PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz.
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ABSTRACT:
SUBMITTER: Crespillo-Casado A
PROVIDER: S-EPMC5429092 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveVanishing white matter (VWM) is a leukodystrophy, characterized by stress-sensitive neurological deterioration and premature death. It is currently without curative treatment. It is caused by bi-allelic pathogenic variants in the genes encoding eukaryotic initiation factor 2B (eIF2B). eIF2B is essential for the regulation of the integrated stress response (ISR), a physiological response to cellular stress. Preclinical studies on VWM mouse models revealed that deregulated ISR is key in the pathophysiology of VWM and an effective treatment target. Guanabenz, an α2-adrenergic agonist, attenuates the ISR and has beneficial effects on VWM neuropathology. The current study aimed at elucidating guanabenz's disease-modifying potential and mechanism of action in VWM mice. Sephin1, an ISR-modulating guanabenz analog without α2-adrenergic agonistic properties, was included to separate effects on the ISR from α2-adrenergic effects.MethodsWild-type and VWM mice were subjected to placebo, guanabenz or sephin1 treatments. Effects on clinical signs, neuropathology, and ISR deregulation were determined. Guanabenz's and sephin1's ISR-modifying effects were tested in cultured cells that expressed or lacked the α2-adrenergic receptor.ResultsGuanabenz improved clinical signs, neuropathological hallmarks, and ISR regulation in VWM mice, but sephin1 did not. Guanabenz's effects on the ISR in VWM mice were not replicated in cell cultures and the contribution of α2-adrenergic effects on the deregulated ISR could therefore not be assessed.InterpretationGuanabenz proved itself as a viable treatment option for VWM. The exact mechanism through which guanabenz exerts its ameliorating impact on VWM requires further studies. Sephin1 is not simply a guanabenz replacement without α2-adrenergic effects.
| S-EPMC9380178 | biostudies-literature
Project description:Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, T cell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific T cells was inhibited by Sephin1. A special TCR + macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.
| S-EPMC9883195 | biostudies-literature
Project description:Integrated stress responses (ISR) may lead to cell death and tissue degeneration via eukaryotic translation initiation factor 2 α (eIF2α)-mediated signaling. Alleviating ISR by modulating eIF2α phosphorylation can reduce the symptoms associated with various diseases. Guanabenz is known to elevate the phosphorylation level of eIF2α and reduce pro-inflammatory responses. However, the mechanism of its action is not well understood. In this study, we investigated the signaling pathway through which guanabenz induces anti-inflammatory effects in immune cells, in particular macrophages. Genome-wide mRNA profiling followed by principal component analysis predicted that colony stimulating factor 2 (Csf2, or GM-CSF as granulocyte macrophage colony stimulating factor) is involved in the responses to guanabenz. A partial silencing of Csf2 or eIF2α by RNA interference revealed that Interleukin-6 (IL6), Csf2, and Cyclooxygenase-2 (Cox2) are downregulated by guanabenz-driven phosphorylation of eIF2α. Although expression of IL1β and Tumor Necrosis Factor-α (TNFα) was suppressed by guanabenz, their downregulation was not directly mediated by eIF2α signaling. Collectively, the result herein indicates that anti-inflammatory effects by guanabenz are mediated by not only eIF2α-dependent but also eIF2α-independent signaling.
| S-EPMC4881500 | biostudies-literature
Project description:The aminoguanidine compound robenidine is widely used as an antibiotic for the control of coccidiosis, a protozoal infection in poultry and rabbits. Interestingly, robenidine is structurally similar to guanabenz (analogs), which are currently undergoing clinical trials as cytoprotective agents for the management of neurodegenerative diseases. Here we show that robenidine and guanabenz protect cells from a tunicamycin-induced unfolded protein response to a similar degree. Both compounds also reduced the tumor necrosis factor ?-induced activation of NF-?B. The cytoprotective effects of guanabenz (analogs) have been explained previously by their ability to maintain eIF2? phosphorylation by allosterically inhibiting protein phosphatase PP1:PPP1R15A. However, using a novel split-luciferase-based protein-protein interaction assay, we demonstrate here that neither robenidine nor guanabenz disrupt the interaction between PPP1R15A and either PP1 or eIF2? in intact cells. Moreover, both drugs also inhibited the unfolded protein response in cells that expressed a nonphosphorylatable mutant (S51A) of eIF2?. Our results identify robenidine as a PP1:PPP1R15A-independent cytoprotective compound that holds potential for the management of protein misfolding-associated diseases.
