Abstract
The North American amphibian, wood frogs, Rana sylvatica are the most studied anuran to comprehend vertebrate freeze tolerance. Multiple adaptations support their survival in frigid temperatures during winters, particularly their ability to produce glucose as natural cryoprotectant. Freezing and its component consequences (anoxia and dehydration) induce multiple stresses on cells. Among these is endoplasmic reticulum (ER) stress, a condition spawned by buildup of unfolded or misfolded proteins in the ER. The ER stress causes the unfolded protein response (UPR) and the ER-associated degradation (ERAD) pathway that potentially could lead to apoptosis. Immunoblotting was used to assess the responses of major proteins of the UPR and ERAD under freezing, anoxia, and dehydration stresses in the liver and skeletal muscle of the wood frogs. Targets analyzed included activating transcription factors (ATF3, ATF4, ATF6), the growth arrest and DNA damage proteins (GADD34, GADD153), and EDEM (ERAD enhancing α-mannosidase-like proteins) and XBP1 (X-box binding protein 1) proteins. UPR signaling was triggered under all three stresses (freezing, anoxia, dehydration) in liver and skeletal muscle of wood frogs with most tissue/stress responses consistent with an upregulation of the primary targets of all three UPR pathways (ATF4, ATF6, and XBP-1) to enhance the protein folding/refolding capacity under these stress conditions. Only frozen muscle showed preference for proteasomal degradation of misfolded proteins via upregulation of EDEM (ERAD). The ERAD response of liver was downregulated across three stresses suggesting preference for more refolding of misfolded/unfolded proteins. Overall, we conclude that wood frog organs activate the UPR as a means of stabilizing and repairing cellular proteins to best survive freezing exposures.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adams CJ, Kopp MC, Larburu N, Nowak PR, Ali MMU (2019) Structure and molecular mechanism of ER stress signaling by the unfolded protein response signal activator IRE1. Front Mol Biosci 6(March):1–12. https://doi.org/10.3389/fmolb.2019.00011
Al-Attar R, Storey KB (2018) Effects of anoxic exposure on the nuclear factor of activated T cell (NFAT) transcription factors in the stress-tolerant wood frog. Cell Biochem Funct 36(8):420–430. https://doi.org/10.1002/cbf.3362
Al-attar R, Zhang Y, Storey KB (2017) Osmolyte regulation by TonEBP/NFAT5 during anoxia-recovery and dehydration–rehydration stresses in the freeze-tolerant wood frog (Rana sylvatica). PeerJ 5(January):e2797. https://doi.org/10.7717/peerj.2797
Almanza A, Carlesso A, Chintha C, Creedican S, Doultsinos D, Leuzzi B, Luís A et al (2019) Endoplasmic reticulum stress signalling – from basic mechanisms to clinical applications. FEBS J 286(2):241–278. https://doi.org/10.1111/febs.14608
Ameri K, Hammond EM, Culmsee C, Raida M, Katschinski DM, Wenger RH, Wagner E et al (2007) Induction of activating transcription factor 3 by anoxia is independent of P53 and the hypoxic HIF signalling pathway. Oncogene 26(2):284–289. https://doi.org/10.1038/sj.onc.1209781
Carrara M, Prischi F, Ali M (2013) UPR signal activation by luminal sensor domains. Int J Mol Sci 14(3):6454–6466. https://doi.org/10.3390/ijms14036454
Chen X, Zhang F, Gong Qi, Cui A, Zhuo S, Zhimin Hu, Han Y et al (2016) Hepatic ATF6 increases fatty acid oxidation to attenuate hepatic steatosis in mice through peroxisome proliferator–activated receptor α. Diabetes 65(7):1904–1915. https://doi.org/10.2337/db15-1637
Costanzo JP, do Amaral MCF, Rosendale AJ, Lee RE (2013) Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog. J Exp Biol 216(18):3461–73. https://doi.org/10.1242/jeb.089342
