scholarly journals Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ai-Ling Tian ◽  
Qi Wu ◽  
Peng Liu ◽  
Liwei Zhao ◽  
Isabelle Martins ◽  
...  
Keyword(s):  
Stress Response ◽  
Immune Responses ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Serine Residue ◽  
Eif2α Phosphorylation ◽  
Lysosomotropic Agents ◽  
Cultured Human Cells

AbstractThe integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.

scholarly journals eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

2020 ◽  
Author(s):  
Michael Schoof ◽  
Morgane Boone ◽  
Lan Wang ◽  
Rosalie Lawrence ◽  
Adam Frost ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Sites ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Rocking Motion

AbstractThe integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2’s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B’s α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

scholarly journals eIF2B conformation and assembly state regulates the integrated stress response

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael Schoof ◽  
Morgane Boone ◽  
Lan Wang ◽  
Rosalie Lawrence ◽  
Adam Frost ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Sites ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Rocking Motion

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

scholarly journals EMBR-32. INTEGRATED STRESS RESPONSE PLAYS A PRO-SURVIVAL ROLE IN MYC-DRIVEN MEDULLOBLASTOMA

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i12-i13
Author(s):  
Sofya Langman ◽  
Alberto Delaidelli ◽  
Yue Zhou Huang ◽  
Poul Sorensen
Keyword(s):  
Stress Response ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Unfolded Proteins ◽  
Treatment Regimens ◽  
Eif2α Phosphorylation ◽  
Cancer Cell Death

Abstract Medulloblastoma (MB) accounts for 20% of diagnosed brain tumors in children. Group 3 (G3) MB subtype is the most aggressive. Molecularly, G3 MB is characterized by MYC overexpression, which drives elevated mRNA translation in tumor cells. PERK is an eukaryotic translation initiation factor 2 (eIF2α) kinase that inhibits mRNA translation under endoplasmic reticulum (ER) stress conditions, such as in response to accumulation of unfolded proteins. When unfolded proteins accumulate in the ER, activated PERK phosphorylates eIF2α. This shuts down global translation and triggers integrated stress response (ISR) to help cells adapt through selective translation of mRNA encoding pro-survival proteins. High mRNA expression of PERK correlates with poor survival in G3 MB patients. In vitro, combination of ER or hypoxic stress with PERK knockdown induces apoptosis in MB cells. ISRIB is an ISR inhibitor that maintains translation rates despite eIF2α phosphorylation. Combining ISRIB with stress such as hypoxia induces apoptosis in MB cells and prevents accumulation of key ISR mediators such as ATF4. In addition, combination of ISRIB and hypoxia induces oxidative stress. Current G3 MB treatment regimens include vincristine, a known ISR inducer. Combination of ISRIB with vincristine amplifies vincristine-induced apoptosis, potentially suggesting novel therapeutic approach for MB. Our findings show that inhibition of ISR in G3 MB represents a powerful inducer of cancer cell death.

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

2020 ◽  
Vol 117 (20) ◽  
pp. 10935-10945 ◽  
Author(s):  
Shanta Karki ◽  
Kathrina Castillo ◽  
Zhaolan Ding ◽  
Olivia Kerr ◽  
Teresa M. Lamb ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nutrient Metabolism ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
The Impact

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.

scholarly journals Proteostasis in dendritic cells is controlled by the PERK signaling axis independently of ATF4

2020 ◽  
Vol 4 (2) ◽  
pp. e202000865
Author(s):  
Andreia Mendes ◽  
Julien P Gigan ◽  
Christian Rodriguez Rodrigues ◽  
Sébastien A Choteau ◽  
Doriane Sanseau ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Stress Response ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Translation Arrest ◽  
Eif2α Phosphorylation ◽  
Signaling Axis ◽  
Stressed Cells ◽  
Cytoskeleton Dynamics

In stressed cells, phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation and gene expression known as the integrated stress response. We show here that DCs are characterized by high eIF2α phosphorylation, mostly caused by the activation of the ER kinase PERK (EIF2AK3). Despite high p-eIF2α levels, DCs display active protein synthesis and no signs of a chronic integrated stress response. This biochemical specificity prevents translation arrest and expression of the transcription factor ATF4 during ER-stress induction by the subtilase cytotoxin (SubAB). PERK inactivation, increases globally protein synthesis levels and regulates IFN-β expression, while impairing LPS-stimulated DC migration. Although the loss of PERK activity does not impact DC development, the cross talk existing between actin cytoskeleton dynamics; PERK and eIF2α phosphorylation is likely important to adapt DC homeostasis to the variations imposed by the immune contexts.

scholarly journals Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Carmela Sidrauski ◽  
Jordan C Tsai ◽  
Martin Kampmann ◽  
Brian R Hearn ◽  
Punitha Vedantham ◽  
...  
Keyword(s):  
Stress Response ◽  
Translational Control ◽  
Control Point ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Eif2α Phosphorylation ◽  
Symmetric Molecule ◽  
Invaluable Tool

The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.

scholarly journals An inhibitor of HIV-1 protease modulates constitutive eIF2α dephosphorylation to trigger a specific integrated stress response

2015 ◽  
Vol 113 (2) ◽  
pp. E117-E126 ◽  
Author(s):  
Aude De Gassart ◽  
Bojan Bujisic ◽  
Léa Zaffalon ◽  
Laurent A. Decosterd ◽  
Antonia Di Micco ◽  
...  
Keyword(s):  
Stress Response ◽  
Stress Responses ◽  
Translation Initiation Factor ◽  
Hiv Protease ◽  
Initiation Factor ◽  
Hiv Protease Inhibitors ◽  
Integrated Stress Response ◽  
Α Subunit ◽  
Eif2α Phosphorylation ◽  
Program Characteristic

