The eIF2 phosphatase: characterization and modulation

Cellular needs are fulfilled by the combined activity of functional proteins. Consequently, cells are equipped with a complex proteostatic network that controls protein production in response to cellular requisites. Thereby, protein synthesis is induced or attenuated depending on the particular cellular conditions. One of the mechanisms to control protein synthesis is the phosphorylation of eIF2, which triggers the so-called Integrated Stress Response (ISR). Kinases that sense stresses induce the phosphorylation of eIF2, which, on the one hand, attenuates global rates of protein synthesis and, on the other hand, activates the expression of specific proteins that help to alleviate the stress. One of the proteins preferentially expressed during the ISR is PPP1R15A, a regulatory subunit of Protein Phosphatase 1 (PP1). The PP1/PPP1R15A holophosphatase dephosphorylates eIF2 and terminates the ISR once the stresses are resolved. Hence, eIF2 kinases and phosphatases work together to control levels of phosphorylated eIF2. Maintaining the right balance between the activity of these kinases and phosphatases is important, as is seen by the correlation between their perturbance and the appearance of certain cellular malfunctions or diseases. However, affecting this balance has been also suggested to have beneficial effects. For example, genetic interference with the PPP1R15A regulatory subunit is proposed to confer protection to mice and cells under ER-stress conditions. This observation led to the search for compounds with the ability to modulate the ISR, in particular, by acting on the eIF2 phosphatases. Three compounds (Salubrinal, Guanabenz and Sephin1) have been proposed as eIF2 phosphatase inhibitors with potential use as therapeutic tools in protein misfolding diseases. However, their precise mechanism of action and their direct effect on the enzyme remains an open question. This thesis focuses on the in vitro reconstitution of the eIF2 phosphatase, which served as a platform for characterizing the enzyme (in terms of its structure, activity and assembly) and for studying the proposed inhibitors. This report details key structural features of the PPP1R15/PP1 holophosphatase, the discovery of a cellular cofactor of the enzyme and the conclusions obtained after analysing the effect of its proposed inhibitors. It also includes the development of several in vitro assays, which could potentially be used to screen libraries of compounds in search for modulators of the enzyme

A Sephin1-insensitive tripartite holophosphatase dephosphorylates translation initiation factor 2α.

The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative small-molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2αP dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.Supported by a Wellcome Trust Principal Research Fellowship to D.R. (Wellcome 200848/Z/16/Z) and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140). M.B. was supported by a Flemish Concerted Research Action (GOA15/016). W.P. was supported by National Institute of Health R01NS091336 and the American Diabetes Association Pathway to Stop Diabetes Grant 1-14-ACN-31. Z.C. is a PhD fellow of the Fund for Scientific Research - Flanders

G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases.

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex.Wellcome Trust, MRC, EU FP7This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.04871.00

Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum

Background: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. Results: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca2+ concentrations approached those normally found in the ER lumen ([Ca2+] K-0.5max = 190 mu M). Conclusions: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.Wellcome Trust [Wellcome 084812/Z/08/Z]; European Commission (EU FP7 Beta-Bat) [277713]; Fundacao para a Ciencia e Tecnologia, Portugal [PTDC/QUI-BIQ/119677/2010]info:eu-repo/semantics/publishedVersio

Mutations in a translation initiation factor identify the target of a memory-enhancing compound

The integrated stress response (ISR) modulates messenger RNA translation to regulate the mammalian unfolded protein response (UPR), immunity, and memory formation. A chemical ISR inhibitor, ISRIB, enhances cognitive function and modulates the UPR in vivo. To explore mechanisms involved in ISRIB action, we screened cultured mammalian cells for somatic mutations that reversed its effect on the ISR. Clustered missense mutations were found at the amino-terminal portion of the delta subunit of guanine nucleotide exchange factor (GEF) eIF2B. When reintroduced by CRISPR-Cas9 gene editing of wild-type cells, these mutations reversed both ISRIB-mediated inhibition of the ISR and its stimulatory effect on eIF2B GEF activity toward its substrate, the translation initiation factor eIF2, in vitro. Thus, ISRIB targets an interaction between eIF2 and eIF2B that lies at the core of the ISR

Severe VWM mutant cells are unable to tolerate a second <i>EIF2S1</i>^<i>S51A</i> mutation.

