Insights into E6AP regulation and function
Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften
(Dr. rer. nat.)
vorgelegt von Simone Kühnle
an der
Mathematisch-Naturwissenschaftliche Sektion Fachbereich für Biologie
Tag der müdlichen Prüfung: 21. Dezember 2011 1.Referent: Prof. Martin Scheffner
2.Referent: Prof. Thomas U. Mayer
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-202339
The greatest glory of living lies not in never falling, but in rising every time we fall.
(Nelson Mandela)
I
I. Table of content
I.
TABLE OF CONTENT ... I
II.
DANKSAGUNG ... V
III.
ABBREVIATIONS ... VI
IV.
SUMMARY ... VII
V.
ZUSAMMENFASSUNG ... IX
1.
INTRODUCTION ... 1
1.1
THE ANGELMAN SYNDROME ... 1
1.2
THE UBIQUITIN PROTEASOME SYSTEM (UPS) ... 2
1.3
E3UBIQUITIN LIGASES ... 5
1.3.1
RING LIGASES ... 5
1.3.2
HECT LIGASES ... 6
1.3.2.1
Nedd4 like ... 7
1.3.2.2
HERC ... 7
1.3.2.3
Single HECT ligases ... 9
1.4
THE UBIQUITIN LIGASE E6AP ... 9
1.4.1
THE ROLE OF E6AP IN CERVICAL CANCER ... 10
1.4.1.1
Human Papillomaviruses (HPV) ... 10
1.4.1.2
The role of E6AP in HPV infected cells ... 11
1.4.1.3
E6-independent functions of E6AP ... 13
1.5
THE GIANT PROTEIN HERC2 ... 15
1.6
STEROID HORMONE SIGNALING ... 17
1.6.1
ESTROGEN RECEPTORS ... 18
2.
AIMS ... 20
3.
MATERIAL AND METHODS ... 21
3.1
MATERIAL ... 21
3.1.1
CHEMICALS ... 21
3.1.2
BUFFERS ... 22
3.1.3
BACTERIAL STRAINS ... 24
3.1.4
MAMMALIAN CELL LINES ... 24
II
3.1.5
CELL CULTURE MATERIAL ... 25
3.1.5.1
Media ... 25
3.1.5.2
Antibiotics ... 25
3.1.5.3
Other materials ... 25
3.1.5.4
Plasticware ... 25
3.1.6
ANTIBODIES ... 25
3.1.6.1
Primary antibodies ... 25
3.1.6.2
Secondary antibodies ... 26
3.1.7
PRIMERS ... 26
3.1.8
PLASMIDS ... 32
3.1.9
MARKER ... 34
3.1.9.1
Protein Standards ... 34
3.1.9.2
DNA Standards ... 34
3.1.10
ENZYMES ... 35
3.1.10.1
Polymerases ... 35
3.1.10.2
Phosphatases ... 35
3.1.11
SOFTWARE ... 35
3.2
METHODS ... 36
3.2.1
TRANSFORMATION OF COMPETENT E.COLI CELLS ... 36
3.2.2
PREPARATION OF PLASMID DNA ... 36
3.2.2.1
Mini Preparation ... 36
3.2.2.2
Midi Preparation ... 36
3.2.3
DETERMINATION OF DNA CONCENTRATION ... 36
3.2.4
RESTRICTION DIGEST ... 37
3.2.5
SEPARATION OF DNA BY ELECTROPHORESIS ... 37
3.2.6
EXTRACTION OF DNA FROM AGAROSE GELS ... 37
3.2.7
LIGATION OF DNA FRAGMENTS ... 37
3.2.8
DNA SEQUENCING ... 37
3.2.9
POLYMERASE-CHAIN-REACTION (PCR) ... 37
3.2.10
PREPARATION OF RNA ... 37
3.2.11
DETERMINATION OF RNA CONCENTRATION ... 38
3.2.12
REVERSE TRANSCRIPTION ... 38
3.2.13
PURIFICATION OF CDNA ... 38
3.2.14
QUANTITATIVE PCR(Q-PCR) ... 38
3.2.15
ANALYSIS OF Q-PCR DATA ... 39
3.2.16
PROTEIN EXPRESSION IN BACTERIA ... 39
3.2.17
AFFINITY PURIFICATION OF GST-FUSION PROTEINS ... 39
3.2.18
ELUTION OF PURIFIED GST FUSION PROTEINS ... 39
3.2.19
PURIFICATION OF BACTERIAL EXPRESSED UBIQUITIN ... 40
III
3.2.20
SDSPAGE ... 40
3.2.21
IN-VITRO UBIQUITINATION ASSAYS ... 40
3.2.22
IN-VITRO THIOESTER ASSAYS ... 40
3.2.23
COOMASSIE-BLUE STAINING ... 41
3.2.24
RADIOGRAPHY ... 41
3.2.25
WESTERN BLOT AND IMMUNODETECTION OF PROTEINS ... 41
3.2.26
DETERMINATION OF PROTEIN CONCENTRATION ... 42
3.2.27
IN-VITRO TRANSLATIION ... 42
3.2.28
IN-VITRO GST-CO-PRECIPITATION ASSAYS (GST-PULLDOWNS) ... 42
3.2.29
CELL CULTURE OF MAMMALIAN CELLS ... 42
3.2.30
TRANSFECTION OF MAMMALIAN CELLS ... 43
3.2.30.1
Lipofection with Lipofectamine 2000 ... 43
3.2.30.2
Lipofection with Turbofect ... 43
3.2.30.3
Nucleofection with Amaxa ... 43
3.2.31
DETERMINATION OF TRANSFECTION EFFICIENCY (ß-GAL ASSAY) ... 43
3.2.32
HARVESTING AND LYSIS OF CELLS ... 44
3.2.32.1
Harvesting of cells ... 44
3.2.32.2
Lysis of cells ... 44
3.2.33
HIS-UBIQUITINATION ASSAYS ... 44
3.2.34
PREPARATION OF CHARCOAL STRIPPED FCS ... 45
3.2.35
SIZE EXCLUSION CHROMATOGRAPHY ... 45
3.2.36
LUCIFERASE ASSAYS ... 45
4.