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.

scholarly journals Quercetin Regulates the Integrated Stress Response to Improve Memory

2019 ◽  
Vol 20 (11) ◽  
pp. 2761 ◽  
Author(s):  
Toshiyuki Nakagawa ◽  
Kazunori Ohta
Keyword(s):  
Stress Response ◽  
Mouse Model ◽  
Therapeutic Potential ◽  
Amyloid Β ◽  
Stress Conditions ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Α Subunit ◽  
Model Of Alzheimer’S Disease

The initiation of protein synthesis is suppressed under several stress conditions, inducing phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2α), thereby inactivating the GTP-GDP recycling protein eIF2B. By contrast, the mammalian activating transcription factor 4 (ATF4, also known as cAMP response element binding protein 2 (CREB2)) is still translated under stress conditions. Four protein kinases (general control nonderepressible-2 (GCN2) kinase, double-stranded RNA-activated protein kinase (PKR), PKR-endoplasmic reticulum (ER)-related kinase (PERK), and heme-regulated inhibitor kinase (HRI)) phosphorylate eIF2α in the presence of stressors such as amino acid starvation, viral infection, ER stress, and heme deficiency. This signaling reaction is known as the integrated stress response (ISR). Here, we review ISR signaling in the brain in a mouse model of Alzheimer’s disease (AD). We propose that targeting ISR signaling with quercetin has therapeutic potential, because it suppresses amyloid-β (Aβ) production in vitro and prevents cognitive impairments in a mouse model of AD.

scholarly journals The Integrated Stress Response and Phosphorylated Eukaryotic Initiation Factor 2α in Neurodegeneration

2020 ◽  
Vol 79 (2) ◽  
pp. 123-143 ◽  
Author(s):  
Sarah Bond ◽  
Claudia Lopez-Lloreda ◽  
Patrick J Gannon ◽  
Cagla Akay-Espinoza ◽  
Kelly L Jordan-Sciutto
Keyword(s):  
Stress Response ◽  
Molecular Mechanisms ◽  
Protein Translation ◽  
Cell Types ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Eukaryotic Initiation Factor ◽  
Pharmacological Agents ◽  
Eif2α Phosphorylation

Abstract The proposed molecular mechanisms underlying neurodegenerative pathogenesis are varied, precluding the development of effective therapies for these increasingly prevalent disorders. One of the most consistent observations across neurodegenerative diseases is the phosphorylation of eukaryotic initiation factor 2α (eIF2α). eIF2α is a translation initiation factor, involved in cap-dependent protein translation, which when phosphorylated causes global translation attenuation. eIF2α phosphorylation is mediated by 4 kinases, which, together with their downstream signaling cascades, constitute the integrated stress response (ISR). While the ISR is activated by stresses commonly observed in neurodegeneration, such as oxidative stress, endoplasmic reticulum stress, and inflammation, it is a canonically adaptive signaling cascade. However, chronic activation of the ISR can contribute to neurodegenerative phenotypes such as neuronal death, memory impairments, and protein aggregation via apoptotic induction and other maladaptive outcomes downstream of phospho-eIF2α-mediated translation inhibition, including neuroinflammation and altered amyloidogenic processing, plausibly in a feed-forward manner. This review examines evidence that dysregulated eIF2a phosphorylation acts as a driver of neurodegeneration, including a survey of observations of ISR signaling in human disease, inspection of the overlap between ISR signaling and neurodegenerative phenomenon, and assessment of recent encouraging findings ameliorating neurodegeneration using developing pharmacological agents which target the ISR. In doing so, gaps in the field, including crosstalk of the ISR kinases and consideration of ISR signaling in nonneuronal central nervous system cell types, are highlighted.

Inhibition of mitochondrial translation overcomes venetoclax resistance in AML through activation of the integrated stress response

2019 ◽  
Vol 11 (516) ◽  
pp. eaax2863 ◽  
Author(s):  
David Sharon ◽  
Severine Cathelin ◽  
Sara Mirali ◽  
Justin M. Di Trani ◽  
David J. Yanofsky ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Agent ◽  
Mitochondrial Protein ◽  
B Cell Lymphoma ◽  
Atp Depletion ◽  
Integrated Stress Response ◽  
Mitochondrial Translation ◽  
A Genome

Venetoclax is a specific B cell lymphoma 2 (BCL-2) inhibitor with promising activity against acute myeloid leukemia (AML), but its clinical efficacy as a single agent or in combination with hypomethylating agents (HMAs), such as azacitidine, is hampered by intrinsic and acquired resistance. Here, we performed a genome-wide CRISPR knockout screen and found that inactivation of genes involved in mitochondrial translation restored sensitivity to venetoclax in resistant AML cells. Pharmacologic inhibition of mitochondrial protein synthesis with antibiotics that target the ribosome, including tedizolid and doxycycline, effectively overcame venetoclax resistance. Mechanistic studies showed that both tedizolid and venetoclax suppressed mitochondrial respiration, with the latter demonstrating inhibitory activity against complex I [nicotinamide adenine dinucleotide plus hydrogen (NADH) dehydrogenase] of the electron transport chain (ETC). The drugs cooperated to activate a heightened integrated stress response (ISR), which, in turn, suppressed glycolytic capacity, resulting in adenosine triphosphate (ATP) depletion and subsequent cell death. Combination treatment with tedizolid and venetoclax was superior to either agent alone in reducing leukemic burden in mice engrafted with treatment-resistant human AML. The addition of tedizolid to azacitidine and venetoclax further enhanced the killing of resistant AML cells in vitro and in vivo. Our findings demonstrate that inhibition of mitochondrial translation is an effective approach to overcoming venetoclax resistance and provide a rationale for combining tedizolid, azacitidine, and venetoclax as a triplet therapy for AML.

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