<p>(A) Experimental design for tracking <i>EIF2S1</i>^<i>S51A</i> mutant cells. A fluorescent-tagged sgRNA/Cas9 plasmid targeting <i>EIF2S1</i> was co-transfected alongside wild type (WT) or <i>EIF2S1</i>^<i>S51A</i> (Mut) templates into CHO-S21 dual reporter cells. Following FACS selection for the transfected cells they were treated with 250 nM thapsigargin (Tg) for 24 hours and reporter expression was analyzed. (B) Flow cytometry analysis of reporter activity in untreated (UT) and thapsigargin-treated (Tg) CHO-S21 cells from the experiment outlined in “A”. Note the emergence of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated cells in the pool offered an <i>EIF2S1</i>^<i>S51A</i> repair template. (C) Flow cytometry analysis of reporter activity in untreated (UT) and thapsigargin-treated (Tg) parental CHO-S21 or indicated VWM mutant cells following targeting of the <i>EIF2S1</i> locus with an <i>EIF2S1</i>^<i>S51A</i> repair template (as described in “A”). Note the lack of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated putative <i>EIF2S1</i>^<i>S51A</i><i>; EIF2B4</i>^<i>A392D</i> or <i>EIF2S1</i>^<i>S51A</i><i>; EIF2B4</i>^<i>R484W</i> double mutant cells (lower right panel). (D) Percentage of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated putative <i>EIF2S1</i>^<i>S51A</i> mutant cells in the indicated population from experiments as in “C”. Shown are means ± S.D. N = 6 (Parent), 5 (<i>EIF2B4</i>^<i>A392D</i>), and 3 (<i>EIF2B4</i>^<i>R484W</i> and <i>EIF2B4</i>^<i>R468W</i>). *** P<0.001, ** P<0.01, n.s. not significant, One way ANOVA followed by Dunnett’s multiple comparisons test.</p

Heightened ISR in <i>EIF2B4</i>^<i>A392D</i> cells.

<p>(A) Flow cytometry analysis of <i>CHOP</i>::<i>GFP</i> and <i>XBP1</i>::<i>Turquoise</i> dual reporter-containing parental CHO-S21 and <i>EIF2B4</i>^<i>A392D</i> mutant cells. The cells were untreated (UT) or stimulated with 250 nM thapsigargin (Tg) or 0.5 mM histidinol (His) for 24 hours. Note the enhanced response of the <i>CHOP</i>::<i>GFP</i> ISR reporter. (B) Bar diagram of the median ± S.D. of the reporter gene activity from experiments as shown in “A”. N = 3, *P = 0.0057 for Tg, *P = 0.037 for His, Unpaired t test. (C) Experimental design for tracking <i>EIF2B4</i>^<i>A392D</i> mutations. A fluorescent protein-marked sgRNA/Cas9 plasmid targeting <i>EIF2B4</i> and a wildtype or <i>EIF2B4</i>^<i>A392D</i> mutant repair template marked by a silent <i>Spe</i>I mutation were co-transfected into CHO-S21 cells. Transfected cells (selected by FACS), were treated with histidinol and divided into four bins (Bin #1 to #4) by level of <i>CHOP</i>::<i>GFP</i> expression. After recovery, genomic DNA was isolated from cells in each bin and the targeted region of <i>EIF2B4</i> was amplified by PCR and digested with <i>Spe</i>I to reveal frequency of targeting by either repair template. (D) PCR fragments digested with <i>Spe</i>I from genomic DNA of the indicated bins, visualized on an agarose gel. Shown is an image of a representative experiment reproduced twice. (E) Plot of the distribution of <i>Spe</i>I digested fragments in the four bins of transduced cells from the experiment in “D”. The band intensities of the digested fragments (reporting on recombination of the wildtype or mutant repair template) were normalized to total PCR product intensity and the distribution of the relative frequency of recombination in the different bins was plotted. Note the enrichment for recombination of the <i>EIF2B4</i>^<i>A392D</i> mutant repair template in the ISR^High bin.</p

Stress-resistance of wildtype and VWM cells.

<p>(A) Schema of experiments to compare the effect of thapsigargin in parental CHO-S21 and VWM mutant cells. Cells were treated with thapsigargin (Tg; 250 nM) for the indicated time, washed free of compounds and allowed to recover before assay. W = WST-1 assay, P = Puromycin labeling. (B) Immunoblot of puromycinylated proteins following a brief pulse of puromycin, reporting on levels of translation under the indicated experimental conditions. Shown is a representative experiment reproduced four times. P and E indicate parental CHO-S21 and <i>EIF2B4</i>^<i>A392D</i> mutant cells, respectively. (C) Quantification of “B”. Signal intensities of puromycinylated proteins were normalized by eIF2α. Shown are means ± SEM of four independent experiments. (D) Cell viability measured by the WST-1 assay in the experiment described in “A”. Shown are the mean ± SEM of four replicates of a representative experiment repeated three (R484W, R468W) to six (A392D) times.</p