RESULTS ... 46
4.1
PHYSICAL AND FUNCTIONAL INTERACTION OF THE HECT UBIQUITIN LIGASES E6AP AND HERC2 ... 46
4.1.1
IDENTIFICATION OF HERC2 AS A NEW INTERACTION PARTNER OF E6AP ... 46
4.1.2
ENDOGENOUS E6AP INTERACTS WITH ENDOGENOUS HERC2 ... 46
4.1.3
ALL THREE E6AP ISOFORMS ARE ABLE TO BIND TO ENDOGENOUS HERC2 ... 48
4.1.4
THE RCC1B DOMAIN OF HERC2 MEDIATES BINDING AN N-TERMINAL REGION OF E6AP ... 49
4.1.5
HERC2 IS NOT A SUBSTRATE OF E6AP ... 51
4.1.6
THE ISOLATED RCC1B DOMAIN STIMULATES E6AP LIGASE ACTIVITY IN-VITRO IN A TRANS- UBIQUITINATION REACTION ... 51
4.1.7
BINDING OF RCC1B ON BOTH SUBSTRATE AND LIGASE SITE OF E6AP IS REQUIRED FOR STIMULATION ... 53
4.1.8
RCC1B STIMULATES E6AP AUTO-UBIQUITINATION AT THE E3 LIGASE LEVEL OF THE UBIQUITINATION CASCADE ... 54
4.1.9
RCC1B STIMULATES UBIQUITINATION OF E6AP SUBSTRATES. ... 56
4.1.10
CLONING OF A FULL-LENGTH HERC2 EXPRESSION CONSTRUCT ... 57
IV
4.1.11
ECTOPIC EXPRESSION OF HERC2 STIMULATES E6AP AUTO-UBIQUITINATION IN CELLS ... 59
4.1.12
ECTOPIC EXPRESSION OF HERC2 STIMULATES SUBSTRATE UBIQUITINATION IN CELLS ... 61
4.1.13
HERC2 KNOCKDOWN HAS NO EFFECT ON E6AP SUBSTRATES IN HPV POSITIVE CELLS ... 62
4.2
E6AP A NEGATIVE REGULATOR OF ESTRADIOL SIGNALING ... 64
4.2.1
ARC IS DEREGULATED IN THE ABSENCE OF E6AP WITHOUT BEING A DIRECT SUBSTRATE .... 64
4.2.2
E6AP BINDS TO THE UBL DOMAIN OF RAD23 ... 65
4.2.3
E6AP BINDS TO THE UBL DOMAIN OF SACSIN ... 67
4.2.4
RAD23 BINDS TO AN N-TERMINAL REGION OF E6AP ... 68
4.2.5
E6AP REGULATES ESTROGEN RECEPTOR-MEDIATED ARC TRANSCRIPTION ... 70
4.2.6
E6AP REPRESSES ESTROGEN RECEPTOR SIGNALING IN LUCIFERASE REPORTER ASSAYS ... 71
4.2.7
ENDOGENOUS E6AP REPRESSES ESTROGEN RECEPTOR SIGNALING IN LUCIFERASE REPORTER ASSAYS ... 73
4.2.8
E6AP NEGATIVELY REGULATES THE TRANSCRIPTION OF POTENTIAL ESTROGEN RESPONSIVE TARGET GENES ... 74
5.
DISCUSSION ... 77
5.1
HERC2 AND E6AP ... 77
5.1.1
HERC2:A NEW E6AP INTERACTION PARTNER ... 77
5.1.2
HERC2 BINDS TO E6AP ... 78
5.1.3
HERC2 STIMULATES E6AP AUTO-UBIQUITINATION ... 80
5.1.4
HERC2 STIMULATES E6AP-MEDIATED SUBSTRATE UBIQUITINATION ... 81
5.1.5
A POTENTIAL ROLE FOR HERC2 IN HPV INFECTED CELLS? ... 83
5.1.6
EFFECT OF E6AP ON HERC2 FUNCTION ... 84
5.2
E6AP AND ESTROGEN RECEPTOR SIGNALING ... 85
5.2.1
E6AP INTERACTS WITH THE UBL DOMAIN CONTAINING PROTEINS RAD23 AND SACSIN ... 86
5.2.2
AN N-TERMINAL REGION OF E6AP MEDIATES BINDING TO THE UBL DOMAIN OF RAD23 AND SACSIN ... 88
5.2.3
ARC TRANSCRIPTION IS ESTROGEN RESPONSIVE AND NEGATIVELY REGULATED BY E6AP .... 88
5.2.4
E6AP ACTS AS A REPRESSOR OF ESTROGEN RECEPTOR SIGNALING IN LUCIFERASE ASSAYS . 90
5.2.5
E6AP REPRESSES ESTRADIOL-MEDIATED SFPQ TRANSCRIPTION ... 92
5.3
FUTURE PERSPECTIVES ... 94
6.
LITERATURE ... 98
7.
EIDESSTATTLICHE ERKLÄRUNG ... 112
8.
PUBLICATIONS ... 113
V
II. Danksagung
An erster Stelle bedanke ich mich ganz besonders bei Professor Martin Scheffner für die Möglichkeit an diesem Projekt zu arbeiten, für seine wissenschaftliche Unterstützung und Diskussionsbereitschaft und nicht zuletzt für die Hilfe bei der Erstellung dieser Arbeit.
Herrn Professor Thomas U. Mayer danke ich für die Begutachtung dieser Arbeit.
Kosta gebührt ein ganz großer Dank, für seine ungebrochene Unterstützung während meines gesamten wissenschaftlichen Werdegangs, die vielen kritischen und lehrreichen Diskussionen und vor allem für seine Freundschaft.
Den aktuellen und ehemaligen Mitgliedern der AG Scheffner möchte ich für die gute Arbeitsatmosphäre sowie die Diskussionsbereitschaft und die schöne Zeit im Labor danken. Ein herzliches Dankeschön an Dana und Elli für die konstruktiven Korrekturen Danke speziell an Gregor, für die Geduld und Hilfe bei so einigen Krisen während des Schreibprozesses.
Professor Heike von Baum möchte ich für die Korrektur dieser Arbeit danken.
Auch bei Armin Günther von Trenzyme GmbH bedanke ich mich für seine Einweisung und Hilfe bei allen q-PCR Experimenten.
All meine Studenten schließe ich, für die Mitarbeit an Projekten und Ihr Engagement, in meinen Dank ein, besonders meinen Diplomanden Benedikt für seine begeisterte Arbeit an Rad23 und Sacsin und meine Masterstudentin Julia für Ihre Selbstständigkeit und ihre Geduld mit einer schreibenden Betreuerin.
Danke an Vijay, Alejandro und Toto, ohne Euch hätten die langen Laborabende nicht halb so viel Spaß gemacht.
Ein großes Dankeschön an Susi, aus deren Bachelor-Arbeit eine langjährige Freundschaft entstanden ist. Danke für deine uneingeschränkte Unterstützung in allen Bereichen und für das kritische Lesen dieser Arbeit.
Ganz besonders danken möchte ich meiner „kleinen“ Schwester Kerstin, die seit 28 Jahren meine größte Unterstützung und beste Freundin ist und ohne die nichts von Alledem möglich gewesen wäre.
Last but not least, danke ich ganz herzlich meinen Eltern, so wie Götz und Angelika, für Euren Glauben an mich, Eure finanzielle und moralische Unterstützung und Eure Liebe.
DANKE!!!
