E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes

Abstract

E6AP was originally identified as the ubiquitin-protein ligase involved in human papillomavirus (HPV) E6-mediated p53 degradation and has since been shown to act as an E3 ubiquitin-protein ligase in the ubiquitination of several other protein substrates. To further define E6AP function at the molecular and cellular levels, a ribozyme-based gene inactivation approach was adopted. A library of hammerhead ribozymes, with randomized arm sequences, was used to screen active molecules along the entire E6AP transcript for ribozyme-cleavable sites. Ligation-anchored PCR was adapted to detect cleavage products, and ribozymes designed to the selected sites were characterized both in vitro and in vivo. Ribozyme-mediated reduction in E6AP expression was found to enhance the apoptotic response of HeLa cells to mitomycin C-induced DNA damage. These findings suggest that E6AP has potential as a drug target, as its suppression can potentiate apoptosis in HPV-positive cells treated with a cytotoxic drug. Cancer Gene Therapy (2003) 10, 707-716. doi:10.1038/sj.cgt.7700623Keywords: E6AP; gene function; apoptosis; ribozyme; in vitro selection T he ubiquitin-dependent proteolytic pathway has emerged as a crucial mechanism in cellular regulation. 1 Protein ubiquitination involves a cascade of cellular enzymes called E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin-protein ligase). Ubiquitin is activated at its C-terminal glycine to form a thiolester with the active site cysteine on E1 in an ATP-dependent reaction. The activated ubiquitin is then transferred to one of a family of E2s, preserving the high-energy thiolester bond. E2 enzymes catalyze isopeptide bond formation between ubiquitin and substrate protein directly or in association with an E3. Multiubiquitinated proteins are recognized by a large multisubunit protease complex, the 26S proteosome and degraded.The 100 kDa cellular protein E6AP, in association with human papillomavirus (HPV) E6, was initially identified as a ubiquitin-protein ligase responsible for p53 degradation. 2 It has since been shown that E6AP acts as an E3 in the ubiquitin-dependent proteolysis of various other cellular proteins, both in the presence and absence of HPV E6. 3-7 E6AP was also the founding member of a family of structurally related E3s carrying a carboxyl terminus that is highly conserved among various organisms, termed the hect domain (homology to E6AP C terminus). 8 Besides its protein ligase function, E6APwas recently shown to act as a coactivator in nuclear hormone receptor-mediated transactivation, 9 and mutations in E6AP have been causally linked with a genetic neurological disorder known as Angelman Syndrome. 10,11 Despite the identification of several E6AP-interacting proteins, the role of E6AP in cellular physiology remains poorly understood. Recent works from several groups suggest that E6AP may be involved in the regulation of important cellular processes such as transcription, signal transduction, cell survival and/or apoptosis, cell-cycle contr...

Oxidative stress regulated expression of Ubiquitin Carboxyl-terminal Hydrolase-L1: Role in cell survival

“…ubiquitin ligase E6AP and SKP2, induced severe apoptosis. 41,42 However, we found that down regulation of UCHL1 had only a minimal effect on apoptotic cell death under our experimental conditions. The reason might be because UCHL1 is not actively involved in the apoptotic pathway but rather in maintaining cell homeostasis.…”

Growth Suppression Induced by Downregulation of E6-AP Expression in Human Papillomavirus-Positive Cancer Cell Lines Depends on p53

“…Taken together, the results described above corroborate previous data indicating that E6-AP is required for both p53 degradation and the viability of HPV-positive cell lines (4,12,15,16,27). However, they do not conclusively address the question as to whether the ability of E6-AP to facilitate p53 degradation in the presence of E6 is essential for the viability of HPV-positive cancer cells or whether other yet uncharacterized functions of E6-AP are involved.…”

“…Similarly, the downregulation of E6-AP expression by antisense approaches or overexpression of a catalytically inactive E6-AP mutant results in the accumulation of p53 in HPV-positive cells but not in HPVnegative cells (4,26). Moreover, ribozyme-mediated reduction of E6-AP expression enhances the apoptotic response of HeLa cells, an HPV-18-positive cell line, to the DNA damage-inducing drug mitomycin C (16). However, since E6-AP has been implicated in E6-mediated degradation of proteins other than p53 (e.g., E6TP1 and hScrib) (9,21), it remains unclear if this apoptosis-enhancing effect is directly linked to the ability of E6-AP to target p53 for degradation in the presence of E6.…”

Role of the ubiquitin ligase E6AP/UBE3A in controlling levels of the synaptic protein Arc

“…S2A). This finding is reminiscent of the interaction of E6AP with p53 in the absence of the E6 oncoprotein: E6AP does not detectably bind to p53 in coprecipitation assays and is not involved in p53 degradation in HPV-negative cells (31)(32)(33)(34), and yet it is ubiquitinated by E6AP in vitro (35) (Fig. S2A).…”