Protein synthesis persists during necrotic cell death

Cell death is an intrinsic part of metazoan development and mammalian immune regulation. Whereas the molecular events orchestrating apoptosis have been characterized extensively, little is known about the biochemistry of necrotic cell death. Here, we show that, in contrast to apoptosis, the induction of necrosis does not lead to the shut down of protein synthesis. The rapid drop in protein synthesis observed in apoptosis correlates with caspase-dependent breakdown of eukaryotic translation initiation factor (eIF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate eIF2-α. In necrosis induced by tumor necrosis factor, double-stranded RNA, or viral infection, de novo protein synthesis persists and 28S ribosomal RNA fragmentation, eIF2-α phosphorylation, and proteolytic activation of PKR are absent. Collectively, these results show that, in contrast to apoptotic cells, necrotic dying cells retain the opportunity to synthesize proteins.

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Double-Stranded RNA-Activated Protein Kinase (PKR) Is Negatively Regulated by 60S Ribosomal Subunit Protein L18

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1116 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1116-1125 ◽

Cited By ~ 61

Author(s):

Kotlo U. Kumar ◽

Sri P. Srivastava ◽

Randal J. Kaufman

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Cell Growth ◽

Ribosomal Subunit ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Cancer Tissue ◽

Double Stranded Rna ◽

Dsrna Binding

ABSTRACT The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2α), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2α in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2α phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.

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Molecular Cross-talk between MEK1/2 and mTOR Signaling during Recovery of 293 Cells from Hypertonic Stress

Journal of Biological Chemistry ◽

10.1074/jbc.m404945200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 46023-46034 ◽

Cited By ~ 41

Author(s):

Susanne Naegele ◽

Simon J. Morley

Keyword(s):

Protein Synthesis ◽

Ribosomal Protein ◽

Signaling Pathways ◽

De Novo ◽

Human Kidney ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Hypertonic Stress ◽

Salt Shock ◽

Ribosomal Protein S6

To investigate the role for initiation factor phosphorylation inde novotranslation, we have studied the recovery of human kidney cells from hypertonic stress. Previously, we have demonstrated that hypertonic shock causes a rapid inhibition of protein synthesis, the disaggregation of polysomes, the dephosphorylation of eukaryotic translation initiation factor (eIF)4E, 4E-BP1, and ribosomal protein S6, and increased association of 4E-BP1 with eIF4E. The return of cells to isotonic medium promotes a transient activation of Erk1/2 and the phosphorylation of initiation factors, promoting an increase in protein synthesis that is independent of a requirement for eIF4E phosphorylation. Asde novotranslation is associated with the phosphorylation of 4E-BP1, we have investigated the role of the signaling pathways required for this event by the use of cell-permeable inhibitors. Surprisingly, although rapamycin, RAD001, wortmannin, and LY294002 inhibited the phosphorylation of 4E-BP1 and its release from eIF4E, they did not prevent the recovery of translation rates. These data suggest that only a small proportion of the available eIF4F complex is required for maximal translation rates under these conditions. Similarly, prevention of Erk1/2 activity alone with low concentrations of PD184352 did not impinge uponde novotranslation until later times of recovery from salt shock. However, U0126, which prevented the phosphorylation of Erk1/2, ribosomal protein S6, TSC2, and 4E-BP1, attenuatedde novoprotein synthesis in recovering cells. These results indicate that the phosphorylation of 4E-BP1 is mediated by both phosphatidylinositol 3-kinase-dependent rapamycin-sensitive and Erk1/2-dependent signaling pathways and that activation of either pathway in isolation is sufficient to promotede novotranslation.

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Murine Cytomegalovirus m142 and m143 Are both Required To Block Protein Kinase R-Mediated Shutdown of Protein Synthesis

Journal of Virology ◽

10.1128/jvi.00908-06 ◽

2006 ◽

Vol 80 (20) ◽

pp. 10181-10190 ◽

Cited By ~ 55

Author(s):

Ralitsa S. Valchanova ◽

Marcus Picard-Maureau ◽

Matthias Budt ◽

Wolfram Brune

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Rna Binding ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Murine Cytomegalovirus ◽