Cullinan SB, Diehl JA (2006) Coordination of ER and oxidative stress signaling: the PERK/Nrf2 signaling pathway. Int J Biochem Cell Biol 38(3):317–332. https://doi.org/10.1016/j.biocel.2005.09.018
Eaton SL, Roche SL, Hurtado ML, Oldknow KJ, Farquharson C, Gillingwater TH, Wishart TM (2013) Total protein analysis as a reliable loading control for quantitative fluorescent western blotting. PLoS ONE 8(8):e72457. https://doi.org/10.1371/journal.pone.0072457
Gerber VEM, Wijenayake S, Storey KB (2016) Anti-apoptotic response during anoxia and recovery in a freeze-tolerant wood frog (Rana Sylvatica). PeerJ 4(March):e1834. https://doi.org/10.7717/peerj.1834
Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D (2000) Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6(5):1099–1108. https://doi.org/10.1016/S1097-2765(00)00108-8
Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397(6716):271–274. https://doi.org/10.1038/16729
Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N et al (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11(3):619–633. https://doi.org/10.1016/S1097-2765(03)00105-9
Hawkins LJ, Storey KB (2020) Advances and applications of environmental stress adaptation research. Comp Biochem Physiol A: Mol Integr Physiol 240(February):110623. https://doi.org/10.1016/j.cbpa.2019.110623
Holczer M, Bánhegyi G, Kapuy O (2016) GADD34 keeps the MTOR pathway inactivated in endoplasmic reticulum stress related autophagy. PLOS ONE 11(12):e0168359. https://doi.org/10.1371/journal.pone.0168359 (Edited by Dong-Yan Jin)
Inaba Y, Furutani T, Kimura K, Watanabe H, Haga S, Kido Y, Matsumoto M et al (2015) Growth arrest and DNA damage-inducible 34 regulates liver regeneration in hepatic steatosis in mice. Hepatology 61(4):1343–1356. https://doi.org/10.1002/hep.27619
Kim R, Emi M, Tanabe K, Murakami S (2006) Role of the unfolded protein response in cell death. Apoptosis 11(1):5–13. https://doi.org/10.1007/s10495-005-3088-0
Kiss AJ, Muir TJ, Lee RE Jr, Costanzo JP (2011) Seasonal variation in the hepatoproteome of the dehydration- and freeze-tolerant wood frog, Rana sylvatica. Int J Mol Sci 12(12):8406–14. https://doi.org/10.3390/ijms12128406
Kozutsumi Y, Segal M, Normington K, Gething M-J, Sambrook J (1988) The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332(6163):462–464. https://doi.org/10.1038/332462a0
Krivoruchko A, Storey KB (2013) Activation of the unfolded protein response during anoxia exposure in the turtle Trachemys scripta elegans. Mol Cell Biochem 374(1–2):91–103. https://doi.org/10.1007/s11010-012-1508-3
Ku H-C, Cheng C-F (2020) Master regulator activating transcription factor 3 (ATF3) in metabolic homeostasis and cancer. Front Endocrinol 11(August):556. https://doi.org/10.3389/fendo.2020.00556
Layne JR, Lee RE (1995) Adaptations of frogs to survive freezing. Climate Res 5:53–59. https://doi.org/10.3354/cr005053
Lee AS (2005) The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35(4):373–381. https://doi.org/10.1016/j.ymeth.2004.10.010
Logan SM, Cheng-Wei Wu, Storey KB (2019) The squirrel with the lagging EIF2: global suppression of protein synthesis during torpor. Comp Biochem Physiol A: Mol Integr Physiol 227(January):161–171. https://doi.org/10.1016/j.cbpa.2018.10.014
Mamady H, Storey KB (2008) Coping with the stress: expression of ATF4, ATF6, and downstream targets in organs of hibernating ground squirrels. Arch Biochem Biophys 477(1):77–85. https://doi.org/10.1016/j.abb.2008.05.006
Mori K (2009) Signalling pathways in the unfolded protein response: development from yeast to mammals. J Biochem 146(6):743–750. https://doi.org/10.1093/jb/mvp166
Nishio N, Isobe K-I (2015) GADD34-deficient mice develop obesity, nonalcoholic fatty liver disease, hepatic carcinoma and insulin resistance. Sci Rep 5(1):13519. https://doi.org/10.1038/srep13519
