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Role of Ubiquitin and the HPV E6 Oncoprotein in E6AP- Mediated Ubiquitination

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Role of Ubiquitin and the HPV E6 Oncoprotein in E6AP- Mediated Ubiquitination Role of ubiquitin and the HPV E6 oncoprotein in E6AP- mediated ubiquitination Franziska Mortensena,b, Daniel Schneidera,c, Tanja Barbica, Anna Sladewska-Marquardta, Simone Kühnlea,1, Andreas Marxb,c, and Martin Scheffnera,b,2 aDepartment of Biology, University of Konstanz, 78457 Konstanz, Germany; bKonstanz Research School Chemical Biology, University of Konstanz, 78457 Konstanz, Germany; and cDepartment of Chemistry, University of Konstanz, 78457 Konstanz, Germany Edited by Aaron Ciechanover, Technion-Israel Institute of Technology, Bat Galim, Haifa, Israel, and approved July 7, 2015 (received for review March 25, 2015) Deregulation of the ubiquitin ligase E6 associated protein (E6AP) autism spectrum disorders (15, 16) and (ii) in mice, results in encoded by the UBE3A gene has been associated with three dif- increased E6AP levels and autistic phenotypes (17). ferent clinical pictures. Hijacking of E6AP by the E6 oncoprotein of The notion that alteration of the substrate spectrum, loss of distinct human papillomaviruses (HPV) contributes to the develop- E3 function, and increased E3 function of E6AP contribute to ment of cervical cancer, whereas loss of E6AP expression or func- the development of distinct disorders indicates that expression tion is the cause of Angelman syndrome, a neurodevelopmental and/or E3 activity of E6AP have to be tightly controlled. Whereas disorder, and increased expression of E6AP has been involved in some mechanisms controlling transcription of the UBE3A gene autism spectrum disorders. Although these observations indicate have been identified (e.g., the paternal allele is silenced by a that the activity of E6AP has to be tightly controlled, only little is UBE3A antisense transcript) (14, 18), only little is known about known about how E6AP is regulated at the posttranslational level. how the E3 activity of E6AP is regulated. We recently reported Here, we provide evidence that the hydrophobic patch of ubiquitin that E6AP binds to HERC2, a member of the HECT E3 family, comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiq- and that HERC2 acts as an allosteric activator of E6AP (19). uitination, whereas it does not affect the catalytic properties of The physiological relevance of this interaction is indicated by the the isolated catalytic HECT domain of E6AP. Furthermore, we show finding that a point mutation in the HERC2 gene, resulting in a that the HPV E6 oncoprotein rescues the disability of full-length mutant HERC2 protein with increased turnover rate and hence E6AP to use a respective hydrophobic patch mutant of ubiquitin decreased protein levels, underlies the development of a neu- for ubiquitination and that it stimulates E6AP-mediated ubiquiti- rodevelopmental disorder with AS-like features (20). nation of Ring1B, a known substrate of E6AP, in vitro and in cells. When analyzing the E6AP–HERC2 interaction, we observed Based on these data, we propose that E6AP exists in at least two that a ubiquitin variant, in which the so-called canonical hydro- different states, an active and a less active or latent one, and that phobic patch of ubiquitin is mutated (Ub_hpI), is only poorly the activity of E6AP is controlled by noncovalent interactions with used by E6AP for ubiquitination and that HERC2 can partially ubiquitin and allosteric activators such as the HPV E6 oncoprotein. rescue this disability of E6AP (19). This observation prompted us E6AP/UBE3A | ubiquitin | human papillomavirus | E6 oncoprotein | to take a closer look at the role of ubiquitin in E6AP-mediated Angelman syndrome ubiquitination. We found that different surface areas of ubiquitin affect the ability of E6AP to catalyze the final transfer of ubiq- uitin to a substrate protein by different mechanisms, although n eukaryotes, posttranslational modification of proteins by they are not critically involved in the preceding steps (interaction Iubiquitin plays a pivotal role in the regulation of many cellular processes, including cell cycle, DNA metabolism (e.g., DNA repair, transcription), and various signal transduction pathways Significance (1–4). The specificity of the ubiquitin-conjugation system is mainly ensured by E3 ubiquitin ligases, which mediate the rec- Deregulation of components of the ubiquitin–proteasome ognition of target proteins. Based on the presence of distinct system contributes to the development of various diseases. A domains and their mode of action, E3 proteins can be grouped prominent example is the ubiquitin ligase E6AP/UBE3A, which into three families, RING/RING-like E3s, RING-in-between- is associated with three disorders: in complex with the E6 RING (RBR) E3s, and HECT E3s (5–7). All E3s have in- oncoprotein of human papillomaviruses, it contributes to cer- teraction sites for both substrate proteins and E2 ubiquitin- vical carcinogenesis; loss of E6AP expression results in the