The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly.

Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss

Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic

Indexación: Web of ScienceTwo ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.https://elifesciences.org/content/4/e0543

The Transmembrane Kinase Ire1p Is a Site-Specific Endonuclease That Initiates mRNA Splicing in the Unfolded Protein Response

AbstractThe endoplasmic reticulum (ER) communicates with the nucleus through the unfolded protein response (UPR), which senses accumulation of unfolded proteins in the ER lumen and leads to increased transcription of genes encoding ER-resident chaperones. As a key regulatory step in this signaling pathway, the mRNA encoding the UPR-specific transcription factor Hac1p becomes spliced by a unique mechanism that requires tRNA ligase but not the spliceosome. Splicing is initiated upon activation of Ire1p, a transmembrane kinase that lies in the ER and/or inner nuclear membrane. We show that Ire1p is a bifunctional enzyme: in addition to being a kinase, it is a site-specific endoribonuclease that cleaves HAC1 mRNA specifically at both splice junctions. The addition of purified tRNA ligase completes splicing; we therefore have reconstituted HAC1 mRNA splicing in vitro from purified components

Cell, Vol. 90, 1031--1039, September 19, 1997, Copyright 1997 by Cell Press

ed upon activation of Ire1p, a transmembrane and HAC1 mRNA splicing is not affected by mutations kinase that lies in the ER and/or inner nuclear mem- that inhibit spliceosome function. Furthermore, yeast brane. We show that Ire1p is a bifunctional enzyme: tRNA ligase (encoded by RLG1) is required for splicing in addition to being a kinase, it is a site-specific endori- of HAC1 mRNA. An allele of RLG1, rlg1-100, completely bonuclease that cleaves HAC1 mRNA specifically at blocks the UPR without affecting splicing of tRNAs (an both splice junctions. The addition of purified tRNA essential function). In this mutant, HAC1 mRNA be- ligase completes splicing; we therefore have reconsti- comes specifically degraded when the pathway is acti- tuted HAC1 mRNA splicing in vitro from purified com- vated. Based on this observation and the known cata- ponents. lytic activity of tRNA ligase, we proposed that tRNA ligase joins the HAC1 mRNA halves that are produced Introduction upon

Cell, Vol. 87, 405--413, November 1, 1996, Copyright 1996 by Cell Press

this reporter, one belonged to a novel complementation RLG1, that restored the induction of KAR2 mRNA tran- group, as diploids heterozygous for this mutation and scription upon Tm treatment (Figure 2A, lanes 7 and 8). either Dire1 or Dhac1 exhibit the ability to induce the The identification of this gene was surprising, because UPR. Additional complemetation tests showed that the RLG1 encodes tRNA ligase, a previously characterized other two isolated mutants were allelic to ire1. For reaessential protein that is required for pre-tRNA splicing sons outlined below, we henceforth refer to the novel (Phizicky et al., 1992). To ascertain whether rlg1-100 is mutant as rlg1-10

Cell, Vol. 99, 691--702, December 23, 1999, Copyright 1999 by Cell Press

tRNA splicing (Sidrauski et al., 1996; Gonzalez et al., by the transmembrane kinase/endoribonuclease Ire1. 1999). Both HAC1 mRNA (u 5 unspliced, uninduced) and We show that yeast HAC1 mRNA is correctly spliced HAC1 mRNA are exported to the cytosol and become in mammalian cells uponUPR induction and that mam- engaged in polyribosomes, but only the spliced form gives malian Ire1 can precisely cleave both splice junctions. rise to Hac1 protein (Chapman and Walter, 1997). Splicing Surprisingly, UPR induction leads to proteolytic cleav- is therefore a key regulatory step in the UPR pathway age of Ire1, releasing fragments containing the kinase (reviewed in Shamu, 1998; Sidrauski et al., 1998). and nuclease domains that accumulate in the nucleus. The recent identification of Ire1p homologs suggests Nuclear localization and UPR induction are reduced that at least some aspects of the UPR are conserved in in presenilin-1 knockout cells. These results suggest higher eukaryo

The EMBO Journal Vol.18 No.11 pp.3119--3132, 1999

this paper were performed under optimal ADP-stimulating conditions. A gain in affinity could result from cooperative binding of Ire1p to the two splice sites and thus lead to increased cleavage. Folding of the intron and/ or base pairing interactions between the two exons may significantly contribute to cooperativity by positioning the two splice sites in an optimal orientation with respect to each othe

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response.

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2αP) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2αP in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2αP by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals