Abstract
Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoan-specific FIC-domain-containing ER-localized enzyme (FICD) rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here we show that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD−/− hamster cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation-defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.
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References
Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).
Wang, M. & Kaufman, R.J. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 529, 326–335 (2016).
Walter, P. & Ron, D. The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081–1086 (2011).
Freiden, P.J., Gaut, J.R. & Hendershot, L.M. Interconversion of three differentially modified and assembled forms of BiP. EMBO J. 11, 63–70 (1992).
Chevalier, M. et al. Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP. J. Biol. Chem. 273, 26827–26835 (1998).
Preissler, S. et al. Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker. eLife 4, e08961 (2015).
Laitusis, A.L., Brostrom, M.A. & Brostrom, C.O. The dynamic role of GRP78/BiP in the coordination of mRNA translation with protein processing. J. Biol. Chem. 274, 486–493 (1999).
Chambers, J.E., Petrova, K., Tomba, G., Vendruscolo, M. & Ron, D. ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load. J. Cell Biol. 198, 371–385 (2012).
Carlsson, L. & Lazarides, E. ADP-ribosylation of the Mr 83,000 stress-inducible and glucose-regulated protein in avian and mammalian cells: modulation by heat shock and glucose starvation. Proc. Natl. Acad. Sci. USA 80, 4664–4668 (1983).
Sanyal, A. et al. A novel link between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response. J. Biol. Chem. 290, 8482–8499 (2015).
Ham, H. et al. Unfolded protein response-regulated Drosophila Fic (dFic) protein reversibly AMPylates BiP chaperone during endoplasmic reticulum homeostasis. J. Biol. Chem. 289, 36059–36069 (2014).
Preissler, S. et al. AMPylation matches BiP activity to client protein load in the endoplasmic reticulum. eLife 4, e12621 (2015).
Yarbrough, M.L. et al. AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 323, 269–272 (2009).
Worby, C.A. et al. The fic domain: regulation of cell signaling by adenylylation. Mol. Cell 34, 93–103 (2009).
Broncel, M., Serwa, R.A., Bunney, T.D., Katan, M. & Tate, E.W. Global profiling of Huntingtin-associated protein E (HYPE)-mediated AMPylation through a chemical proteomic approach. Mol. Cell. Proteomics 2, 715–725 (2015).
Engel, P. et al. Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins. Nature 482, 107–110 (2012).
Bunney, T.D. et al. Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions. Structure 22, 1831–1843 (2014).
Anderson, W.B. & Stadtman, E.R. Glutamine synthetase deadenylation: a phosphorolytic reaction yielding ADP as nucleotide product. Biochem. Biophys. Res. Commun. 41, 704–709 (1970).
Xu, Y., Carr, P.D., Vasudevan, S.G. & Ollis, D.L. Structure of the adenylylation domain of E. coli glutamine synthetase adenylyl transferase: evidence for gene duplication and evolution of a new active site. J. Mol. Biol. 396, 773–784 (2010).
Neunuebel, M.R. et al. De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila. Science 333, 453–456 (2011).
Garcia-Pino, A., Zenkin, N. & Loris, R. The many faces of Fic: structural and functional aspects of Fic enzymes. Trends Biochem. Sci. 39, 121–129 (2014).
Roy, C.R. & Cherfils, J. Structure and function of Fic proteins. Nat. Rev. Microbiol. 13, 631–640 (2015).
Harms, A., Stanger, F.V. & Dehio, C. Biological diversity and molecular plasticity of FIC domain proteins. Annu. Rev. Microbiol. 70, 341–360 (2016).
Paton, A.W. et al. AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443, 548–552 (2006).
Bertolotti, A., Zhang, Y., Hendershot, L.M., Harding, H.P. & Ron, D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat. Cell Biol. 2, 326–332 (2000).
Pincus, D. et al. BiP binding to the ER-stress sensor Ire1 tunes the homeostatic behavior of the unfolded protein response. PLoS Biol. 8, e1000415 (2010).
