Towards understanding the role of the ubiquitinligase E6AP in human disease

Die Ubiquitin Ligase E6AP (E6 assoziiertes Protein) stellt das Gründungsmitglied der Familie der HECT (homolog zum E6AP C-Terminus) Ubiquitin Ligasen dar. Das zugehörige Gen UBE3A befindet sich auf Chromosom 15 in der Region q11-13. Interessanterweise kann die Deregulation der E6AP Aktivität mit zwei völlig unterschiedlichen Krankheitsbildern beim Menschen assoziiert werden. Vermutlich stellt E6AP damit zurzeit das wichtigste Beispiel für die Tatsache dar, dass nicht physiologische Modulation des Ubiquitin Konjugationssystems zur Ausbildung von humanen Krankheitsbildern führen kann. So kann z.B. nicht physiologische Aktivierung von E6AP zur Entwicklung von Gebärmutterhalskrebs beitragen, während Inaktivierung von E6AP kausativ zur Entstehung der schweren neurologischen Erkrankung Angelman Syndrom führt. Zusammen genommen bedeutet dies, dass eine genaue Charakterisierung der Signalwege an denen E6AP beteiligt ist, unumgänglich ist, da bis zum heutigen Tag hierzu sehr wenig bekannt ist. Deshalb sollte im Rahmen dieser Arbeit ein Beitrag zur Aufklärung der E6AP Physiologie geleistet werden.Während der experimentellen Arbeiten zu dieser wurde jedoch klar, dass die gängigen Säugerzellkultursysteme zur Überexpression oder für RNA Interferenz vermittelte Depletion von E6AP nicht hinreichend verwendbar waren. Der Grund hierfür war das unerwartete Ergebnis aus Zellkulturexperimenten, dass die ektopische Modulation von E6AP Proteinleveln hochgradig zytotoxisch war, so dass entweder Totalverlust der jeweiligen Kultur oder unzulängliche Expression von E6AP bzw. ineffizienter "knockdown" von E6AP beobachtet wurde. Aus diesen Gründen wurden neuartige Zellkultursystem entwickelt, bei denen die Überexpression bzw. der "knockdown" stringent an einen selektierbaren Marker gekoppelt wurde, so dass die üblichen Fallstricke umgangen werden konnten. Für die Überexpression konnte dies durch eine Ubiquitin-Fusionsstrategie erreicht werden, bei der die Antibiotikumsresistenz Puromycin N-Acetlytransferase an Ubiquitin, gefolgt von dem zu exprimierenden Protein fusioniert wurde. Hierbei wird die Tatsache ausgenutzt wird, dass Proteine, die an den C-Terminus von Ubiquitin fusioniert wurden, in Eukaryonten effizient durch zelleigene Ubiquitin spezifische Protease gespalten werden. Ergebnisse aus Experimenten, bei denen das beschrieben Konstrukt verwendet wurde, haben gezeigt, dass wie erwartet, jede transfizierte und Puromycin resistente Zelle das zu exprimierende Protein exprimierte. Eine ähnliche Strategie wurde verwendet um ein RNAi-basierte Knockdownsystem zu etablieren, dass die gleichen Eigenschaften zeigt, wie das Ubiquitin-Fusionssystem. Darüber hinaus wurden bei Systemen zu induzierbaren Systemen weiter entwickelt, die im Folgenden für diverse Fragestellungen verwendet wurden. Aus diesen Experimenten lies sich ableiten, dass die Deregulation von E6AP Expression in beide Richtungen zu drastischen zytotoxischen Effekten in Säugerzellen führt, welche sich nur durch Einstellen des normalen Expressionslevels von E6AP revertieren ließen. Darüber hinaus konnte durch Überexpressionsexperimente mit E6AP Mutanten gezeigt werden, dass der beobachtete zytotoxische Effekt von der Liagseaktivität von E6AP abhängt.Die entwickelten Expressionssysteme wurden weiterführend vor allem dazu verwendet, die Einflüsse von E6AP Expression auf das zelluläre Proteom und Ubiquitom zu untersuchen. Ergänzt wurden diese Experimente durch klassische Affinitätsansätze, mit denen nach E6AP Interaktionspartnern gesucht wurde. Daten aus all diesen Experimenten lieferten drei Proteine, deren Funktion möglicherweise von E6AP beinflusst sein kann. Diese waren Herc2, Nesprin-2 und die schwere Kette des nicht-Muskle Myosins IIA (MYH9). Ein weiterführende Charakterisierung der Kandidaten ergab, dass es sich bei dem HECT Protein Herc2 um einen direkten Interaktionspartner von E6AP handelt, während sich keine Effekt durch E6AP auf MYH9 duch Co-Überexpressionsanalysen in Zellen nachweisen ließ und dies obwohl MYH9 in vitro sehr effizient von E6AP ubiquitiniert wurde. Ähnlich verhielt es sich für das 800 kD große Nesprin-2, ein Protein der Kernmembran. Auch hier konnte keine Interaction mit E6AP nachgewiesen werden. Überraschenderweise jedoch, führt die Überexpression einer inaktiven Mutante von E6AP zu einer quantitativen Relokalisierung von Nesprin-2 aus der Kernmembran hinaus ins Zytosol. Zusätzlich konnte gezeigt werden, dass in Purkinje Neuronen von AS Mäusen, mit den verwendeten Methoden, kein Nesprin-2 in der Kernmembran detektiert werden kann während die Nesprin-2 Lokalisation in wild typischen Kontrollmäusen nicht verändert war. Außerdem konnte für E6AP, Herc2 und Nesprin-2 in humanen und murinen Embryonen eine überlappende Gewebeexpression gezeigt werden (Nebennierenmark, Testis u. spinale Neuronen)