VI
III. Abbreviations
AS Angelman Syndrome ORF Open reading frame
βGal beta-Galactosidase p.A. Pro analysi
cDNA coding DNA PBS Phosphate buffered saline
βGal beta-Galactosidase PCR Polymerase chain reaction
CMV Cytomegalovirus Puro Puromycine resistance gene
DMSO Dimethylsulphoxide RNAi RNA interference
DTT Dithiothreitol RT Reverse transcription
EDTA Ethylenediamine tetraacetic acid nt Nucleotide
FCS Fetal calf serum OD Optical density
GST Glutathione-S-transferase ONPG Orthonitrophenyl-b-D- Galactosidase
HPV Human Papillomavirus ORF Open reading frame
IP Immunoprecipitation p.A. Pro analysi
mRNA Messenger RNA PBS Phosphate buffered saline
Mdm Murine double minute PCR Polymerase chain reaction Ni-NTA Nickel-nitrilotriacetic acid Puro Puromycine resistance gene
nt Nucleotide shRNA Small hairpin RNA
OD Optical density siRNA Small interfering RNA
ONPG Orthonitrophenyl-b-D- Galactosidase
wt Wild type
VII
IV. Summary
Angelman Syndrome is a severe neurological disorder caused by mutations in the UBE3A gene, which encodes the HECT E3 ubiquitin ligase E6AP. Since loss-of-function of E6AP is the monogenetic cause for Angelman Syndrome development, the identification of E6AP interaction partners and substrates is crucial to understand the physiology of this disease. Although several E6AP substrates have been identified in the meantime, their stabilization does not or only partially provide explanations for the symptoms observed in Angelman patients. Therefore, the identification of pathways, which are deregulated in the absence of E6AP, is a prerequisite in order to understand E6AP function and its role in Angelman Syndrome physiology. Several approaches have been performed in our lab over the past few years to identify new substrates and interaction partners of E6AP. Among others, we found the giant HECT E3 ligase Herc2 as a potential new E6AP interaction partner. In the course of this project, it was possible to confirm the interaction of E6AP with Herc2 and furthermore map the binding sites required for interaction on both proteins. Moreover, E6AP ubiquitin ligase activity was stimulated upon binding to Herc2, indicating that this interaction has functional consequences. In conclusion, we were able to show that Herc2 does not serve as a substrate of E6AP, but rather acts as an allosteric activator of E6AP, which could probably be important for Angelman Syndrom physiology.
In addition to the identification of E6AP interaction partners, previous proteomics- based approaches performed in our group identified several proteins that showed increased protein levels in the absence of E6AP, therefore representing putative E6AP substrates. Two of the candidates that have been identified in this manner are the proteins Arc and SFPQ. However, E6AP-mediated ubiquitination and degradation of these two proteins could not be demonstrated, indicating that Arc and SFPQ protein levels are regulated via pathways other than the ubiquitin-proteasome system. The facts that E6AP is a known modulator of steroid receptor signaling and that the promoters of Arc and SFPQ, respectively, contain putative estrogen-responsive elements, led us to investigate the role of E6AP in the transcription of estrogen responsive genes. Indeed, E6AP displayed a negative effect on estrogen receptor activity in luciferase reporter assays, indicating that E6AP acts as a repressor in this type of assay. Moreover, estrogen receptor signaling stimulated transcription of endogenous Arc and SFPQ genes and thus both could be classified as estrogen responsive genes. In addition, E6AP knockdown resulted in increased transcription of the Arc and SFPQ genes. Based on these results a model is proposed in this thesis, in which E6AP acts as a repressor of estrogen receptor mediated transcription. This offers a new perspective for the physiology of Angelman Syndrome, as global
VIII
deregulation of estrogen responsive genes can potentially explain a variety of symptoms observed in Angelman patients.
IX
V. Zusammenfassung
Angelman Syndrom ist eine schwere neurologische Erkrankung, welche durch Mutationen im UBE3A Gen ausgelöst wird. Dieses Gen kodiert für die HECT E3 Ubiquitin-Ligase E6AP. Da Angelman Syndrom eine monogenetische Erkrankung ist, wird der Verlust von E6AP-Aktivität als Grund für die Erkrankung angenommen. Die Identifizierung neuer Interaktionspartner und Substrate von E6AP ist daher eforderlich um die grundlegenden physiologischen Mechanismen der Erkrankung aufzuklären.
Obwohl mittlerweile einige Substrate von E6AP beschrieben wurden, konnten diese mit Symptomen von Angelman-Patienten nicht, bzw. nur sehr eingeschränkt in Verbindung gebracht werden. Daher ist die Identifizierung der zellulären Signalwege, an welchen E6AP funktionell beteiligt ist, weiterhin wichtig, um die physiologischen Auswirkungen des Verlustes der E6AP Expression in Angelman-Patienten zu verstehen.
In unserer Arbeitsgruppe wurden in den vergangenen Jahren verschiedene experimentelle Ansätze durchgeführt, um neue E6AP Interaktionspartner zu identifizieren. Eines der Proteine, welches als potentielles E6AP-bindendes Protein entdeckt wurde, ist die HECT-E3-Ligase Herc2. Im Laufe dieser Arbeit wurde die Interaktion von E6AP mit Herc2 genauer untersucht und bestätigt. Außerdem konnten die für die Interaktion notwendigen Bindestellen auf beiden Proteinen identifiziert werden. Weiterhin konnte gezeigt werden, dass die Interaktion von Herc2 mit E6AP funktionelle Konsequenzen hat, da die Bindung von Herc2 an E6AP dessen Ligaseaktivität beeinflusst. Herc2 ist kein Substrat für E6AP, stattdessen stellt es einen allosterischen Aktivator für E6AP dar, was potentiell zum Verständis der Physiologie des Angelman Syndroms beitragen könnte.
Zusätzlich zur Identifikation neuer E6AP Interaktionspartner wurden in den vergangenen Jahren verschiedene „Proteomics“-basierte Experimente durchgeführt um Proteine zu identifizieren, welche in Abwesenheit von E6AP erhöhte Proteinkonzentrationen aufweisen und daher potentielle E6AP-Substrate darstellen.
Zwei Proteine, welche in diesem Zusammenhang identifiziert wurden, sind die Proteine Arc und SFPQ. Es konnte jedoch weder eine Ubiquitinierung noch ein E6AP- vermittelter Abbau von Arc und SFPQ beobachtet werden. Daher wurde nach anderen Möglichkeiten gesucht, um die vorhandene Deregulation der Proteinkonzentration zu erklären. Da E6AP bereits vor einigen Jahren als Regulator der Steroid-Rezeptor- vermittelten Signaltransduktion beschrieben wurde und in den Promotoren der für Arc und SFPQ kodierenden Gene potentielle Östrogen-responsive Elemente gefunden werden konnten, wurde im Folgenden der Effekt von E6AP auf Östrogen-vermittelte Transkription untersucht. Tatsächlich konnte in Luciferase-Reporter basierenden Experimenten ein negativer Effekt von E6AP auf die Östrogen-Rezeptor Aktivität
X
beobachtet werden. Dieses Ergebnis lässt vermuten, dass es sich bei E6AP um einen Repressor von Östrogen Rezeptor vermittelter Transkription handelt. Weiterhin konnte gezeigt werden, dass die Transkription der Gene für Arc und SFPQ durch Östrogen Zugabe stimuliert werden kann und es sich daher um Östrogen-responsive Gene handelt. Da „Knockdown“ von E6AP unter Östrogen-Stimulation zu einer erhöhten Transkriptionsrate der Arc und SFPQ Gene führte, kann vermutet werden, dass E6AP auch als Repressor für die endogene Östrogen-vermittelte Transkription agiert.
Basierend auf diesen Daten wird in dieser Arbeit ein Modell vorgestellt, in welchem E6AP als Repressor für Östrogen-Rezeptor vermittelte Transkription dient. Dieses Modell könnte eine neue Perspektive für die Physiologie von Angelman Syndrom bieten, da eine globale Deregulation der Östrogen-vermittelten Transkription eine Möglichkeit bietet, das breite Spektrum der Symptome dieser Erkrankung zu erklären.
Introduction 1
1. Introduction
1.1 The Angelman Syndrome
Approximately 45 years ago, the pediatrician Dr. Harry Angelman first described two patients with severe developmental delays, seizures, jerky movements, and a happy demeanor (Angelman, 1965). In reference to a painting he once saw while visiting a museum in Verona, he named the new syndrome „The Happy Puppet Syndrome“.
