Cell Stress and Chaperones

, Volume 24, Issue 1, pp 149–158 | Cite as

Targeting HPV-16 antigens to the endoplasmic reticulum induces an endoplasmic reticulum stress response

  • David H. Martínez-Puente
  • José J. Pérez-Trujillo
  • Yolanda Gutiérrez-Puente
  • Humberto Rodríguez-Rocha
  • Aracely García-García
  • Odila Saucedo-Cárdenas
  • Roberto Montes-de-Oca-Luna
  • María J. Loera-AriasEmail author
Original Paper


Very promising results have been observed with a deoxyribonucleic acid (DNA) vaccine based on human papillomavirus type-16 (HPV-16) antigen retention and delivery system in the endoplasmic reticulum (ER). However, the mechanism by which these antigens are processed once they reach this organelle is still unknown. Therefore, we evaluated whether this system awakens a stress response in the ER. Different DNA constructs based on E6 and E7 mutant antigens fused to an ER signal peptide (SP), a signal for retention in the ER (KDEL), or both signals (SPK), were transfected into HEK-293 cells. Overexpression of E6 and E7 antigens targeted to the ER (SP, and SPK constructs) induced ER stress, which was indicated by an increase of the ER-stress markers GRP78/BiP and CHOP. Additionally, the ER stress response was mediated by the ATF4 transcription factor, which was translocated into the nucleus. Besides, the overexpressed antigens were degraded by the proteasome. Through a cycloheximide-chase assay, we demonstrated that when both protein synthesis and proteasome were inhibited, the overexpressed antigens were degraded. Interestingly, when proteasome was blocked autophagy was increased and the ER stress response decreased. Taken together, these results indicate that the antigens are initially degraded by the ERAD pathway, and autophagy degradation pathway can be induced to compensate the proteasome inhibition. Therefore, we provided a new insight into the mechanism by which E6 and E7 mutant antigens are processed once they reach the ER, which will help to improve the development of more effective vaccines against cancer.


E7 Stress response Signal peptide KDEL signal ER targeting GRP78/BiP 


Funding information

This study was supported through grants from the Programa de Apoyo a la Investigacion Cientifica y Tecnologica (grant no. SA179-15) from the Universidad Autonoma de Nuevo Leon and the National Council for Science and Technology (CONACYT; grant no. CB10-158509). DHMP was the recipient of a scholarship from CONACYT.

Supplementary material

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Supplementary Fig. 1

HEK-293 cells were transfected with the SPK construct at different DNA concentrations; 24 h later they detected with an anti E7. (PNG 31 kb)

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Supplementary Fig. 2

ATF4 nuclear translocation. a) Expression of the ATF4 protein in the nucleus in those cells transfected with the constructs of interest. In BFA, CRT SPK and SP, a greater translocation to the nucleus of the transcription factor ATF4 than in the rest of the constructs can be observed, as indicated by red arrows. b) DAPI was used for nuclear staining, anti E7 detected in the red channel and ATF4 in the green channel. The cells were photo-documented with the QImage program for further analysis. The red boxes on the green channel of ATF4 represent the zoom zone of incise a). 400 X, Scale bar = 40 μm. (PNG 107 kb)

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12192_2018_952_Fig9_ESM.png (131 kb)

(PNG 131 kb)

12192_2018_952_MOESM3_ESM.tiff (1.5 mb)
High-resolution image (TIFF 1521 kb)


