2A protein is involved not only in primary but also in secondary processing of EMCV-1 capsid proteins.
... Global translational block occurs in response to a variety of stimuli, including ER stress (Ron, 2002), iron deficiency (Han et al., 2001), amino acid starvation (Dever et al., 1992), infection (Chakrabarti et al., 2012), and hypoxia (Staudacher et al., 2015). This makes sense from a metabolic standpoint, as ribosome synthesis and translation are extremely energy-intensive processes. ...
Diverse acute and chronic injuries induce damage responses in the gastrointestinal (GI) system, and numerous cell types in the gastrointestinal tract demonstrate remarkable resilience, adaptability, and regenerative capacity in response to stress. Metaplasias, such as columnar and secretory cell metaplasia, are well-known adaptations that these cells make, the majority of which are epidemiologically associated with an elevated cancer risk. On a number of fronts, it is now being investigated how cells respond to injury at the tissue level, where diverse cell types that differ in proliferation capacity and differentiation state cooperate and compete with one another to participate in regeneration. In addition, the cascades or series of molecular responses that cells show are just beginning to be understood. Notably, the ribosome, a ribonucleoprotein complex that is essential for translation on the endoplasmic reticulum (ER) and in the cytoplasm, is recognized as the central organelle during this process. The highly regulated management of ribosomes as key translational machinery, and their platform, rough endoplasmic reticulum, are not only essential for maintaining differentiated cell identity, but also for achieving successful cell regeneration after injury. This review will cover in depth how ribosomes, the endoplasmic reticulum, and translation are regulated and managed in response to injury (e.g., paligenosis), as well as why this is essential for the proper adaptation of a cell to stress. For this, we will first discuss how multiple gastrointestinal organs respond to stress through metaplasia. Next, we will cover how ribosomes are generated, maintained, and degraded, in addition to the factors that govern translation. Finally, we will investigate how ribosomes and translation machinery are dynamically regulated in response to injury. Our increased understanding of this overlooked cell fate decision mechanism will facilitate the discovery of novel therapeutic targets for gastrointestinal tract tumors, focusing on ribosomes and translation machinery.
... Four eIF2 kinases (GCN2, HRI, PERK, and PKR) are responsible for the phosphorylation of eIF2α. [38][39][40][41] To elucidate the upstream regulator of eIF2α in CisR cells, we determined whether siGDF15 and siG- UV irradiation, ATP-competitive kinase inhibitors, and oxidative stress. 19,41,[44][45][46][47] Moreover, GDF15 overexpression might increase the oxidative stress. ...
Gastric cancer is a common cancer worldwide, particularly in East Asia. Chemotherapy is used in adjuvant or palliative therapies for gastric cancer. However, subsequent chemoresistance often develops. Growth differentiation factor 15 (GDF15) links to several cancers, but its effect on chemoresistance in gastric cancer remains unclear. Here, we analyzed clinical samples from genetic databases and included patients with gastric cancer. We dissected the regulatory mechanism underlying GDF15-mediated resistance of cisplatin in human gastric cancer cells. We showed that GDF15 serum levels might be a valuable biomarker for predicting prognosis in gastric cancer. The expressions of GDF15 and its receptor glial cell-derived neurotrophic factor family receptor a-like (GFRAL) in gastric tumors are important for malignant progression. Moreover, GDF15 expression is increased in gastric cancer cells with cisplatin resistance, resulting from elevated intracellular glutathione (GSH) and antioxidant activities. Upregulated GDF15 could increase intracellular GSH content by activating the GFRAL-GCN2-eIF2α-ATF4 signaling, enhancing cystine-uptake transporter xCT expression, and contributing biosynthesis of GSH in human gastric cancer cells. In conclusion, our results indicate that GDF15 could induce chemoresistance by upregulating xCT expression and GSH biosynthesis in human gastric cancer cells. Targeting GDF15 could be a promising treatment method for gastric cancer progression.
... Phosphorylation levels of eIF2α in cells cultured with Def 3 ·Fe 3+ were increased compared to addition of hTf, but lower than in presence of aTf. eIF2α can be phosphorylated by the heme-regulated eIF2α-kinase (HRI), which is activated under heme-limited conditions in erythroid cells 18 . As hemoglobin levels were maintained in Def 3 ·Fe 3+ -treated cells, it is possible that the observed increase in p-eIF2α is due to other mechanisms, such as an increase in intracellular oxidative stress levels due to iron overload. ...