| S-EPMC6737217 | biostudies-literature
Project description:Sephin1 is the selective inhibitor of Protein Phosphatase 1 Regulatory Subunit 15A (PPP1R15A), which is an important factor in the integrated stress response (ISR) of mammalian cells. Sephin1 can promote ISR by inhibiting the dephosphorylation of eIF2α, and preventing neurodegenerative disease occurrence in mice. However, we found that Sephin1 plays a protumorigenic role in mouse tumor models by suppressing the antitumor immune activities, which was highly relevant to the function of PPP1R15A. To fully understand the mechanism of Sephin1 to the antitumor immunity, we designed this experiment to explore the function of PPP1R15A and Sephin1 in the single-cell expression level. To understand effect of injection of Sephin1 to the immune cells in blood and tumor microenvironment in mice, we injected Sephin1 to mice intraperitoneally, and then subcutaneously inoculated with B16F1 cells. Blood before/after the tumor development and tumor tissues were collected and immune cells were isolated for single-cell sequencing and bioinformatics analysis afterwards. We found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in several tumor-killing immune cell populations, including CD8+ T cells, NK cells and NKT cells. The expression levels of cytotoxicity-related genes in lymphocytes were altered upon Sephin1 treatment. Additionally, TCR repertoire analysis demonstrated that the clonal expansion of tumor-specific T cells was inhibited by Sephin1, resulting in a compromised antitumor immune response. A special TCR+ macrophage subtype was identified to be significantly depleted upon Sephin1 treatment, implying that this type of macrophage plays a key antitumor role in the tumor microenvironment. The effects of Sephin1 were also verified in in vitro experiments and studies of other tumor cell lines. Taken together, these data suggest that inhibiting PPP1R15A promotes tumorigenesis by inducing adverse immune suppression. PPP1R15A has the potential to be an effective target for tumor therapy. Besides, a macrophage subtype, the TCR+ macrophages, played an important role in the anti-tumor immunity and can also be significantly inhibited by Sephin1.
2022-12-14 | GSE220656 | GEO
Project description:Acid-sensing ion channels (ASICs) are voltage-independent cation channels that detect decreases in extracellular pH. Dysregulation of ASICs underpins a number of pathologies. Of particular interest is ASIC3, which is recognised as a key sensor of acid-induced pain and is important in the establishment of pain arising from inflammatory conditions, such as rheumatoid arthritis. Thus, the identification of new ASIC3 modulators and the mechanistic understanding of how these compounds modulate ASIC3 could be important for the development of new strategies to counteract the detrimental effects of dysregulated ASIC3 activity in inflammation. Here, we report the identification of novel ASIC3 modulators based on the ASIC3 agonist, 2-guanidine-4-methylquinazoline (GMQ). Through a GMQ-guided in silico screening of Food and Drug administration (FDA)-approved drugs, 5 compounds were selected and tested for their modulation of rat ASIC3 (rASIC3) using whole-cell patch-clamp electrophysiology. Of the chosen drugs, guanabenz (GBZ), an ?2-adrenoceptor agonist, produced similar effects to GMQ on rASIC3, activating the channel at physiological pH (pH 7.4) and potentiating its response to mild acidic (pH 7) stimuli. Sephin1, a GBZ derivative that lacks ?2-adrenoceptor activity, has been proposed to act as a selective inhibitor of a regulatory subunit of the stress-induced protein phosphatase 1 (PPP1R15A) with promising therapeutic potential for the treatment of multiple sclerosis. However, we found that like GBZ, sephin1 activates rASIC3 at pH 7.4 and potentiates its response to acidic stimulation (pH 7), i.e. sephin1 is a novel modulator of rASIC3. Furthermore, docking experiments showed that, like GMQ, GBZ and sephin1 likely interact with the nonproton ligand sensor domain of rASIC3. Overall, these data demonstrate the utility of computational analysis for identifying novel ASIC3 modulators, which can be validated with electrophysiological analysis and may lead to the development of better compounds for targeting ASIC3 in the treatment of inflammatory conditions.