Several years later, the syndrome was given the more appropriate designation
„Angelman Syndrome“ (Williams and Frias, 1982). In 1987, a deletion of the chromosomal region 15q11.2-q13 was linked directly to Angelman Syndrome (Kaplan et al., 1987; Magenis et al., 1987) and during the following decade it was shown that disruption of a single gene, UBE3A, is responsible for the development of the disease (Kishino et al., 1997).
Later on it was demonstrated that the disruption of the chromosomal region, in every case of Angelman Syndrome, occurs on the maternal chromosome and it became clear that Angelman Syndrome belongs to the rather rare cases of imprinting disorders. The paternal UBE3A allele is silenced by methylation in a tissue specific manner. While the UBE3A gene is biallelicly expressed in the majority of tissues, it is only expressed from the maternal allele in specific brain regions, including CA3-neurons of the hippocampus, purkinje neurons, and cells of the olfactory bulb (Rougeulle et al., 1997).
The genetic mechanisms that can result in Angelman Syndrome are versatile, including the above-mentioned chromosomal deletion as well as intragenic UBE3A mutations, including frameshift and point mutations, uniparental paternal disomy and imprinting defects (Fang et al., 1999).
Since its initial description, the widely accepted core features of the syndrome, which occur in 100% of Angelman-individuals and enable distinction from other similar neurological diseases, are a combination of: severe developmental delay, behavioral uniqueness, severe speech impairment, and movement or balance disorder (Williams et al., 1995; Williams et al., 2006). However, the severity of phenotypes correlates with the molecular conditions of the patient. For instance, patients with deletions in the 15q11-13 region tend to have a more severe phenotype (e.g., greater developmental delay and more severe seizures) than individuals that bear uniparental paternal disomy or imprinting defects (Lossie et al., 2001; Moncla et al., 1999).
The fact that Angelman Syndrome was directly linked to mutations of a single gene, namely UBE3A, allowed the development of an Angelman Syndrome mouse model.
Introduction 2
The characterization of Ube3a knockout mice and mice deficient for the maternal Ube3a allele revealed that these mice display phenotypes that are similar to the symptoms of Angelman patients, like impaired spatial learning and motor function, ataxia as well as abnormal dendritic spine morphology and hippocampal EEG recordings (Cheron et al., 2005; Dindot et al., 2008; Miura et al., 2002). Thus, the mouse models of Angelman Syndrome are very likely eminently suitable tools to study the physiology of Angelman Syndrome. In addition to the mouse models, an Angelman model has been established in Drosophila melanogaster (Wu et al., 2008). Despite the high evolutionary distance of humans and flies, dube3a knockout flies display some features that coincide with the symptoms of Angelman Syndrome patients and the phenotypes observed in mouse models. Flies deficient for dube3a display abnormal locomotive behavior and impaired long-term memory as well as predestination for ataxia.
An important breakthrough was accomplished when the disease was directly linked to the gene UBE3A and its gene product E6AP. At that time, E6AP had already been reported to function as E3 ubiquitin ligase and Angelman Syndrome turned out to be the first described single gene disorder of the ubiquitination pathway (Kishino et al., 1997; Matsuura et al., 1997). The fact that loss of an E3 ubiquitin ligase results in the development of Angelman Syndrome strongly indicates that deregulation of one or more E6AP substrates cause the observed phenotypes in Angelman Syndrome.
Although several substrates have been identified over the last decade (see 1.4.1.3), the physiological relevance of these substrates for the development of the disease still remains uncertain.
1.2 The Ubiquitin Proteasome System (UPS)
Maintanance of proteostasis is crucial for cellular survival; this includes regulation of protein synthesis as well as protein turnover. One of the major pathways involved in regulated protein turnover is the ubiquitin proteasome system (UPS). Its deregulation is associated with various human diseases, ranging from cancer to several neurological disorders, including Angelman Syndrome and Parkinson’s disease (reviewed in (Ciechanover and Schwartz, 2004)).
Ubiquitin is a small protein consisting of 76 amino acids, which can be covalently attached to substrate proteins in an enzymatic reaction, termed ubiquitination (Hershko and Ciechanover, 1998). Ubiquitin is found in all eukaryotes and highly conserved during evolution. The process of the ubiquitination reaction is an enzymatic cascade requiring the sequential activity of three (or sometimes four) classes of enzymes (Figure 1). In the first step, the ubiquitin-activating enzyme (E1) activates ubiquitin in an ATP dependent reaction, by formation of an ubiquitin-adenylate
Introduction 3
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K48 / K11 K63
Endocytosis Signal transduction
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Figure 1 The ubiquitination cascade. Ubiquitination involves the subsequent reaction of three enzymes.
In a first step, ubiquitin is activated by the E1 enzyme in an ATP-‐dependent reaction and is transferred to a catalytic cysteine of the E1 enzyme upon formation of a thioester bond with the C-‐terminal glycine of ubiquitin. The E1 transfers the ubiquitin to the second enzyme of the cascade (E2) again under thioester bond formation. The third step involves an E3 ligase that transfers ubiquitin to a substrate protein.
Dependent on the class of E3 ligase, different mechanisms are involved. While RING E3 ligases bring ubiquitin-‐loaded E2 and substrate into close proximity and enable transfer of ubiquitin from the E2 to an internal lysine residue of the substrate protein, HECT ligases take ubiquitin over from the E2, again under thioester bond formation and, transfer ubiquitin directly to the substrate protein. Ubiquitin contains seven internal lysine residues that can be used for ubiquitin chain formation. Dependent on the modality of ubiquitination, this can result in very different consequences for the substrate protein.
Introduction 4
intermediate. Then, ubiquitin is transferred to the catalytic cysteine in the active center of the E1 enzyme. This occurs under formation of a thioester bond in-between the catalytic cysteine of the E1 enzyme and the C-terminal glycine residue of Ubiquitin. In the next step, ubiquitin is transferred from the E1’s active site cysteine to the catalytic cysteine of ubiquitin-conjugating enzymes (E2), again via formation of a thioester bond. The third step involves the activity of ubiquitin ligases (E3) which catalyze the transfer of ubiquitin to the substrate (Hershko et al., 1983). In some cases, a fourth class of enzymes (E4) may be involved in building up ubiquitin chains (Hoppe, 2005). Different classes of E3 ubiquitin ligases utilize different mechanisms of ubiquitin transfer. Ubiquitin ligases can be divided into two main groups, RING and HECT E3 ligases (see 1.3.1 and 1.3.2). RING E3 ligases arrange the ubiquitin-loaded E2 enzyme and the substrate protein in close proximity and therefore allow transfer of ubiquitin to the substrate without including enzymatic activity of the E3 itself (Lorick et al., 1999). In contrast, ubiquitination reactions performed by HECT ligases involve an additional enzymatic step. HECT ligases accept ubiquitin from the E2 enzyme under formation of a thioester bond between ubiquitin and a catalytic cysteine in the HECT domain of the enzyme and consequently transfer ubiquitin directly to the substrate protein (Huibregtse et al., 1995).