Novoa I, Zeng H, Harding HP, Ron D (2001) Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of EIF2α. J Cell Biol 153(5):1011–1022. https://doi.org/10.1083/jcb.153.5.1011
Oslowski CM, Urano F (2011) Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Methods Enzymol 71–92. https://doi.org/10.1016/B978-0-12-385114-7.00004-0
Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11(4):381–389. https://doi.org/10.1038/sj.cdd.4401373
Ozcan L, Tabas I (2012) Role of endoplasmic reticulum stress in metabolic disease and other disorders. Annu Rev Med 63(1):317–328. https://doi.org/10.1146/annurev-med-043010-144749
Rider MH, Hussain N, Horman S, Dilworth SM, Storey KB (2006) Stress-induced activation of the AMP-activated protein kinase in the freeze-tolerant frog Rana sylvatica. Cryobiology 53(3):297–309. https://doi.org/10.1016/j.cryobiol.2006.08.001
Roufayel R, Biggar KK, Storey KB (2011) Regulation of cell cycle components during exposure to anoxia or dehydration stress in the wood frog, Rana sylvatica. J Exp Zool A: Ecol Genet Physiol 315a(8):487–94. https://doi.org/10.1002/jez.696
Rubinsky B, Wong ST, Hong JS, Gilbert J, Roos M, Storey KB (1994) 1H Magnetic resonance imaging of freezing and thawing in freeze-tolerant frogs. Am J Physiol-Regul Integr Comp Physiol 266(6):R1771–R1777. https://doi.org/10.1152/ajpregu.1994.266.6.R1771
Rutkowski DT (2019) Liver function and dysfunction – a unique window into the physiological reach of ER stress and the unfolded protein response. FEBS J 286(2):356–378. https://doi.org/10.1111/febs.14389
Senft D, Ronai ZA (2015) UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci 40(3):141–48. https://doi.org/10.1016/j.tibs.2015.01.002
Shen J, Prywes R (2004) Dependence of site-2 protease cleavage of ATF6 on prior site-1 protease digestion is determined by the size of the luminal domain of ATF6. J Biol Chem 279(41):43046–43051. https://doi.org/10.1074/jbc.M408466200
Shuda M, Kondoh N, Imazeki N, Tanaka K, Okada T, Mori K, Hada A et al (2003) Activation of the ATF6, XBP1 and Grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis. J Hepatol 38(5):605–614. https://doi.org/10.1016/S0168-8278(03)00029-1
Singh G, Storey KB (2020) MondoA:MLX complex regulates glucose-dependent gene expression and links to circadian rhythm in liver and brain of the freeze-tolerant wood frog, Rana Sylvatica. Mol Cell Biochem 473(1–2):203–216. https://doi.org/10.1007/s11010-020-03820-9
Storey JM, Storey KB (1985) Triggering of cryoprotectant synthesis by the initiation of ice nucleation in the freeze tolerant frog, Rana sylvatica. J Comp Physiol B 156(2):191–195. https://doi.org/10.1007/BF00695773
Storey JM, Storey KB (2019) In defense of proteins: chaperones respond to freezing, anoxia, or dehydration stress in tissues of freeze tolerant wood frogs. J Exp Zool A: Ecol Integr Physiol 331(7):392–402. https://doi.org/10.1002/jez.2306
Storey KB (1998) Survival under stress: molecular mechanisms of metabolic rate depression in animals. S Afr J Zool 33(2):55–64. https://doi.org/10.1080/02541858.1998.11448454
Storey KB, Storey JM (1984) Biochemical adaption for freezing tolerance in the wood frog, Rana sylvatica. J Comp Physiol B 155(1):29–36. https://doi.org/10.1007/BF00688788
Storey KB, Storey JM (2005) Biochemical adaptation to extreme environments. In: Walz W (ed) Integrative physiology in the proteomics and post-genomics age. Humana Press, Totowa, pp 169–200. https://doi.org/10.1385/1-59259-925-7:169
Storey KB, Storey JM (2013) Molecular biology of freezing tolerance. In: Comprehensive physiology. Hoboken, John Wiley & Sons, Inc. https://doi.org/10.1002/cphy.c130007