Ubiquitin ligase E6-AP and its role in human disease

The ubiquitin ligase E6-AP (E6-associated protein) represents a prime example for the notion that deregulated modification of proteins with ubiquitin contributes to the development of human disease: loss of E6-AP function by mutation is responsible for the development of AS (Angelman syndrome), a neurological disorder, and unscheduled activation of E6-AP by complex formation with the E6 oncoprotein of HPVs (human papillomaviruses) contributes to cervical carcinogenesis. However, while there is a considerable amount of data concerning the oncogenic properties of the E6-E6-AP complex, only little is known about the function(s) of E6-AP in neurons. This is mainly due to the fact that although some E6-AP substrates have been identified, it is at present unclear whether deregulated modification/degradation of these proteins is involved in the pathogenesis of AS. Similarly, the cellular pathways involving E6-AP remain enigmatic. To obtain insights into the physiological functions of E6-AP, we are currently employing several strategies, including quantitative affinity proteomics and RNA interference approaches. The results obtained will eventually allow the introduction of E6-AP into functional protein networks and so reveal potential targets for molecular approaches in the treatment of E6-AP-associated diseases

Ubiquitin-fusion protein system: a powerful tool for ectopic protein expression in mammalian cells

Clonal cell lines stably expressing a protein of interest are frequently used in the characterization of the potential physiological functions of mammalian proteins, but the generation of such cell lines is laborious and time-consuming. Here, we made use of the fact that fusion proteins consisting of ubiquitin linked to the N terminus of a protein of interest are efficiently processed by ubiquitin-specific proteases into their respective free proteins within eukaryotic cells, and designed an expression system that allows easy and highly efficient selection of mammalian cells ectopically expressing a protein of interest

The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins including the human homologs of Dlg and Scribble for degradation via the ubiquitin-proteasome system. Similar to p53 degradation, E6-induced degradation of Scribble is mediated by the ubiquitin ligase E6-AP. In contrast, degradation of Dlg in vitro and within cells has been reported to be independent of E6-AP, suggesting that the E6 oncoprotein has the ability to interact with ubiquitin ligases other than E6-AP. Furthermore, the ability of the E6 oncoprotein to interact with these yet unidentified ubiquitin ligases may be shared by the E6 protein of so-called low risk human papillomaviruses that are not associated with cervical cancer. In this study, we used the RNA interference technology and mouse embryo fibroblasts derived from E6-AP-deficient mice to obtain information about the identity of the ubiquitin ligase(s) involved in E6-mediated degradation of Dlg. We report that, within cells, E6-mediated degradation of Dlg depends on the presence of functional E6-AP and provide evidence that the E6 protein of low risk human papillomaviruses functionally interacts with E6-AP. Based on these data, we propose that, in general, the proteolytic properties of human papillomavirus E6 proteins are mediated by interaction with E6-AP