Ubiquitination of substrate proteins mainly occurs via attachment of the C-terminal glycine residue of ubiquitin to the
ε
-amino group of an internal lysine residue of the substrate protein under formation of an isopeptide bond (Hershko and Ciechanover, 1998). However, attachment of ubiquitin to other amino acid residues, including serine, threonine and cysteine residues as well as head-to-tail linkage of ubiquitin to the α-amino group of the N-terminus of substrate proteins, has been described (Breitschopf et al., 1998; Ciechanover and Ben-Saadon, 2004; Shimizu et al., 2010).Ubiquitin itself has seven internal lysine residues (K6, K11, K27, K29, K33, K48, K63) that can be used for the attachment of additional ubiquitin moieties and thus the formation of ubiquitin chains (poly-ubiquitination). The fate of ubiquitinated proteins is determined by the modality of ubiquitination. While mono-ubiquitination (attachment of a single ubiquitin moiety to a substrate protein) plays an important role in DNA damage response and endocytosis, multi-ubiquitination (multiple monoubiquitination of the substrate protein) was described in association with endocytosis and cellular localization (Hicke, 2001; Spence et al., 1995). Additionally substrates modified with ubiquitin chains have very different fates dependent on the chain type. K48-linked chains that contain four or more ubiquitin moieties are shuttled to the 26S proteasome, followed by subsequent proteolysis (Chau et al., 1989; Thrower et al., 2000). In contrast ubiquitin chains that are linked via K63 play an important non- proteolytic role in intracellular signaling, DNA repair and endocytosis (Deng et al., 2000; Duncan et al., 2006; Spence et al., 1995). K48 and K63 ubiquitin chains appear
Introduction 5
to be the most abundant and thus extensively studied and best-characterized types of ubiquitin chains. However, in the meantime ubiquitination involving K11-linked chains has also been associated with proteolysis by the 26S proteasome in the cellular pathway of ER-associated degradation (ERAD) (Xu et al., 2009) and cell cycle regulation (Jin et al., 2008). In contrast to K48 and K63 ubiquitin chains, much less is known about the function of chains linked by K6, K29 or K33.
Like many other postranslational modifications (e.g., phosphorylation or acetylation) ubiquitination is a reversible mechanism of protein modification. Removal of ubiquitin moieties is referred to as de-ubiquitination and performed via an enzymatic reaction involving hydrolysis of the isopeptide bonds by deubiquitinating enzymes (DUBs).
Different DUBs have been described to be specific for different types of ubiquitin chains (Pickart, 2001). Taken together, ubiquitination of proteins is a highly dynamic system, which can result in very diverse consequences for the modified substrate protein (reviewed in (Komander, 2009)).
1.3 E3 Ubiquitin Ligases
The ubiquitination cascade has a hierarchical structure. The human genome encodes for two activating enzymes (E1) (Jin et al., 2007; McGrath et al., 1991; Pelzer et al., 2007), followed by approximately 40 conjugating enzymes (E2) and possibly 600- 1000 ubiquitin ligases (E3) (Ye and Rape, 2009) . The hierarchical structure and the fact that almost every protein in the cell is targeted by ubiquitination, indicates that the substrate specificity is displayed by the huge number of E3 ligases. As mentioned before, E3 ligases can be mainly divided into two major groups, RING and HECT E3 ubiquitin ligases. However, additional E3 ligases that are structurally related to RING ligases have been described, such as PHD (plant homology domain) proteins and U- box domain proteins (Coscoy and Ganem, 2003; Hatakeyama et al., 2001).
1.3.1 RING ligases
In contrast to HECT ligases, RING E3 ligases facilitate ubiquitination of substrates by mediating the transfer from the E2 enzyme to the substrate in an indirect manner by acting as scaffold proteins that bring substrate and ubiquitin-loaded E2 into close proximity. Nevertheless, it has to be mentioned that very recently so-called RING/HECT hybrids have been described which display thioester formation of ubiquitin within the RING domain and thus perform a direct transfer of ubiquitin to the substrate. This mechanism was described for the RING ligases HHARI and Parkin (Wenzel et al., 2011). With approximately 600 encoding genes, the class of RING ligases is the largest group of E3 ligases and consists of a variety of different structured ligases: single proteins that display ubiquitin ligase activity, for example Mdm2 or Ring1b, or RING ligases that function in large protein complexes, like the
Introduction 6
APC/C (anaphase-promoting) or the SCF (SKP1-CUL1-F-box protein) complexes (summarized in (Deshaies and Joazeiro, 2009)). Although the family of E3 RING ligases harbors members of very different shapes, all of them share a RING (“Really interesting new gene”) domain that can bind to the ubiquitin-loaded E2 and allows the transfer of ubiquitin to substrate proteins. RING domains are composed of the basic sequence Cys-X2 -Cys-X(9-39) -Cys-X(1-3) -His- X(2-3) -Cys-X2 -Cys-X(4-48) -Cys- X2 –Cys ( where X can be any amino acid). The conserved histidine and cysteine residues are buried in the core of the structure and are stabilized by coordination of two Zn atoms (Freemont et al., 1991). Several different RING domains have been described so far and it was shown that in some RING domains cysteine and histidin residiues can be interchanged and can additionally be replaced by other amino acids that are able to coordinate Zn atoms, for example arginine (Zheng et al., 2002). The U-Box domain of U-Box ligases and the PHD domain of PHD ligases are structurally related to the RING domain and are, at least in some cases, able to mediate ubiquitination of substrate proteins (Coscoy and Ganem, 2003; Hatakeyama et al., 2001). It is assumed that binding of RING ubiquitin ligases to their substrates and binding to the ubiquitin-loaded E2 enzyme occurs at distant sites. In single subunit RING E3 ligases the two binding sites by definition have to be located on the same protein. Several RING ligases are however composed of multi subunits where one subunit holds the RING domain and therefore the E3 ligase activity, while another subunit recognizes the substrate protein (Deshaies and Joazeiro, 2009). Additionally some RING E3 ligases do not display E3 ligase activity on their own, but need to form heterodimeric complexes with a second RING protein, leading to the activation of E3 ligase activity. Examples are the Mdm2/MdmX and the Ring1b/Bmi1 heterodimers. In these complexes Mdm2 and Ring1b contain an active RING domain while MdmX and Bmi1 do not. Upon formation of the complexes, Bmi1 and MdmX stimulate the E3 ligase activity of the binding partner (Linares et al., 2003; Wang et al., 2004).
1.3.2 HECT ligases
Members of the HECT family of ligases display a direct catalytic role in the transfer of ubiquitin to substrates. HECT ligases contain a C-terminal HECT (Homologous to E6AP Carboxyl Terminus) domain common to all family members and named after the founding member of the HECT family, E6AP (Huibregtse et al., 1995). HECT domains are bilobal domains consisting of a C-terminal part (C lobe) that contains the catalytic cysteine residue and an N-terminal part (N lobe) that allows binding to the ubiquitin- loaded E2. It is believed that flexible linkers connect N and C lobe and that binding of the N lobe to a ubiquitin-loaded E2 results in a conformational change within the HECT domain. This is assumed to bring the catalytic cysteine residue into close proximity of the E2 and allows transfer of ubiquitin to a conserved catalytic cysteine residue in the
Introduction 7
HECT domain, resulting in formation of a thioester bond with the C-terminal Glycin of ubiquitin (Huang et al., 1999; Ogunjimi et al., 2005; Verdecia et al., 2003).
