Human Papillomavirus E7 Requires the Protease Calpain to Degrade the Retinoblastoma Protein

Grant A Darnell,Wayne A Schroder,Toni M Antalis,Eleanore Lambley,Lee Major,Joy Gardner,Geoff Birrell,Angel Cid-Arregui,Andreas Suhrbier

doi:10.1074/jbc.m706860200

Abstract

Cervical cancers transformed by high risk human papilloma virus (HPV) express the E7 oncoprotein, which accelerates the degradation of the retinoblastoma protein (Rb). Here we show that the E7-mediated degradation of Rb requires the calcium-activated cysteine protease, calpain. E7 bound and activated mu-calpain and promoted cleavage at Rb(810), with mutation of this residue preventing E7-mediated degradation. The calpain cleavage product, Rb(1-810), was unable to mediate cell cycle arrest but retained the ability to repress E6/E7 transcription. E7 also promoted the accelerated proteasomal degradation of Rb(1-810). Calpain inhibitors reduced the viability of HPV-transformed cells and synergized with cisplatin. Calpain, thus, emerges as a central player in E7-mediated degradation of Rb and represents a potential new drug target for the treatment of HPV-associated lesions.

Highlights

Infection with high risk human Papillomavirus (HPV)2 types is responsible for virtually all cases of cervical cancer, which is currently the second most common cause of death from cancer among women worldwide (1)
Calpain Is Required for E7-mediated Degradation of retinoblastoma protein (Rb)— Calpains exist as heterodimers comprising a large 80-kDa catalytic subunit and a smaller 28-kDa regulatory subunit
A plasmid encoding FLAG-tagged codon-optimized HPV-16 E7 was transfected into CAPN4Ϫ/Ϫ murine embryonic fibroblasts, and Rb levels were determined by Western blotting

Summary

Introduction

Infection with high risk human Papillomavirus (HPV)2 types is responsible for virtually all cases of cervical cancer, which is currently the second most common cause of death from cancer among women worldwide (1). Calpain inhibitors are being developed for a number of diseases including cancer (16, 17) and were able to inhibit E7-mediated degradation of Rb. They reduced the viability of HPV-transformed cells through up-regulation of p53 and synergized with cisplatin, a drug frequently used to treat cervical cancer. Incubation of HeLa and Caski cell lines with the proteasomal inhibitor lactacystin resulted in a substantial increase in the levels of this Ϸ95-kDa Rb species but not the 110-kDa Rb species (Fig. 1C, top panel, lanes 4 and 6).

Results

Conclusion