Iron, supplemented as iron-loaded transferrin (holotransferrin), is an essential nutrient in mammalian cell cultures, particularly for erythroid cultures. The high cost of human transferrin represents a challenge for large scale production of red blood cells (RBCs) and for cell therapies in general. We evaluated the use of deferiprone, a cell membrane-permeable drug for iron chelation therapy, as an iron carrier for erythroid cultures. Iron-loaded deferiprone (Def3·Fe³⁺, at 52 µmol/L) could eliminate the need for holotransferrin supplementation during in vitro expansion and differentiation of erythroblast cultures to produce large numbers of enucleated RBC. Only the first stage, when hematopoietic stem cells committed to erythroblasts, required holotransferrin supplementation. RBCs cultured in presence of Def3·Fe³⁺ or holotransferrin (1000 µg/mL) were similar with respect to differentiation kinetics, expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Replacement of holotransferrin supplementation by Def3·Fe³⁺ was also successful in cultures of myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). Thus, iron-loaded deferiprone can partially replace holotransferrin as a supplement in chemically defined cell culture medium. This holds promise for a significant decrease in medium cost and improved economic perspectives of the large scale production of red blood cells for transfusion purposes.
... Under normal conditions, HRI binds hemin, which inhibits its protein kinase activity. Heme deficiency relieves inhibition and stimulates kinase activity, activating the ISR (18,21). GCN2 is activated by binding to deacylated transfer RNAs (tRNAs) through its histidyl-tRNA synthetase-related domain, which accumulates in response to amino acid deprivation (22). ...
... HRI is activated by iron deprivation in erythroid cells (21) and by oxidative stress (40), heat shock, osmotic stress, and nitric oxide, among others (41). There were no differences in HRI transcript (p = 0.20) and protein levels (p = 0.22) between controls and SS-patients (Supplementary Figures S1A-C). ...
Introduction
Primary Sjögren’s syndrome (SS) is an autoimmune exocrinopathy that affects the structure and function of salivary and lachrymal glands. Labial salivary gland (LSG) acinar cells from SS patients lose cellular homeostasis and experience endoplasmic reticulum and oxidative stress. The integrated cellular stress response (ISR) is an adaptive pathway essential for restoring homeostasis against various stress-inducing factors, including pro-inflammatory cytokines, and endoplasmic reticulum and oxidative stress. ISR activation leads eIF2α phosphorylation, which transiently blocks protein synthesis while allowing the ATF4 expression, which induces a gene expression program that seeks to optimize cellular recovery. PKR, HRI, GCN2, and PERK are the four sentinel stress kinases that control eIF2α phosphorylation. Dysregulation and chronic activation of ISR signaling have pathologic consequences associated with inflammation.
Methods
Here, we analyzed the activation of the ISR in LSGs of SS-patients and non-SS sicca controls, determining the mRNA, protein, and phosphorylated-protein levels of key ISR components, as well as the expression of some of ATF4 targets. Moreover, we performed a qualitative characterization of the distribution of ISR components in LSGs from both groups and evaluated if their levels correlate with clinical parameters.
Results
We observed that the four ISR sensors are expressed in LSGs of both groups. However, only PKR and PERK showed increased expression and/or activation in LSGs from SS-patients. eIF2α and p-eIF2α protein levels significantly increased in SS-patients; meanwhile components of the PP1c complex responsible for eIF2α dephosphorylation decreased. ATF4 mRNA levels were decreased in LSGs from SS-patients along with hypermethylation of the ATF4 promoter. Despite low mRNA levels, SS-patients showed increased levels of ATF4 protein and ATF4-target genes involved in the antioxidant response. The acinar cells of SS-patients showed increased staining intensity for PKR, p-PKR, p-PERK, p-eIF2α, ATF4, xCT, CHOP, and NRF2. Autoantibodies, focus score, and ESSDAI were correlated with p-PERK/PERK ratio and ATF4 protein levels.
Discussion
In summary, the results showed an increased ISR activation in LSGs of SS-patients. The increased protein levels of ATF4 and ATF4-target genes involved in the redox homeostasis could be part of a rescue response against the various stressful conditions to which the LSGs of SS-patients are subjected and promote cell survival.