While the HECT domain is necessary for E2 binding and ubiquitin complex formation, the N terminus of HECT E3 ligases is thought to be responsible for substrate specificity (Huibregtse et al., 1995b). Based on specific motifs present in the N terminus of the HECT ligases, family members can be assigned into three different subfamilies. HECT domain containing proteins that hold WW motifs are classified as Nedd4-like E3 ligases. Family members that contain one or more Rcc1-like domains (RLDs) in their N-terminal extensions are termed HERC proteins (HECT and Rcc1-like). The last group includes HECT ligases with none of the above-mentioned motifs and are thus referred to as the group of single-HECT E3s (Scheffner and Staub, 2007).
1.3.2.1 Nedd4 like
Nedd4-like HECT ligases are characterized by the presence of an N-terminal calcium- dependent phospholipid binding C2 domain that allows the association of family members with the plasma membrane, endosomes or multivesicular bodies (Dunn et al., 2004; Plant et al., 2000). In addition, Nedd4-like ligases contain two to four WW- domains that allow interaction with PY motifs (Pro/Leu-Pro-X-Tyr) of substrate proteins (Staub et al., 1996). Membrane-associated Nedd4-like HECT ligases, like Nedd4-1 or Nedd4-2, play a role in ubiquitination of membrane-bound proteins and in ubiquitin-dependent shuttling of proteins to endosomes. For example, Nedd4-2 was described to interact with the Na+ channel ENaC via its PY motif resulting in ENaC endocytosis and subsequent proteolysis. Deletion of the PY motifs in ENaC are associated with a disease called Liddle Syndrome that is characterized by enhanced Na+ signaling (Abriel et al., 1999; Staub et al., 1996). Several family members, like ITCH or Nedd4 have also been associated with functions in immune response (Fang et al., 2002; Gao et al., 2004).
1.3.2.2 HERC
The HERC subfamily of HECT ligases is characterized by the presence of one or more Rcc1-like (Regulator of chromosome condensation 1) repeats in the N terminus. The HERC protein family consists of six family members, which can be further divided into the large HERC proteins (Herc1 and Herc2) that contain more than one Rcc1-like domain (RLDs) and several additional domains and the small HERC proteins (Herc3-6) that are defined by the presence of only one RLD (Figure2). The Rcc1-like domains are named after their homology to the Regulator of chromosome condensation 1 protein (Rcc1). Rcc1 is composed of mostly seven 51-68 amino acid repeats which display a seven-bladed propeller structure (Renault et al., 2001; Renault et al., 1998).
Functionally, the Rcc1 protein acts as a regulator of chromosome condensation and
Introduction 8
further studies revealed a function as guanine-nucleotide exchange factor for the small GTPase Ran (Bischoff and Ponstingl, 1991; Ohtsubo et al., 1987). The propeller domain of Rcc1 was shown to display two distinct functions: while one face of the propeller is responsible for the GEF activity, the other enables binding to the histones H2A and H2B, therefore tethering Rcc1 to chromatin (Nemergut et al., 2001; Renault et al., 2001). Due to the fact that Rcc1 displays GEF activity, it was not surprising when the large HERC protein Herc1 with its RLD1 was described to be a guanine- nucleotide releasing factor for small G proteins like Arf and Rab (Garcia-Gonzalo et al., 2005; Rosa et al., 1996). However, similar functions have not yet been described for other HERC family members. Besides its function as a GEF, not much is known about the physiological functions of Herc1. Although it has been shown that Herc1 is able to form thioester bonds with ubiquitin in the presence of UbcH5 (Schwarz et al., 1998), only one Herc1 substrate has been identified so far, the tuberous sclerosis complex protein TSC2 (Chong-Kopera et al., 2006). The second large HERC protein Herc2 is of great interest for this work and thus will be described in a separate chapter (1.5).
Small members of the HERC family, including Herc3-6, have been described to interact with each other and colocalize to late endosomes and lysosomes. However, even though they share the same cellular distribution they yet have different interaction partners and display distinct functions. While Herc3 was reported to interact with the ubiquitin-like domain containing proteins h-PLIC1 and h-PLIC2 without targeting these proteins for ubiquitination, Herc5 was identified as the E3 ligase that targets the metastasis suppressor Nm23B for ubiquitination and proteasomal degradation (Hochrainer et al., 2008). However, additionally to its role as E3 ubiquitin ligase, Herc5 was described as the major ligase for conjugation of the ubiquitin-like protein
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Figure 2 The Herc subfamily of HECT ligases. The six Herc proteins can be subdivided into two groups. The large Herc proteins ( 1 and 2) and the small Herc proteins (3-‐6). All Herc proteins hold one or more Rcc1-‐like domain. Additonal individual domains can be found in the large Herc proteins (Rosa, 2005).
Introduction 9
ISG15, thereby regulating the innate antiviral response of cells to interferon (Dastur et al., 2006).
1.3.2.3 Single HECT ligases
The subfamily of Single HECT E3 ligases subsumes all proteins containing a HECT domain but none of the above-mentioned additional protein domains. Thus the single HECT ligases are a very diverse group of ligases with family members, including E6AP, EDD or HUWE1. Although these single-HECT ligases do not contain WW, C2 or Rcc1- like domains, they still have N-terminal protein interaction domains that are crucial for their function. The single Hect E3 ligase EDD (E3 identified by differential display) for example contains an amino-terminal ubiquitin-associated domain (UBA), a PABC domain, and an UBR1-like Zn-finger motif and was described as an E3 ligase for the topoisomerase beta II binding protein TOPBP1 and for ß-Catenin (Hay-Koren et al., 2011; Honda et al., 2002). A second example for a single HECT E3 ubiquitin ligase is E6AP, which will be described in detail in the following chapter (1.4).
1.4 The Ubiquitin Ligase E6AP
E6AP is the founding member of the family of HECT ligases and its name “E6- associated protein” is derived from its ability to bind to the E6 protein of human papillomaviruses (HPV) (Huibregtse et al., 1991). The gene UBE3A, located on chromosome 15q11-13, encodes E6AP (Kishino et al., 1997). Three isoforms are expressed in cells, as the result of alternative splicing and which only differ at their very N termini. The N termini of isoform2 and isoform3 are elongated by 20 and 23 amino acids, respectively (Yamamoto et al., 1997). If these three E6AP isoforms have distinct functions, e.g., regarding substrate specificity or tissue distribution, is still unknown. E6AP belongs to the subfamily of single-HECT ligases and except for the C- terminal HECT domain it does not contain further known protein-protein interaction domains. However, specific amino acid regions of E6AP were shown to mediate binding to interaction partners, such as the HPV E6 oncoprotein. The binding site for E6 was shown to be located between amino acids 378 and 395 of E6AP (isoform1), an 18 amino acid long helical region (Chen et al., 1998; Huibregtse et al., 1993) (Figure3).
As already mentioned, loss of E6AP expression due to UBE3A mutations or deletions, results in development of the severe neurological disorder Angelman Syndrome.
However, in addition to its involvement in Angelman Syndrome, E6AP has also been implicated in HPV-induced cervical carcinogenesis. The basic mechanisms involved in the development of these two diseases are totally different (Figure 3). While loss of E6AP expression in specific brain regions and thus a loss-of-function mechanism is responsible for Angelman Syndrome, a “gain-of-function” mechanism of E6AP, upon
Introduction 10
interaction with the viral E6 oncoprotein, is the major trigger in the development of HPV-induced cervical carcinogenesis.