Castro-Roa, D. et al. The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Nat. Chem. Biol. 9, 811–817 (2013).
Xiao, J., Worby, C.A., Mattoo, S., Sankaran, B. & Dixon, J.E. Structural basis of Fic-mediated adenylylation. Nat. Struct. Mol. Biol. 17, 1004–1010 (2010).
Luong, P. et al. Kinetic and structural insights into the mechanism of AMPylation by VopS Fic domain. J. Biol. Chem. 285, 20155–20163 (2010).
Khater, S. & Mohanty, D. In silico identification of AMPylating enzymes and study of their divergent evolution. Sci. Rep. 5, 10804 (2015).
Petrova, K., Oyadomari, S., Hendershot, L.M. & Ron, D. Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3. EMBO J. 27, 2862–2872 (2008).
Gaut, J.R. & Hendershot, L.M. Mutations within the nucleotide binding site of immunoglobulin-binding protein inhibit ATPase activity and interfere with release of immunoglobulin heavy chain. J. Biol. Chem. 268, 7248–7255 (1993).
Avezov, E. et al. Lifetime imaging of a fluorescent protein sensor reveals surprising stability of ER thiol redox. J. Cell Biol. 201, 337–349 (2013).
Scorsone, K.A., Panniers, R., Rowlands, A.G. & Henshaw, E.C. Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis. J. Biol. Chem. 262, 14538–14543 (1987).
Acknowledgements
We thank R. Antrobus (CIMR mass spectrometry), R. Schulte and the CIMR flow cytometry team for assistance; H.P. Harding, N. Amin-Wetzel and J. Chambers (CIMR) for advice and comments on the manuscript; and C. Flandoli (Cambridge, UK) for the cartoon. Supported by Wellcome Trust Principal Research Fellowship to D.R. (Wellcome 200848/Z/16/Z), a UK Medical Research Council PhD studentship to L.A.P. and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140).
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S.P. conceived, designed and led the project; conducted in vitro experiments, analysis and interpretation of data; and drafted and revised the article. C.R. designed, conducted and interpreted the in vivo experiments and contributed to drafting and revising the article. L.A.P. contributed to protein purification and fluorescence polarization experiments and revised the manuscript. V.S. provided valuable insights and discussions and revised the manuscript. D.R. oversaw the project conception and design, construction of plasmid DNA, analysis and interpretation of data and drafting and revising of the article.
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Integrated supplementary information
Supplementary Figure 1 AMPylation with ATP-FAM generates BiP specifically labeled on its substrate-binding domain.
(a) SDS-PAGE gel of AMPylated BiP incubated without or with SubA protease. BiP was AMPylated in vitro with FICDE234G in presence of a fluorescently labeled ATP derivative (ATP-FAM) and the resulting AMPylated BiP, with a fluorescent AMP attached (BiPT518-AMP-FAM) was re-purified (as in Fig. 2d). After prolonged treatment without or with SubA (4 hours at 30°C) the samples were denatured and applied to SDS-PAGE. The fluorescence signals of the fluorophore in the gel were detected (excitation: 488 nm, emission: 526 nm; upper panel) and the proteins were visualized by staining with Coomassie (CBB; lower panel). Uncleaved full-length BiPT518-AMP-FAM (FL), the nucleotide binding domain (NBD), the substrate binding domain (SBD), FICD, and SubA are indicated.
(b) Time-dependent plot of fluorescence polarization (FP) of BiP AMPylated with FAM-labeled AMP (BiPT518-AMP-FAM, from the sample shown in “a” above) after incubation without (black line) or with wildtype FICD protein (red line). The decrease in the FP signal reflects release of the fluorophore from BiP.
Uncropped gel images are shown in Supplementary Data Set 1.
Supplementary Figure 2 Absorbance spectra of ion pair chromatography elution profiles.
