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...

Cardiac myosins and their subunit compositions were studied in ten species of marsupial mammals. Using native gel electrophoresis, ventricular myosin in macropodoids showed three isoforms, V(1), V(2) and V(3), and western blots using specific anti-alpha- and anti-beta-cardiac myosin heavy chain (MyHC) antibodies showed their MyHC compositions to be alphaalpha, alphabeta and betabeta, respectively. Atrial myosin showed alphaalpha MyHC composition but differed from V(1) in light chain composition. Small marsupials (Sminthopsis crassicaudata, Antechinus stuartii, Antechinus flavipes) showed virtually pure V(1), while the larger (1-3 kg) Pseudocheirus peregrinus and Trichosurus vulpecula showed virtually pure V(3). The five macropodoids (Bettongia penicillata, Macropus eugenii, Wallabia bicolour, M. rufus and M. giganteus), ranging in body mass from 2 to 66 kg, expressed considerably more alpha-MyHC (22.8%) than expected for their body size. These results show that cardiac myosins in marsupial mammals are substantially the same as their eutherian counterparts in subunit composition and in the correlation of their expression with body size, the latter feature underlies the scaling of resting heart rate and cardiac cross-bridge kinetics with specific metabolic rate. The data from macropodoids further suggest that expression of cardiac myosins in mammals may also be influenced by their metabolic scope.

The masseter muscle of eutherian grazing mammals typically express beta or slow myosin heavy chain (MyHC). Myosins in the masseter of 4 species of kangaroos and a slow limb muscle of one of them were compared with their cardiac myosin by pyrophosphate and sodium dodecyl sulphate (SDS) gel electrophoresis, immunoblotting and immunohistochemistry. It was found that ventricular muscle contains three isoforms homologous to V1 (alpha-MyHC homodimer), V2 (heterodimer) and V3 (beta-MyHC homodimer) of eutherian cardiac muscle, and that the masseter contained V1, with traces of V2 and V3, in great contrast to eutherian ruminants, which express only V3. A polyclonal antibody (anti-KJM) was raised in rabbits against red kangaroo masseter myosin. After cross-absorption against limb muscle myofibrils, anti-KJM specifically reacted in Westerns with MyHCs from masseter but not limb muscles, and immunohistochemically with masseter, but not limb muscle fibers. In pyrophosphate Western blots, anti-KJM reacted with V1 but not with V3. However, a monoclonal antibody specific for eutherian slow myosin stained all kangaroo slow muscle fibers but only weakly stained scattered fibers in the masseter. The SDS-PAGE revealed that light chain composition of masseter and ventricular myosins is identical, but isoforms of both light chains of kangaroo limb slow myosin were observed. These results confirm that kangaroo jaw muscle express alpha-MyHC rather than beta-MyHC. The difference in MyHC gene expression between marsupial and eutherian grazers may be related to the fact that kangaroos are not ruminants, and have only a single chance to comminute food into fine particles, hence the need for the greater speed and power of muscle contraction associated with V1 containing muscle fibers.

Ventricular myosin in eutherian mammals undergoes a perinatal change in response to a sharp rise in thyroid hormone levels during development. In this investigation, changes in ventricular myosin heavy chains (MyHCs) of the tammar wallaby (Macropus eugenii) from early pouch life to adulthood were analysed using native gel electrophoresis, SDS-PAGE and western blotting. Adult wallaby ventricle showed three myosin isoenzymes, V(1), V(2) and V(3); western blots using specific anti-alpha-MyHC and anti-beta-MyHC antibodies showed their MyHC compositions to be alphaalpha, alphabeta and betabeta, respectively. Ventricular muscle in early pouch joeys expressed predominantly beta-MyHC. Up to 200 days, the time of initial pouch exit, alpha-MyHC content was around 5%. Thereafter, there was a sharp increase of alpha-MyHC expression to 35% by 242 days of age, eventually falling back to 23% in the adult. These changes correlate with known surges in plasma levels of thyroid hormones around pouch exit. The results suggest that ventricular myosins in a marsupial mammal also undergo a developmental change, and that marsupial ventricular myosins are thyroid responsive as in eutherians. The increased alpha-MyHC expression empowers the heart to meet the enhanced cardiovascular demands of out-of-pouch activity and the thermogenic action of thyroid hormones.