Deletion Mutants ◽

Sequence Motifs ◽

Protein Kinase R ◽

Double Stranded Rna

ABSTRACT Cytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse functions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential for virus replication, but their functions have remained unknown. In the present study, we deleted the essential US22 family genes, m142 and m143, from the MCMV genome and propagated the mutant viruses on complementing cells. The m142 and the m143 deletion mutants were both unable to replicate in noncomplementing cells at low and high multiplicities of infection. In cells infected with the deletion mutants, viral immediate-early and early proteins were expressed, but viral DNA replication and synthesis of the late-gene product glycoprotein B were inhibited, even though mRNAs of late genes were present. Global protein synthesis was impaired in these cells, which correlated with phosphorylation of the double-stranded RNA-dependent protein kinase R (PKR) and its target protein, the eukaryotic translation initiation factor 2α, suggesting that m142 and m143 are necessary to block the PKR-mediated shutdown of protein synthesis. Replication of the m142 and m143 knockout mutants was partially restored by expression of the human cytomegalovirus TRS1 gene, a known double-stranded-RNA-binding protein that inhibits PKR activation. These results indicate that m142 and m143 are both required for inhibition of the PKR-mediated host antiviral response.

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Oleanolic acid rich solubilized triterpene extracts from mistletoe induce apoptotic and necrotic cell death of murine B16. F10 melanoma cells

Planta Medica ◽

10.1055/s-0029-1234600 ◽

2009 ◽

Vol 75 (09) ◽

Author(s):

CM Strüh ◽

S Jäger ◽

CM Schempp ◽

T Jakob ◽

A Scheffler ◽

...

Keyword(s):

Cell Death ◽

Oleanolic Acid ◽

Melanoma Cells ◽

Necrotic Cell Death ◽

Necrotic Cell

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KRT8 (keratin 8) attenuates necrotic cell death by facilitating mitochondrial fission-mediated mitophagy through interaction with PLEC (plectin)

Autophagy ◽

10.1080/15548627.2021.1897962 ◽

2021 ◽

pp. 1-18

Author(s):

Ahruem Baek ◽

Sumin Son ◽

Yu Mi Baek ◽

Dong-Eun Kim

Keyword(s):

Cell Death ◽

Mitochondrial Fission ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Keratin 8

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Correction of eIF2-dependent defects in brain protein synthesis, synaptic plasticity, and memory in mouse models of Alzheimer’s disease

Science Signaling ◽

10.1126/scisignal.abc5429 ◽

2021 ◽

Vol 14 (668) ◽

pp. eabc5429

Author(s):

Mauricio M. Oliveira ◽

Mychael V. Lourenco ◽

Francesco Longo ◽

Nicole P. Kasica ◽

Wenzhong Yang ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Protein Synthesis ◽

Synaptic Plasticity ◽

Translation Initiation ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Postmortem Brain Tissue ◽

Phosphorylated Eif2α

Neuronal protein synthesis is essential for long-term memory consolidation, and its dysregulation is implicated in various neurodegenerative disorders, including Alzheimer’s disease (AD). Cellular stress triggers the activation of protein kinases that converge on the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which attenuates mRNA translation. This translational inhibition is one aspect of the integrated stress response (ISR). We found that postmortem brain tissue from AD patients showed increased phosphorylation of eIF2α and reduced abundance of eIF2B, another key component of the translation initiation complex. Systemic administration of the small-molecule compound ISRIB (which blocks the ISR downstream of phosphorylated eIF2α) rescued protein synthesis in the hippocampus, measures of synaptic plasticity, and performance on memory-associated behavior tests in wild-type mice cotreated with salubrinal (which inhibits translation by inducing eIF2α phosphorylation) and in both β-amyloid-treated and transgenic AD model mice. Thus, attenuating the ISR downstream of phosphorylated eIF2α may restore hippocampal protein synthesis and delay cognitive decline in AD patients.

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Targeting Necrosis: Elastase-like Protease Inhibitors Curtail Necrotic Cell Death Both In Vitro and in Three In Vivo Disease Models

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.0c01683 ◽

2021 ◽

Vol 64 (3) ◽

pp. 1510-1523

Author(s):

Boris Khalfin ◽

Alexandra Lichtenstein ◽

Amnon Albeck ◽

Ilana Nathan

Keyword(s):

Cell Death ◽

Protease Inhibitors ◽

Disease Models ◽

Necrotic Cell Death ◽

Necrotic Cell

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The Leader Protein of Theiler's Virus Prevents the Activation of PKR

Journal of Virology ◽

10.1128/jvi.01010-19 ◽

2019 ◽

Vol 93 (19) ◽

Cited By ~ 4

Author(s):

Fabian Borghese ◽

Frédéric Sorgeloos ◽

Teresa Cesaro ◽

Thomas Michiels

Keyword(s):

Stress Granule ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Wild Type Virus ◽

Granule Formation ◽

Double Stranded Rna ◽

Eif2α Phosphorylation ◽

Leader Protein ◽

Infected Cells ◽

L Proteins

ABSTRACT Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler’s murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA. IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler’s virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.

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