scholarly journals Higher-order phosphatase–substrate contacts terminate the integrated stress response

2021 ◽  
Vol 28 (10) ◽  
pp. 835-846
Author(s):  
Yahui Yan ◽  
Heather P. Harding ◽  
David Ron
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Higher Order ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Composite Surface ◽  
Regulatory Subunits

AbstractMany regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c–PPP1R15A–G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR.

scholarly journals Higher order phosphatase-substrate contacts terminate the Integrated Stress Response

2021 ◽  
Author(s):  
Yahui Yan ◽  
Heather P Harding ◽  
David Ron
Keyword(s):  
Stress Response ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Higher Order ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Composite Surface ◽  
Regulatory Subunits ◽  
Eif2α Kinase

Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits are known to engage PP1c's surface, locally affecting flexible phosphopeptides access to the active site. However, catalytic efficiency of holophosphatases towards their natively-folded phosphoprotein substrates is largely unexplained. Here we present a Cryo-EM structure of the tripartite PP1c/PPP1R15A/G-actin holophosphatase that terminates signalling in the Integrated Stress Response (ISR) in pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin's role in eIF2α dephosphorylation is supported crystallographically by the structure of the binary PPP1R15A-G-actin complex, and by biochemical and genetic confirmation of the essential role of PPP1R15A-G-actin contacts to eIF2αP dephosphorylation. In the pre-dephosphorylation CryoEM complex, G-actin aligns the catalytic and regulatory subunits, creating a composite surface that engages eIF2α's N-terminal domain to position the distant phosphoserine-51 at the active site. eIF2α residues specifying affinity for the holophosphatase are confirmed here to make critical contacts with the eIF2α kinase PERK. Thus, a convergent process of higher-order substrate recognition specifies functionally-antagonistic phosphorylation and dephosphorylation in the ISR.

scholarly journals Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Schoof ◽  
Lan Wang ◽  
J. Zachery Cogan ◽  
Rosalie E. Lawrence ◽  
Morgane Boone ◽  
...  
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Viral Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Double Stranded Rna ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor Eif2

AbstractViral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2’s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five ‘aromatic fingers’. NSs binding preserves eIF2B activity by maintaining eIF2B’s conformation in its active A-State.

scholarly journals Viral Evasion of the Integrated Stress Response Through Antagonistic eIF2-P Mimicry

2021 ◽  
Author(s):  
Michael Schoof ◽  
Lan Wang ◽  
J Zachery Cogan ◽  
Rosalie Lawrence ◽  
Morgane Boone ◽  
...  
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Viral Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Double Stranded Rna ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor Eif2

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2's dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 43 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα; mediated by five 'aromatic fingers'. NSs binding preserves eIF2B activity by maintaining eIF2B's conformation in its active A-State.;

scholarly journals Mutagenesis screen uncovers lifespan extension through integrated stress response inhibition without reduced mRNA translation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maxime J. Derisbourg ◽  
Laura E. Wester ◽  
Ruth Baddi ◽  
Martin S. Denzel
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Lifespan Extension ◽  
Protein Homeostasis ◽  
Integrated Stress Response ◽  
Mutagenesis Screen

AbstractProtein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B. However, the role of the ISR during aging remains poorly understood. Using a genomic mutagenesis screen for longevity inCaenorhabditis elegans, we define a role of eIF2 modulation in aging. By inhibiting the ISR, dominant mutations in eIF2B enhance protein homeostasis and increase lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolong lifespan. Lifespan extension through impeding the ISR occurs without a reduction in overall protein synthesis. Instead, we observe changes in the translational efficiency of a subset of mRNAs, of which the putative kinasekin-35is required for lifespan extension. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging.

scholarly journals eIF2B extends lifespan through inhibition of the integrated stress response

2020 ◽  
Author(s):  
Maxime Derisbourg ◽  
Laura Wester ◽  
Ruth Baddi ◽  
Martin S. Denzel
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Protein Homeostasis ◽  
Integrated Stress Response ◽  
Nucleotide Exchange

AbstractProtein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B. However, the role of the ISR during aging remains unexplored. Using a genomic screen in Caenorhabditis elegans, we discovered a role of eIF2B and the eIF2 kinases in longevity. By limiting the ISR, these mutations enhanced protein homeostasis and increased lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolonged lifespan. Lifespan extension through ISR inhibition occurred without changes in overall protein synthesis, and depended on enhanced translational efficiency of the kinase KIN-35. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging.

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 TgIF2K-B is an eIF2α kinase in Toxoplasma gondii that responds to oxidative stress and optimizes pathogenicity

2020 ◽  
Author(s):  
Leonardo Augusto ◽  
Jennifer Martynowicz ◽  
Parth H. Amin ◽  
Kenneth R. Carlson ◽  
Ronald C. Wek ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Toxoplasma Gondii ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Oxidative Stresses ◽  
Oxidative Balance

AbstractToxoplasma gondii is an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered by Toxoplasma is suggested to impair parasite replication and dissemination. However, the strategies by which Toxoplasma mitigates oxidative stress are not completely clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eIF2. Here, we show that the Toxoplasma eIF2 kinase, TgIF2K-B, is activated in response to oxidative stress and affords protection. Knockout of TgIF2K-B, Δtgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in an in vivo model of infection. Our results establish that TgIF2K-B is essential for Toxoplasma responses to oxidative stress, which are important for the parasite’s ability to establish persistent infection in its host.IMPORTANCEToxoplasma gondii is a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor, TgIF2α. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasite’s oxidative stress response and its ability to persist in the host as a latent infection.

scholarly journals Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection

2021 ◽  
Vol 22 (11) ◽  
pp. 5646
Author(s):  
Qing Chun Zhu ◽  
Shumin Li ◽  
Li Xia Yuan ◽  
Rui Ai Chen ◽  
Ding Xiang Liu ◽  
...  
Keyword(s):  
Stress Response ◽  
Mrna Expression ◽  
Translation Initiation Factor ◽  
Interleukin 8 ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Activator Protein 1 ◽  
Diarrhea Virus ◽  
Cytokines And Chemokines ◽  
Coronavirus Infection

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.

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