Abstract
The unfolded protein response (UPR) in the endoplasmic reticulum (ER) is a highly conserved protein-quality-control mechanism critical for cells to make survival-or-death decisions under ER-stress conditions. However, how UPR sensors are activated remains unclear. Here, we report that ER luminal protein canopy homolog 2 (CNPY2) is released from grp78 upon ER stress. Free CNPY2 then engages protein kinase R-like ER kinase (PERK) to induce expression of the transcription factor C/EBP homologous protein (CHOP), thereby initiating the UPR. Indeed, deletion of CNPY2 blocked the PERK–CHOP pathway and protected mice from UPR-induced liver damage and steatosis. Additionally, CNPY2 is transcriptionally upregulated by CHOP in a forward-feed loop to further enhance UPR signaling. These findings demonstrate the critical roles of CNPY2 in ER stress and suggest that CNPY2 is a potential new therapeutic target for UPR-related diseases such as metabolic disorders, inflammation and cancer.
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Acknowledgements
Authors thank S. Olson who provided the pET28b vector and protocol for purification of His-tagged CNPY2 from the inclusion bodies. We also thank the MUSC protein core for providing the service for the ITC experiments, D. Gewirth, E. Ansa-Addo and S. Parnham for critical reading of the manuscript. The current study was supported by grants from National Institutes of Health (NIH) to Z.L. (R01DK105033, P01CA186866, R01CA213290, R01CA188419 and R01AI070603). Z.L. is supported by the Abney Foundation and the South Carolina SmartState Centers of Economic Excellence.
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F.H. and Z.L. designed experiments, analyzed the data and wrote the manuscript. B.L. initiated the project and contributed to generation of Cnpy2 KO mice and data analysis. F.H. performed most of the experiments. B.W. helped in the purification of GST-eif2α and GST-eif2αS51A. J.M. assisted with in vitro experiments. B.R. and C.D. subcloned His-tagged mouse Cnpy2 into the pHGK vector. S.S. performed histological analysis. A.D. provided important reagents. All the authors discussed the data and approved the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Generation of Cnpy2 KO mice
(a) Schematic of the targeting vector in the Cnpy2 locus and the expected sequence of Cnpy2 loci after cre-mediated recombination. Neo: neomycin phosphotransferase. (b) Immunoblotting showing efficient knockout of Cnpy2 in the indicated organs. (c) H&E staining for liver sections of Cnpy2 Het and KO mice. Scale bar: 50 μm. (d) Levels of serum ALT in Cnpy2 Het (n=15) and KO (n=15) mice. (e) Body weight changes of Cnpy2 Het and KO mice after 24 h-treatment with tunicamycin. Het (n=12) and KO (n=12) mice were used for each experiment. Data are shown as the mean ± s.d. of three independent experiments. *P < 0.05 by Student’s t-test (e). NS: Not Significant.
Supplementary Figure 2 Role of CNPY2 on IRE1α/XBP1 pathway.
(a) Xbp1 splicing levels in livers were measured by qRT-PCR. The mRNA level of β-actin is shown as a quantity control. (b) The levels of Xbp1s downstream molecules, Edem1 and Erdj4, were measured by qRT-PCR. (c) Primary hepatocytes isolated from Cnpy2 Het and KO mice were treated with tunicamycin for 16 h. The transcript levels of Xbp1s were measured by qRT-PCR. The mRNA level of β-actin was shown as a quantity control. Dot plots show quantification of qRT-PCR for three independent experiments. No significant changes were observed among all groups (a-c).
Supplementary Figure 3 CNPY2 interacts with grp78.
(a) RAW264.7 cells were treated with DMSO, Thapsigargin (TG), or Tunicamycin (Tu) for 16 h. Immunoblots of CNPY2, CHOP, grp78, and β-actin were performed. (b) Co-immunoprecipitation of CNPY2 and grp78 using anti-CNPY2 antibody. Results represent two independent experiments.
Supplementary Figure 4 Isothermal titration calorimetry (ITC) negative control.
PERK-LD was injected into PBS buffer (a) or CNPY2 6C-A mutant protein (b). The generated heat was measured by ITC. PERK: CNPY2 6C-A stoichiometry (n) = 230.1 ± 4.3. Result represents three independent experiments. "n" means number of binding sites.
Supplementary Figure 5 Stability of overexpressed CNPY2 WT and mutant proteins.
(a) CNPY2 WT and mutant-overexpressing MEFs were treated with cycloheximide (CHX) at the indicated times, and then Immunoblotting was carried out for CNPY2 and β-actin. Result represents two independent experiments. (b) Empty vector or FLAG-tagged C3-A CNPY2 mutant retrovirus was transduced into wild type MEFs. Immunoblotting was performed for the indicated proteins. Result represents two independent experiments.
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Hong, F., Liu, B., Wu, B. et al. CNPY2 is a key initiator of the PERK–CHOP pathway of the unfolded protein response. Nat Struct Mol Biol 24, 834–839 (2017). https://doi.org/10.1038/nsmb.3458
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DOI: https://doi.org/10.1038/nsmb.3458
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