(a) 3D absorbance plots of the nucleotide standard (upper panel) and the ‘BiP-AMP + FICD’ de-AMPylation sample (lower panel) shown in Fig. 3c (grey and red traces therein). The elution profiles between 6 and 13 minutes are shown. Note that the absorbance characteristics of the AMP standard and the de-AMPylation product (both eluting at ~11.1 minutes) are qualitatively indistinguishable with an absorbance maximum at ~260 nm (arrows).
(b) Direct comparison of the absorbance spectra at 11.14 minutes of the profiles shown in “a”.
Supplementary Figure 3 Analysis of FICD overexpression in cells.
(a) FICD immunoblot of FICD-deficient (-/-) CHO-K1 cells transfected with plasmids encoding the indicated FICD derivatives. The eIF2α below serves as a loading control. Note the higher protein levels of the weaker AMPylation active/de-AMPylation defective FICDE234V/L/Q/K mutants compared to the AMPylation hyperactive FICDE234G.
(b) Flow cytometry source data from a representative experiment (one of three) used to generate the plot in Fig. 6b.
Uncropped blot images are shown in Supplementary Data Set 1.
Supplementary Figure 4 Analysis of FICD overexpression by flow cytometry and native PAGE.
(a) Flow cytometry source data from one of three independent repeats plotted in Fig. 6c.
(b) Immunoblot of endogenous BiP from wildtype and FICD-deficient (-/-) CHO-K1 cells resolved by native-PAGE. The cells were co-transfected with the indicated pairs of plasmids as in Fig. 6c and allowed to grow for 36 hours. The AMPylated ‘B’ form of BiP is indicated (as are the other major species, see Fig. 1 legend). Immunoblots of the same samples resolved by SDS-PAGE report on FICD, total BiP and total eIF2α (which also serves as a loading control). Data representative of two independent experiments are shown.
Note the absence of AMPylated BiP (‘B’ form) in cells co-transfected with wildtype FICD and the de-AMPylation defective/AMPylation active FICDE234G.
Uncropped blot images are shown in Supplementary Data Set 1.
Supplementary Figure 5 Wild-type and FICD–/– cells respond indistinguishably to unfolded protein stress in the ER.
(a) Flow cytometry analysis of wildtype and FICD-deficient (-/-) CHO-K1 CHOP::GFP UPR reporter cells treated with the UPR-inducing compounds, tunicamycin (2.5 μg/ml) or thapsigargin (0.5 μM), for 16 hours before analysis. Note the equal accumulation of CHOP::GFP-positive cells in tunicamycin- or thapsigargin-treated wildtype and FICD-/- cells.
(b) Plot of the median values ± SD of the GFP fluorescent signal of the samples described in “a” from three independent experiments (fold change relative to untreated wildtype cells).
(c) Flow cytometry analysis of wildtype and FICD-deficient (-/-) CHO-K1 CHOP::GFP UPR reporter cells transiently transfected with plasmids encoding the Cas9 nuclease and single guide RNAs targeting hamster BiP. Note the similar levels of UPR signaling in wildtype and FICD-/- cells.
(d) Plot of the median values ± SD of the CHOP::GFP fluorescent signal in the transfected subpopulation of the cells shown in “c” from three independent experiments. Transfected cells were identified by co-expression of a mCherry marker (not shown) carried by the Cas9 plasmid (fold change relative to wildtype cells transfected with plasmid DNA encoding Cas9 and mCherry only).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Note (PDF 1785 kb)
Supplementary Table 1
Description of plasmids used for the study. (XLSX 44 kb)
Supplementary Data Set 1
Uncropped blot, autoradiograph and gel images. (PDF 15392 kb)
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Preissler, S., Rato, C., Perera, L. et al. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP. Nat Struct Mol Biol 24, 23–29 (2017). https://doi.org/10.1038/nsmb.3337
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DOI: https://doi.org/10.1038/nsmb.3337
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