conjugating enzymes. However, whereas in the case of RBR E3s development of Angelman syndrome; and increased E6AP ex- and HECT E3s, ubiquitin is transferred from the E3 to sub- pression has been associated with autism spectrum disorders. strates, RING/RING-like E3s function as adaptors between This indicates that E6AP has to be tightly controlled; however, substrates and E2s (i.e., ubiquitin is transferred from the E2 to only little is known about how this is achieved. By analyzing the substrate). the role of ubiquitin and the E6 oncoprotein in E6AP-mediated E6AP, the founding member of the HECT E3 family, was ubiquitination, we provide evidence that E6AP exists in an originally identified as an interacting protein of the E6 onco- active state and a latent state and that its activity is controlled protein of cancer-associated human papillomaviruses (HPVs) (8, by allosteric effectors. 9). The E6–E6AP complex targets the tumor suppressor p53 and — Author contributions: F.M., D.S., S.K., and M.S. designed research; F.M., D.S., T.B., A.S.-M., other proteins which in the absence of E6 are not targeted by and S.K. performed research; F.M., D.S., T.B., A.S.-M., S.K., A.M., and M.S. analyzed data; E6AP—for ubiquitination and degradation thereby contributing and F.M., D.S., S.K., A.M., and M.S. wrote the paper. to HPV-induced cervical carcinogenesis (10, 11). In 1997, it was The authors declare no conflict of interest. UBE3A recognized that alterations in the gene, which encodes This article is a PNAS Direct Submission. E6AP, resulting in loss of E6AP expression or in the expression 1Present address: Department of Microbiology and Immunobiology, Harvard Medical of E6AP variants with compromised E3 activity, are the cause of School, Boston, MA 02115. the Angelman syndrome (AS), a neurodevelopmental disorder 2To whom correspondence should be addressed. Email: [email protected]. – (12 14). Recently, it was reported that amplification of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. UBE3A gene (i) is found in a certain percentage of patients with 1073/pnas.1505923112/-/DCSupplemental. 9872–9877 | PNAS | August 11, 2015 | vol. 112 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1505923112 Downloaded by guest on September 28, 2021 of E6AP with cognate E2 enzymes, thioester complex formation The Hydrophobic Patches of Ubiquitin Are Not Required for Cognate of E6AP with ubiquitin). Furthermore, we show that the HPV E2s to Bind to E6AP. A possible explanation for the requirement of E6 oncoprotein rescues the disability of E6AP to use Ub_hpI, intact patches I and II for E6AP-mediated ubiquitination is that demonstrating that E6 does not only alter the substrate spectrum E6AP interacts noncovalently with (one of) these patches, when of E6AP but also acts as a potent allosteric activator of E6AP. ubiquitin is in thioester complex with E2 enzymes. If this is the case (i.e., E6AP contacts ubiquitin and E2 simultaneously), it can Results be predicted that the ubiquitin loading status of cognate E2s The Hydrophobic Patches of Ubiquitin Are Critical for E6AP-Mediated affects their affinity for E6AP. Due to both the inherent ther- Ubiquitination. Genetic analyses have shown that three distinct modynamic instability of thioester bonds and the fact that acti- surface areas of ubiquitin are essential for viability of Saccha- vated ubiquitin is rapidly transferred from E2 to E6AP, it is romyces cerevisiae (21). Two of these “hydrophobic patches”— notoriously difficult to analyze the binding of E2 enzymes in involving Leu-8 and Ile-44 (termed patch I in the following) and thioester complex with ubiquitin to E6AP. It was recently shown Ile-36, Leu-71, and Leu-73 (termed patch II) (Fig. 1A)—have been that a UbcH5a variant, in which the catalytic Cys residue was more closely examined for their role in ubiquitination mediated replaced by Lys, can be efficiently loaded with ubiquitin via by distinct E2 ubiquitin-conjugating enzymes and E3 enzymes isopeptide bond formation and that the resulting complex is a (e.g., refs. 22–31). Notably, patch II but not patch I was shown reasonable structural mimic of the thioester complex of UbcH5a to be required for covalent attachment of ubiquitin to substrate with ubiquitin (28). Thus, we adopted this strategy to study the interaction of E6AP with UbcH7 and UbcH5b (Fig. S1), which proteins catalyzed by the HECT E3s Rsp5 and NEDD4L (22, 30). both support E6AP-mediated ubiquitination in vitro (19, 35). As In addition, the HECT domains of Rsp5, NEDD4L, NEDD4, and shown by isothermal titration calorimetry and size exclusion SMURF2, which are all members of the NEDD4 subfamily of chromatography, the ubiquitin loading status of UbcH7 does not HECT E3s, harbor a noncovalent binding site for a second ubiq- have a major influence on the ability of UbcH7 to interact with uitin molecule (that is not in thioester complex with the HECT the isolated HECT domain of E6AP (Fig. S2), confirming pre- domain) (23, 24, 32, 33). This interaction is mediated by patch I, viously published data on E6AP–UbcH7 interaction (36).
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