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

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 calciumactivated cysteine protease, calpain. E7 bound and activated -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 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). Vaccination will likely have a substantial impact; however, current modeling suggests that it may take 40 -50 years to reach an Ϸ50% reduction in mortality (2). Integration of part of the HPV genome is believed to be central to the transformation process (3) and often results in the increased expression of the two viral oncoproteins, E6 and E7. A principle activity of E6 and E7 is promotion of the accelerated degradation of p53 and the retinoblastoma protein (Rb), respectively (4, 5). Hypophosphorylated Rb binds the E2F transcription factor, resulting in repression of E2F-dependent gene transcription and G 1 cell cycle arrest (6). The promotion of Rb degradation mediated by E7 is believed to involve a two-step process (7). First, E7 binds hypophosphorylated Rb (8), which leads to the displacement of E2F and entry into the cell cycle. How E2F is displaced by E7 remains unclear as E7 and E2F binding sites on Rb have recently been shown to be quite distinct (9). The second step involves E7 targeting Rb for accelerated degradation via the proteasome (10 -12). A new insight into the mechanism of E7-mediated degradation of Rb was recently provided by our observation that the serine protease inhibitor SerpinB2 was able to inhibit E7-mediated degradation of Rb in HeLa cells (13,14). There are no reports that SerpinB2 inhibits the proteasome, and it has recently emerged that SerpinB2 inhibits calpain-mediated degradation of Rb. 3Calpains represent a group of calcium-activated cysteine proteases, two of which, -calpain and m-calpain, are ubiquitously expressed and found both in the cytoplasm and the nucleus (15). Herein we provide evidence that the first step in the E7-mediated degradation of Rb involves calpain cleavage. E7 was shown to bind and activate -calpain and promoted cleavage of full-length Rb 1-928 to yield Rb . Rb 1-810 was unable to promote cell cycle arrest, suggesting that this cleavage event was sufficient to displace E2F from Rb. The second step involved the promotion by E7 of the proteasomal degradation of Rb . 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 also reduced the viability of HPV-tr...

Gene expression profile regulated by the HPV16 E7 oncoprotein and estradiol in cervical tissue

“…The HPV16 E7 is considered the major transforming protein given its ability, in cell culture, to immortalize human epithelial cells (Halbert et al, 1991), and, in the context of genetically engineered mice expressing E7, to induce cervical tumors [in cooperation with 17β-estradiol (E 2 )] as well as to maintain precancerous lesions and cancers once they have arisen (Arbeit et al, 1996; Jabbar et al, 2009; Riley et al, 2003). E7's oncogenic potential is only partially attributed to its inactivation of the retinoblastoma tumor suppressor protein (pRb) (Darnell et al, 2007) given the observation that inactivation of pRb is not sufficient to explain all the oncogenic effects caused by E7 (Balsitis et al, 2006). It is well documented that the transforming activities of the E7 protein are related to the binding of a large number of cellular targets (McLaughlin-Drubin and Munger, 2009).…”

Role of Calpain in the Formation of Human Papillomavirus Type 16 E1^E4 Amyloid Fibers and Reorganization of the Keratin Network

“…Two calpains, -and m-, are found ubiquitously in vertebrates and are so called because of their dependence on M and mM concentrations of Ca 2ϩ respectively. Interestingly, HPV16 E7 was shown to reduce the calcium requirement of -calpain, suggesting that HPV infection might lead to calpain activation (5). We decided to test whether calpain is capable of cleaving off the N terminus of 16E1 E4.…”

The biological properties of E6 and E7 oncoproteins from human papillomaviruses

“…Thus, the ability to promote pRb degradation appears to be an exclusive property of the high-risk HPV types. Two recent studies have provided further insights on the mechanisms involved in HPV16 E7-mediated pRb degradation, which appears to be mediated by the cullin 2 ubiquitin ligase complex and calcium-activated cysteine protease, calpain [96,97].…”