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

Inactivation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with development of the Angelman syndrome. Recently, it was reported that in mice, loss of E6AP expression results in increased levels of the synaptic protein Arc and a concomitant impaired synaptic function, providing an explanation for some phenotypic features of Angelman syndrome patients. Accordingly, E6AP has been shown to negatively regulate activity-regulated cytoskeleton-associated protein (Arc) and it has been suggested that E6AP targets Arc for ubiquitination and degradation. In our study, we provide evidence that Arc is not a direct substrate for E6AP and binds only weakly to E6AP, if at all. Furthermore, we show 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

Protein tyrosine phosphatase H1 is a target of the E6 oncoprotein of high-risk genital human papillomaviruses

The E6 proteins of high-risk genital human papillomaviruses (HPV), such as HPV types 16 and 18, possess a conserved C-terminal PDZ-binding motif, which mediates interaction with some cellular PDZ domain proteins. The binding of E6 usually results in their ubiquitin-mediated degradation. The ability of E6 to bind to PDZ domain proteins correlates with the oncogenic potential. Using a yeast two-hybrid system, GST pull-down experiments and coimmunoprecipitations, we identified the protein tyrosine phosphatase H1 (PTPH1/PTPN3) as a novel target of the PDZ-binding motif of E6 of HPV16 and 18. PTPH1 has been suggested to function as tumour suppressor protein, since mutational analysis revealed somatic mutations in PTPH1 in a minor fraction of various human tumours. We show here that HPV16 E6 accelerated the proteasome-mediated degradation of PTPH1, which required the binding of E6 to the cellular ubiquitin ligase E6-AP and to PTPH1. The endogenous levels of PTPH1 were particularly low in HPV-positive cervical carcinoma cell lines. The reintroduction of the E2 protein into the HPV16-positive cervical carcinoma cell line SiHa, known to lead to a sharp repression of E6 expression and to induce growth suppression, resulted in an increase of the amount of PTPH1. Our data suggest that reducing the level of PTPH1 may contribute to the oncogenic activity of high-risk genital E6 proteins

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organspecific regulation of ubiquitin activation

E6AP promotes the degradation of the PML tumor suppressor

The promyelocytic leukemia (PML) tumor suppressor is essential for the formation of PML nuclear bodies (NBs). PML and PML-NBs have been implicated in the regulation of growth inhibition, senescence and apoptosis. PML is activated in response to stress signals and is downregulated in certain human cancers. However, the factors mediating PML stability are incompletely understood. Here we demonstrate that a catalytically active form of the mammalian E3 ligase E6AP (HPV E6-associated protein) acts to reduce the half-life of the PML protein by promoting its degradation in the proteasome. E6AP mediates the ubiquitination of PML in an in vitro ubiquitination assay. E6AP and PML interact at physiological levels and colocalize in PML-NBs. Importantly, PML protein expression is elevated in multiple organs and cell types from E6AP null mice and in lymphoid cells is associated with increased number and intensity of PML-NBs. This PML elevation is enhanced in response to DNA damage. Our results identify E6AP as an important regulator of PML and PML-NBs

Mutation of HERC2 causes developmental delay with Angelman-like features

Background: deregulation of the activity of the ubiquitin ligase E6AP (UBE3A) is well recognised to contribute to the development of Angelman syndrome (AS). The ubiquitin ligase HERC2, encoded by the HERC2 gene in patients with Angelman/Prader-Willi syndrome, is thought to be a key regulator of E6AP.Methods and results: using a combination of autozygosity mapping and linkage analysis, we studied an autosomal-recessive neurodevelopmental disorder with some phenotypic similarities to AS, found among the Old Order Amish. Our molecular investigation identified a mutation in HERC2 associated with the disease phenotype. We establish that the encoded mutant HERC2 protein has a reduced half-life compared with its wild-type counterpart, which is associated with a significant reduction in HERC2 levels in affected individuals.Conclusions: our data implicate a model in which disruption of HERC2 function relates to a reduction in E6AP activity resulting in neurodevelopmental delay, suggesting a previously unrecognised role of HERC2 in the pathogenesis of A