  1. B’chir W, Maurin A-C, Carraro V, Averous J, Jousse C, Muranishi Y, Parry L, Stepien G, Fafournoux P, Bruhat A (2013) The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression. Nucleic Acids Res 41:7683–7699. CrossRefGoogle Scholar
  2. Bonifacino JS, Weissman AM (1998) Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu Rev Cell Dev Biol 14:19–57. CrossRefGoogle Scholar
  3. Brostrom CO, Brostrom MA (1998) Regulation of translational initiation during cellular responses to stress. Prog Nucleic Acid Res Mol Biol 58:79–125CrossRefGoogle Scholar
  4. Christianson JC, Ye Y (2014) Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nat Struct Mol Biol 21:325–335. CrossRefGoogle Scholar
  5. Coe H, Bedard K, Groenendyk J, Jung J, Michalak M (2008) Endoplasmic reticulum stress in the absence of calnexin. Cell Stress Chaperones 13:497–507. CrossRefGoogle Scholar
  6. Comber JD, Philip R (2014) MHC class I antigen presentation and implications for developing a new generation of therapeutic vaccines. Ther Adv Vaccines 2:77–89. CrossRefGoogle Scholar
  7. Ding W-X, Ni H-M, Gao W, Yoshimori T, Stolz DB, Ron D, Yin XM (2007) Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am J Pathol 171:513–524. CrossRefGoogle Scholar
  8. Donaldson JG, Finazzi D, Klausner RD (1992) Brefeldin a inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature 360:350–352. CrossRefGoogle Scholar
  9. Emmerich CH, Cohen P (2015) Optimising methods for the preservation, capture and identification of ubiquitin chains and ubiquitylated proteins by immunoblotting. Biochem Biophys Res Commun 466:1–14. CrossRefGoogle Scholar
  10. Granados DP, Tanguay P-L, Hardy M-P, Caron É, de Verteuil D, Meloche S, Perreault C (2009) ER stress affects processing of MHC class I-associated peptides. BMC Immunol 10:10. CrossRefGoogle Scholar
  11. 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 6:1099–1108CrossRefGoogle Scholar
  12. Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13:89–102. CrossRefGoogle Scholar
  13. Kaufman RJ (2004) Regulation of mRNA translation by protein folding in the endoplasmic reticulum. Trends Biochem Sci 29:152–158. CrossRefGoogle Scholar
  14. Leach MR, Williams DB (2013) Calnexin and Calreticulin, Molecular Chaperones of the Endoplasmic Reticulum. Landes BioscienceGoogle Scholar
  15. Lee D, Singaravelu G, Park B-J, Ahnn J (2007) Differential requirement of unfolded protein response pathway for calreticulin expression in Caenorhabditis elegans. J Mol Biol 372:331–340. CrossRefGoogle Scholar
  16. Leifert JA, Rodriguez-Carreno MP, Rodriguez F, Whitton JL (2004) Targeting plasmid-encoded proteins to the antigen presentation pathways. Immunol Rev 199:40–53. CrossRefGoogle Scholar
  17. Loera-Arias MJ, Martínez-Pérez AG, Barrera-Hernández A, Ibarra-Obregón ER, González-Saldívar G, Martínez-Ortega JI, Rosas-Taraco A, Villanueva-Olivo A, Esparza-González SC, Villatoro-Hernandez J, Saucedo-Cárdenas O, Montes-de-Oca-Luna R (2010) Targeting and retention of HPV16 E7 to the endoplasmic reticulum enhances immune tumour protection. J Cell Mol Med 14:890–894. CrossRefGoogle Scholar
  18. Mak BC, Wang Q, Laschinger C, Lee W, Ron D, Harding HP, Kaufman RJ, Scheuner D, Austin RC, McCulloch CA (2008) Novel function of PERK as a mediator of force-induced apoptosis. J Biol Chem 283:23462–23472.
  19. Mizushima N, Yoshimorim T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326. CrossRefGoogle Scholar
  20. Nishitoh H (2012) CHOP is a multifunctional transcription factor in the ER stress response. J Biochem (Tokyo) 151:217–219. CrossRefGoogle Scholar
  21. Oosterhuis K, Aleyd E, Vrijland K, Schumacher TN, Haanen JB (2012) Rational design of DNA vaccines for the induction of human papillomavirus type 16 E6- and E7-specific cytotoxic T-cell responses. Hum Gene Ther 23:1301–1312. CrossRefGoogle Scholar
