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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
  • 47 Downloads

Abstract

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.

Keywords

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

Notes

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)

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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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