RNA inteference and "in cellulo" reconstitution assays as tools to uncover the cellular functions of the ubiquitin-protein ligase E6-AP
Final Report Abstract
In eukaryotes, posttranslational modification of proteins by ubiquitin plays a pivotal role in the regulation of many cellular processes, including cell cycle, DNA metabolism (e.g., DNA repair), and signal transduction pathways. Thus not surprisingly, deregulation of components of the ubiquitin system contributes to the development of various diseases. A prominent example is the ubiquitin ligase E6AP, which is encoded by the UBE3A gene and associated with three distinct clinical pictures: in complex with the E6 oncoprotein of human papillomaviruses, it contributes to cervical carcinogenesis; loss of E6AP expression results in the development of Angelman syndrome (AS); and increased E6AP expression has been associated with autism spectrum disorders. Ubiquitin ligases mediate the recognition of substrate proteins thereby ensuring the specificity of the ubiquitin system. Thus, to understand the role of E6AP in human disease, it is essential to identify the substrate spectrum of E6AP and the mechanisms, by which E6AP activity is controlled. In this study, we showed that the giant protein HERC2, which is like E6AP a member of the HECT family of ubiquitin ligases, binds to E6AP. Furthermore, we provided evidence that HERC2 stimulates the ubiquitin ligase activity of E6AP in vitro and within cells and that this stimulatory effect does not depend on the ubiquitin ligase activity of HERC2. Together with other findings from our group, the data indicate that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by allosteric activators. Evidence for the physiological relevance of the ability of HERC2 to activate E6AP was subsequently provided in a collaborative study with the group of A.H. Crosby (St George's University of London, UK). The group of A.H. Crosby identified a missense mutation in the HERC2 gene as the putative cause of a rare neurodevelopmental disorder with AS-like features. We showed that the missense mutation results in a mutant HERC2 protein with a decreased half-life compared to the wild-type protein, and this appears to be mirrored in decreased HERC2 levels in individuals with this disorder. Thus, we propose that due to the HERC2 mutation, the activity of E6AP is compromised resulting in neurodevelopmental delay. During this study, it was reported that in mice, loss of E6AP expression results in increased levels of the synaptic protein Arc - which mediates endocytosis of AMPA receptors at excitatory synapses - and concomitantly in impaired synaptic function, providing an explanation for some phenotypic features of AS patients. Furthermore, it was shown that E6AP negatively regulates Arc protein levels, and it was suggested that E6AP targets Arc for ubiquitination and degradation. In our study, we provided evidence that Arc is not a direct substrate for E6AP and binds only weakly to E6AP, if at all. Furthermore, we showed that down-regulation of E6AP expression stimulates estradiol-induced transcription of the Arc gene. Thus, we propose that Arc protein levels are controlled by E6AP at the transcriptional rather than at the posttranslational level. The mechanism(s), by which E6AP affects estradiol signaling, remain(s) to be determined.
Publications
- (2008). Ubiquitin ligase E6-AP and its role in human disease. Biochem Soc Trans 36, 797-801
Matentzoglu K, Scheffner M
- (2011). Physical and functional interaction of the HECT ubiquitin-protein ligases E6AP and HERC2. J Biol Chem 286, 19410-19416
Kühnle S, Kogel U, Glockzin S, Marquardt A, Ciechanover A, Matentzoglu K, Scheffner M
- (2013). Mutation of HERC2 causes developmental delay with Angelman-like features. J Med Genet 50, 65-73
Harlalka GV, Baple EL, Cross H, Kühnle S, Cubillos-Rojas M, Matentzoglu K, Patton MA, Wagner K, Coblentz R, Ford DL, Mackay DJG, Chioza BA, Scheffner M, Rosa JL, Crosby AH
(See online at https://doi.org/10.1136/jmedgenet-2012-101367) - (2013). The role of the ubiquitin ligase E6AP/UBE3A in controlling levels of the synaptic protein Arc. Proc Natl Acad Sci USA 110, 8888-8893
Kühnle S, Mothes B, Matentzoglu K, Scheffner M
(See online at https://doi.org/10.1073/pnas.1302792110)