  22. Oslowski CM, Urano F (2011) Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Methods Enzymol 490:71–92. CrossRefGoogle Scholar
  23. Perez-Trujillo JJ, Garza-Morales R, Barron-Cantu JA, Figueroa-Parra G, Garcia-Garcia A, Rodriguez-Rocha H, Garcia-Juarez J, Muñoz-Maldonado GE, Saucedo-Cardenas O, Montes-de-Oca-Luna R, Loera-Arias MDJ (2017) DNA vaccine encoding human papillomavirus antigens flanked by a signal peptide and a KDEL sequence induces a potent therapeutic antitumor effect. Oncol Lett 13:1569–1574. CrossRefGoogle Scholar
  24. Pérez-Trujillo JJ, Robles-Rodríguez OA, Garza-Morales R, García-García A, Rodríguez-Rocha H, Villanueva-Olivo A, Segoviano-Ramírez JC, Esparza-González SC, Saucedo-Cárdenas O, Montes-de-Oca-Luna R, Loera-Arias MJ (2018) Antitumor response by endoplasmic reticulum-targeting DNA vaccine is improved by adding a KDEL retention signal. Nucleic Acid Ther 28:252–261.
  25. Reinstein E, Scheffner M, Oren M, Ciechanover A, Schwartz A (2000) Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue. Oncogene 19:5944–5950. CrossRefGoogle Scholar
  26. Ruggiano A, Foresti O, Carvalho P (2014) ER-associated degradation: protein quality control and beyond. J Cell Biol 204:869–879. CrossRefGoogle Scholar
  27. Sano R, Reed JC (2013) ER stress-induced cell death mechanisms. Biochim Biophys Acta 1833:3460–3470. CrossRefGoogle Scholar
  28. Schiller JT, Castellsagué X, Garland SM (2012) A review of clinical trials of human papillomavirus prophylactic vaccines. Vaccine 30. Supplement 5:F123–F138. Google Scholar
  29. Schrag JD, Bergeron JJ, Li Y et al (2001) The structure of calnexin, an ER chaperone involved in quality control of protein folding. Mol Cell 8:633–644CrossRefGoogle Scholar
  30. Senft D, Ronai ZA (2015) UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci 40:141–148. CrossRefGoogle Scholar
  31. Sherritt M, Cooper L, Moss DJ, Kienzle N, Altman J, Khanna R (2001) Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters. Int Immunol 13:265–271CrossRefGoogle Scholar
  32. Shi W, Bu P, Liu J, Polack A, Fisher S, Qiao L (1999) Human papillomavirus type 16 E7 DNA vaccine: mutation in the open reading frame of E7 enhances specific cytotoxic T-lymphocyte induction and antitumor activity. J Virol 73:7877–7881Google Scholar
  33. Tanaka K, Matsuda N (2014) Proteostasis and neurodegeneration: the roles of proteasomal degradation and autophagy. Biochim Biophys Acta 1843:197–204. CrossRefGoogle Scholar
  34. Wang M, Wey S, Zhang Y, Ye R, Lee AS (2009) Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders. Antioxid Redox Signal 11:2307–2316. CrossRefGoogle Scholar
  35. Wu J, Kaufman RJ (2006) From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ 13:374–384. CrossRefGoogle Scholar
  36. Yewdell JW (2001) Not such a dismal science: the economics of protein synthesis, folding, degradation and antigen processing. Trends Cell Biol 11:294–297CrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2019

Authors and Affiliations

  • David H. Martínez-Puente
    • 1
  • José J. Pérez-Trujillo
    • 1
  • Yolanda Gutiérrez-Puente
    • 2
  • Humberto Rodríguez-Rocha
    • 1
  • Aracely García-García
    • 1
  • Odila Saucedo-Cárdenas
    • 1
    • 3
  • Roberto Montes-de-Oca-Luna
    • 1
  • María J. Loera-Arias
    • 1
    Email author
  1. 1.Departamento de Histología, Facultad de MedicinaUniversidad Autonoma de Nuevo LeonMonterreyMéxico
  2. 2.Departamento de Química, Facultad de Ciencias BiológicasUniversidad Autonoma de Nuevo LeonSan Nicolás de los GarzaMéxico
  3. 3.Departamento de Genética Molecular, Centro de Investigación Biomédica del Noreste, Delegación Nuevo LeónInstituto Mexicano del Seguro SocialMexico CityMexico

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