1.4.1 The role of E6AP in cervical cancer
1.4.1.1 Human Papillomaviruses (HPV)
The cottontail rabbit papillomavirus was the first described papillomavirus that induces benign cutaneous papillomas in rabbits that can progress to invasive squamous cell carcinomas (Rous and Beard, 1935; Shope and Hurst, 1933). Subsequently, papillomaviruses have been isolated from different vertebrates. By today, more than a hundred different types of human papillomaviruses (HPVs) have been described.
Approximately 30 of these HPV types infect the genital tract and are sexually transmitted (de Villiers et al., 2004). Due to the type of lesions that arise upon infection with these genital HPV types, a distinction between the classes of “high-risk”
and “low-risk” types is made. While high-risk HPVs, like HPV16 and HPV18 are associated with almost all cases of cervical cancers, low risk HPV types, such as HPV6 or HPV11 cause genital warts, which do not progress to malignant lesions (zur Hausen, 2002). Only two viral genes are generally expressed in HPV-positive cervical carcinomas, the early gene 6 (E6) and the early gene 7 (E7). The products of these two genes act as oncoproteins that are responsible for the carcinogenic potential of
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Figure 3 E6AP in human diseases. Two different mechanisms involving the E3 ubiquitin ligase activity of E6AP result in human diseases. Activation of E6AP by interaction with the E6 oncoprotein of high-‐risk HPVs results in cervical carcinogenesis, while loss of E6AP expression in specific brain regions results in the severe neurological disorder Angelman Syndrome. Figure derived and modified from (Matentzoglu and Scheffner, 2008)
Introduction 11
high-risk HPV types. By interaction with cellular proteins E6 and E7 inactivate two important tumor supressors, namely p53, the retinoblastoma protein (Rb) and Rb- related proteins. While inactivation of p53 hinders cells to achieve cell cycle arrest, enter apoptosis or senescence, inactivation of Rb and Rb-related proteins drives cells into cell division. Thus, the E6 and E7 oncoproteins cooperate regarding to carcinogenesis (Munger et al., 2004). The facts that cervical cancer is the second most frequent cancer amongst woman worldwide and that 99% of cervical cancers are accompanied by HPV infections point out how important it is to understand the mechanisms of HPV action (Pisani et al., 1999). Thus a lot of effort has been put into molecular and clinical HPV studies to identify the major pathways and participating proteins that are involved in HPV-induced carcinogenesis. Moreover vaccination schemes against HPV infections, including vaccines against the most prominent high- risk HPV types, have been developed and are now commonly available. A cure for already infected women however cannot be realized yet and is probably currently and in future the most interesting research area in the HPV field.
1.4.1.2 The role of E6AP in HPV infected cells
Due to its critical role in the regulation of pro-apoptotic genes and senescence, p53 is probably the most important tumor supressor in cells and thus is referred to as “the guardian of the genome” (Lane, 1992; Vousden and Lane, 2007). Under normal cellular conditions, p53 is kept at low levels by its major regulator, the E3 ubiquitin ligase Hdm2 (Mdm2 in mice). Upon cellular stress, p53 levels increase and p53 operates as inhibitor of cell-cycle progression and as a pro-apoptotic protein (Oliner et al., 1992). Inactivation of p53 in HPV-infected cells is therefore assumed to be a crucial step during carcinogenesis. This step involves the viral E6 oncoprotein, which hijacks the cellular E3 ubiquitin ligase E6AP. The interaction of E6AP with the HPV E6 protein results in a gain-of-function mechanism with respect to E6AP ligase activity, as in complex with high risk E6 proteins E6AP can form a ternary complex with the tumor suppressor p53, inducing p53 ubiquitination and degradation by the 26S proteasome.
Neither E6 nor E6AP alone can bind to p53, thus it is assumed that upon complex formation of E6 and E6AP a binding surface for p53 is formed (Huibregtse et al., 1991;
Scheffner et al., 1993; Scheffner et al., 1990) (Figure 4). P53 is not the only substrate of the E6/E6AP complex, as other substrates have been described in the meantime (Scheffner and Staub, 2007). High risk HPV E6 proteins have been shown to bind a number of PDZ domain-containing proteins, such as h-Dlg and h-Scrib and bring them into complex with E6AP. Like p53, the formation of a ternary complex of E6/E6AP and several PDZ domain-containing proteins results in ubiquitination and subsequent proteasomal degradation of these proteins. However, in contrast to p53, PDZ proteins are able to bind to high-risk HPV E6 proteins in the absence of E6AP. This observation
Introduction 12
is due to the fact that high-risk E6 proteins contain a C-terminal PDZ binding motif, which is not present in low-risk E6 types (Kuballa et al., 2007; Nakagawa and Huibregtse, 2000). A similar effect was observed for p53, as low-risk E6 proteins, such as HPV11E6, are not able to target p53 for degradation, which in part explains why low-risk HPV types are non-carcinogenic. Although it was initially assumed that low- risk HPV E6 proteins are not able to bind to E6AP, more recent data show that this is not the case (Kuballa et al., 2007), i.e low-risk E6 proteins can also bind E6AP without being able to target p53 for ubiquitination and proteasomal degradation.
While the importance of p53 destablization for HPV induced cervical carcinogenesis is commonly accepted, the relevance of PDZ protein degradation is not well established, although it is quite likely that destabilization of these proteins contributes to carcinogenesis. It should also be mentioned that E6 expression does not only alter E6AP substrate specificity, but E6/E6AP interaction additionally stimulates E6AP auto- ubiquitination and turnover (Kao et al., 2000). The effect of E6AP destabilization, or of a switch in E6AP substrate specificity on regular E6AP targets in HPV positive cells is not yet elucidated. The reason for that is, above all, the fact that only very few E6- independent E6AP substrates have yet been identified and the physiological relevance for the majority of E6AP substrates still remains unclear.
Figure 4 (A) Schematic structure of the HECT E3 ligase E6AP. The HECT domain with the catalytic cysteine and the E6 binding site are depicted. Numbers display amino acid residues of E6AP isoform 1. LXXL motifs display potential steroid hormone receptor bindings sites. (B) E6-‐dependent E6AP functions. High-‐risk HPVE6 alters E6AP substrate specificity. The E6/E6AP complex targets p53 and PDZ domain containing proteins for proteasomal degradation. HPVE6 stimulates the auto-‐ubiquitination of E6AP (cis-‐mechanism) and targets it for degradation.
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Introduction 13
1.4.1.3 E6-independent functions of E6AP
It is well established that loss of maternal E6AP expression in specific brain regions results in Angelman Syndrome and significant efforts have been made to gain insights into E6AP function in HPV-negative cells (Matsuura et al., 1997; Rougeulle et al., 1997). However, until now the physiological background of Angelman Syndrome remains unclear. Several E6AP substrates have been identified, including the Calcium/phospholipid binding protein Annexin A1, which is involved in exocytosis, the PML tumor supressor, the cell-cycle regulator CDKN1, and Peroxiredoxin1 (Louria- Hayon et al., 2009; Mishra et al., 2009a; Nasu et al., 2010; Shimoji et al., 2009).