Storey KB, Storey JM (2017) Molecular physiology of freeze tolerance in vertebrates. Physiol Rev 97(2):623–665. https://doi.org/10.1152/physrev.00016.2016
Szereszewski KE, Storey KB (2018) Translational regulation in the anoxic turtle, Trachemys scripta elegans. Mol Cell Biochem 445(1–2):13–23. https://doi.org/10.1007/s11010-017-3247-y
Tessier SN, Storey KB (2010) Expression of myocyte enhancer factor-2 and downstream genes in ground squirrel skeletal muscle during hibernation. Mol Cell Biochem 344(1–2):151–162. https://doi.org/10.1007/s11010-010-0538-y
Tsuru A, Imai Y, Saito M, Kohno K (2016) Novel mechanism of enhancing IRE1α-XBP1 signalling via the PERK-ATF4 pathway. Sci Rep 6(1):24217. https://doi.org/10.1038/srep24217
Vabulas RM, Raychaudhuri S, Hayer-Hartl M, Ulrich Hartl F (2010) Protein folding in the cytoplasm and the heat shock response. Cold Spring Harb Perspect Biol 2(12):a004390–a004390. https://doi.org/10.1101/cshperspect.a004390
Vitale A, Cereiotti A, Denecke J (1993) The role of the endoplasmic reticulum in protein synthesis, modification and intracellular transport. J Exp Bot 44(9):1417–1444. https://doi.org/10.1093/jxb/44.9.1417
Wolfgang CD, Chen BP, Martindale JL, Holbrook NJ, Hai T (1997) Gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3. Mol Cell Biol 17(11):6700–6707. https://doi.org/10.1128/MCB.17.11.6700
Woods AK, Storey KB (2006) Vertebrate freezing survival: regulation of the multicatalytic proteinase complex and controls on protein degradation. Biochim Biophys Acta (BBA) - Gen Subj 1760(3):395–403. https://doi.org/10.1016/j.bbagen.2005.12.015
Wu C-W, Tessier SN, Storey KB (2018) Stress-induced antioxidant defense and protein chaperone response in the freeze-tolerant wood frog Rana sylvatica. Cell Stress Chaperones 23(6):1205–1217. https://doi.org/10.1007/s12192-018-0926-x
Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M, Mori K (2000) ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol Cell Biol 20(18):6755–6767. https://doi.org/10.1128/MCB.20.18.6755-6767.2000
Yoshida H, Haze K, Yanagi H, Yura T, Mori K (1998) Identification of the cis -acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. J Biol Chem 273(50):33741–33749. https://doi.org/10.1074/jbc.273.50.33741
Zhang J, Gupta A, Storey KB (2021) Freezing stress adaptations: critical elements to activate Nrf2 related antioxidant defense in liver and skeletal muscle of the freeze tolerant wood frogs. Comp Biochem Physiol B: Biochem Mol Biol 254(June):110573. https://doi.org/10.1016/j.cbpb.2021.110573
Zhang J, Storey KB (2012) Cell cycle regulation in the freeze tolerant wood frog, Rana Sylvatica. Cell Cycle 11(9):1727–1742. https://doi.org/10.4161/cc.19880
Zhang J, Storey KB (2013) Akt signaling and freezing survival in the wood frog, Rana sylvatica. Biochim Biophys Acta Gen Subj 1830(10):4828–37. https://doi.org/10.1016/j.bbagen.2013.06.020
Zhu G, Lee AS (2015) Role of the unfolded protein response, GRP78 and GRP94 in organ homeostasis. J Cell Physiol 230(7):1413–1420. https://doi.org/10.1002/jcp.24923
Acknowledgements
We would like to thank J.M. Storey for editorial review of manuscript.
Funding
Research was supported by a Discovery grant to KBS from the Natural Sciences and Engineering Research Council of Canada (#6793); KBS holds the Canada Research Chair in Molecular Physiology.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Niles, J., Singh, G. & Storey, K.B. Role of unfolded protein response and ER-associated degradation under freezing, anoxia, and dehydration stresses in the freeze-tolerant wood frogs. Cell Stress and Chaperones 28, 61–77 (2023). https://doi.org/10.1007/s12192-022-01307-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12192-022-01307-8