| S-EPMC7068650 | biostudies-literature
Project description:Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inducer of cancer cell death that holds promise in cancer therapy. Cancer cells are more susceptible than normal cells to the cell-death-inducing effects of TRAIL. However, a variety of cancer cells are resistant to TRAIL through complex mechanisms. Here, we investigate the effects of inhibition of eukaryotic initiation factor 2 subunit α (eIF2α) dephosphorylation on TRAIL-induced apoptosis in hepatoma cells. Treatment of hepatoma cells with salubrinal, an inhibitor of eIF2α dephosphorylation, enhances TRAIL-induced eIF2α phosphorylation, CCAAT/enhancer-binding protein homologous protein (CHOP) expression and caspase activation. Salubrinal enhances TRAIL-induced apoptosis, which could be abrogated by caspase inhibitor. Overexpression of phosphomimetic eIF2α (S51D) enhances TRAIL-induced CHOP expression, caspase 7 and PARP cleavage and apoptosis. By contrast, overexpression of phosphodeficient eIF2α (S51A) abrogates the stimulation of TRAIL-induced apoptosis by salubrinal. Moreover, knockdown of growth arrest and DNA damage-inducible protein 34 (GADD34), which recruits protein phosphatase 1 to dephosphorylate eIF2α, enhances TRAIL-induced eIF2α phosphorylation, CHOP expression, caspase activation and apoptosis. Furthermore, the sensitization of hepatoma cells to TRAIL by salubrinal is dependent on CHOP. Knockdown of CHOP abrogates the stimulation of TRAIL-induced caspase activation and apoptosis by salubrinal. Combination of salubrinal and TRAIL leads to increased expression of Bim, a CHOP-regulated proapoptotic protein. Bim knockdown blunts the stimulatory effect of salubrinal on TRAIL-induced apoptosis. Collectively, these findings suggest that inhibition of eIF2α dephosphorylation may lead to synthetic lethality in TRAIL-treated hepatoma cells.
| S-EPMC3944242 | biostudies-literature
Project description:Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x(1-2)Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.
| S-EPMC4500263 | biostudies-other
Project description:We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.
| S-EPMC9462064 | biostudies-literature
Project description:Inhibitors of the HIV aspartyl protease [HIV protease inhibitors (HIV-PIs)] are the cornerstone of treatment for HIV. Beyond their well-defined antiretroviral activity, these drugs have additional effects that modulate cell viability and homeostasis. However, little is known about the virus-independent pathways engaged by these molecules. Here we show that the HIV-PI Nelfinavir decreases translation rates and promotes a transcriptional program characteristic of the integrated stress response (ISR). Mice treated with Nelfinavir display hallmarks of this stress response in the liver, including α subunit of translation initiation factor 2 (eIF2α) phosphorylation, activating transcription factor-4 (ATF4) induction, and increased expression of known downstream targets. Mechanistically, Nelfinavir-mediated ISR bypassed direct activation of the eIF2α stress kinases and instead relied on the inhibition of the constitutive eIF2α dephosphorylation and down-regulation of the phophatase cofactor CReP (Constitutive Repressor of eIF2α Phosphorylation; also known as PPP1R15B). These findings demonstrate that the modulation of eIF2α-specific phosphatase cofactor activity can be a rheostat of cellular homeostasis that initiates a functional ISR and suggest that the HIV-PIs could be repositioned as therapeutics in human diseases to modulate translation rates and stress responses.
| S-EPMC4720296 | biostudies-literature