Identification of the human homologues of yeast Rad23 (hHR23A and hHR23B) as E6AP substrates indicated a role of E6AP in DNA damage signaling and/or a direct link to proteasomal degradation (Kumar et al., 1999). Besides its function in DNA damage repair, Rad23 shuttles K48-ubiquitinated proteins to the 26S proteasome. It holds two ubiquitin-associated domains (UBA) that can bind to K48 ubiquitin chains and an ubiquitin-like domain (UBL) which allows direct interaction with the proteasome (Dantuma et al., 2009). In another study, the Ring E3 ligase Ring1b was identified as an E6AP substrate (Zaaroor-Regev et al., 2010). Ring1b functions in complex with a second Ring ligase Bmi-1, and is part of the polycomb group repressive complex (PcG). PcG complexes modify nucleosomal histones and in this manner regulate transcriptional repression epigenetically (Wang et al., 2004). It was shown that Ring1b mono-ubiquitinates Histone H2A and is therefore involved in the initiation and maintenance of silencing PcG target genes. Auto-ubiquitination of Ring1b, in contrast to other ligases, is not involved in regulation of Ring1b turnover but instead leads to activation of E3 ligase activity towards histone H2A (de Napoles et al., 2004). The turnover of Ring1b was shown to be partially dependent on E6AP but other yet unknown E3 ligases seem to contribute to Ring1b degradation. Thus, it was speculated that loss of E6AP expression and subsequent increase of Ring1b levels would lead to increased global repressive transcriptional activity, which could contribute to the pathogenesis of Angelman Syndrome (Zaaroor-Regev et al., 2010). However this hypothesis remains to be proven. A further quite recently identified E6AP substrate is the activity-regulated cytoskeleton associated protein 3.1 (Arc 3.1) (Greer et al., 2010). Due to the fact that Arc is known to be a major regulator of synaptic plasticity, it is a very promising E6AP target. Arc mRNA is transported to synaptic spines, where local protein translation takes place . In synaptic spines Arc regulates the endocytosis of glutamate receptors of the AMPAR subtype on postsynaptic membranes and is therefore directly responsible for the number of AMPAR receptors expressed (Chowdhury et al., 2006; Rial Verde et al., 2006; Shepherd et al., 2006). If Arc is an E6AP substrate, loss of E6AP would result in increased Arc levels and thus to a decrease in the number of AMPAR receptors exposed, which could at least in parts
Introduction 14
explain the phenotype observed in Angelman patients (reviewed in (Bramham et al., 2008)).
None of the reported targets alone is likely responsible for the complex phenotype of Angelman patients. Thus, it must be assumed that either a yet unknown E6AP target plays an important role in disease development or that the totality of deregulated E6AP substrates results in the observed phenotype. Moreover, a third possibility would be that other, E3-independent functions of E6AP contribute to or are responsible for the Angelman phenotype. A hint for the third possibility is the fact that E6AP was reported to act as a regulator of steroid hormone signaling. E6AP positively affects the activity of the estrogen receptor (ER), the Progesterone receptor (PR), the glucocorticoid receptor (GR), the thyroid hormone receptor (TR), the androgen receptor (AR), and the retinoic acid receptor α (RAR-α) (Nawaz et al., 1999), indicating that E6AP functions as a co-activator for these steroid hormone receptors.
Since E6AP ligase activity does not appear to be required for the co-activator function, ubiquitination of target proteins does not seem to be involved in this process. Thus, it was concluded that E6AP displays two distinct functions: as an E3 ubiquitin ligase and a co-activator of steroid hormone signaling.
E6AP itself holds three LXXLL motifs (see Figure 4 A), which represent potential steroid receptor binding sites. Two of these motifs are located in the N-terminal region of E6AP (one in the E6 binding domain) and the third one in the HECT domain (Ramamoorthy and Nawaz, 2008). Although ubiquitination of steroid hormone receptors is not required for E6AP co-activator function, several proteins participating in steroid hormone signaling have been reported as E6AP substrates, e.g. AIB1 (amplified in breast cancer 1) and PR-B (Progesterone receptor beta) (Mani et al., 2006; Ramamoorthy et al., 2010). Since it was proposed that loss of E6AP ligase activity is responsible for the development of Angelman Syndrome, deregulation of steroid hormone signaling was not brought into connection with the pathology of Angelman Syndrome so far (Ramamoorthy and Nawaz, 2008). However, the reproduction phenotype as well as learning deficits in Ube3a-/- mice might be explained by deregulation of sex hormone signaling after all (Jiang et al., 1998).
A big step towards understanding the molecular and cellular basis of Angelman Syndrome was done in 2007, when the phenotype of the disease was directly linked to a decreased activity of the Calcium and Calmodulin dependent Kinase (CamKIIα) (van Woerden et al., 2007). Decreased CamKII activity in Ube3a-/- mice due to an increased inhibitory phosphorylation on Thr305 and Thr306, was the underlying observation for this conclusion. This molecular alteration could be linked to the observed phenotypes in Angelman mice, in that these phenotypes could be rescued by introduction of a constitutive active CamKII mutant, by crossing Ube3a-/- mice with CamKII mutant mice (Thr305Val, Thr306Ala, mice referred to as CamKII305/6+/-).
Introduction 15
This mutant is unable to undergo inhibitory phosphorylation and is therefore constitutively active. Characterization of Ube3a-/- CamKIT305/306+/- mice revealed a rescue of all tested Angelman Syndrome phenotypes, including learning deficiency, seizures, motor dysfunction and impaired long-term-potentiation (van Woerden et al., 2007). Thus, a functional connection between E6AP and CamKII activity could be demonstrated. Additionally the data indicate that E6AP acts upstream of CamKII in the same pathway. Although these data are promising, the direct link between E6AP ligase activity and CamKII activity remains to be uncovered.
Taken together, although intensive research efforts have been made to elucidate the pathways deregulated in Angelman Syndrome, the data obtained so far have not uncovered the physiology behind loss of E6AP expression. Studies to identify new E6AP interaction partners and substrates and thus the involved cellular pathways seem to be necessary to find the missing pieces in this puzzle. One of the proteins identified as a new E6AP interacting protein in our laboratory, is the HECT E3 ligase Herc2.
1.5 The giant protein Herc2
Herc2 is a giant protein of approximately 528kDa and belongs to the HERC subfamily of HECT E3 ligases (see 1.3.2.2). The gene encoding for Herc2 was first mapped to the “pink eyed deletion” locus (p-locus) on chromosome seven in mice. Loss of this chromosomal region results in ocular albinism (Lyon et al., 1992). Later it was shown that an intron in the Herc2 gene contains the promoter for the adjacent OCA2 gene and thus this intron is involved in transcriptional regulation of OCA2, which is responsible for eye color. Thus, the Herc2 protein is not involved in eye color formation (White and Rabago-Smith, 2011). However, mutations in the Herc2 locus in mice were also associated with a second phenotype, referred to as the “juvenile development and fertility“(jdf) or the “runty junky sterile” (rjs) phenotype (Lehman et al., 1998). These mice show reduced growth, impairments in movement and fertility, and disruption of pigmentation. Herc2 shows a ubiquitous tissue distribution, however the highest Herc2 levels were found in brain and testis (Lehman et al., 1998), which in combination with the jdf/rjs phenotypes indicates a role for Herc2 in fertility and in the central nervous system. In addition to the C-terminal HECT domain, Herc2 holds a variety of other protein-protein interaction domains, including a Z-Z-Zinc finger motif (ZZ), a cytochrome b5 like domain (cytb5), a DOC domain, an M-H domain and three Rcc1-like domains (Rcc1a-c) (Garcia-Gonzalo and Rosa, 2005) (Figure 5). The precise function of these domains, except for the HECT domain, remains elusive. However, based on its similarity to other proteins, it was speculated that Rcc1-like domains may
