Investigation of the FAT10 conjugation pathway
Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
an der Universität Konstanz (Fachbereich Biologie)
vorgelegt von
Stella Ryu
Tag der mündlichen Prüfung: 19.04.12
1. Referent: Prof. Dr. Marcus Groettrup, Universität Konstanz 2. Referent: Prof. Dr. Elke Deuerling, Universität Konstanz
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-193793
Table of contents
1
Danksagung 6
2
Zusammenfassung / Summary 7
2.1 Deutsch 7
2.2 English 9
3
Introduction 12
3.1 The Ubiquitin conjugation system 12
3.1.1 E1 ubiquitin activating enzymes 15
3.1.2 The ubiquitin-like modifier activating enzyme 6 (UBA6) 16
3.1.3 E2 conjugating enzymes 17
3.1.4 E3 Ligases 17
3.1.4.1 HECT E3 ligases 18
3.1.4.2 RING finger E3 ligases 18
3.1.5 The tripartite motif (TRIM) protein family 21
3.1.6 TRIM11 22
3.2 Ubiquitin-like proteins (UBLs) 24
3.2.1 Ubiquitin like modifier (ULM): an overview 24
3.2.2 The ubiquitin like modifier FAT10 26
3.2.3 FAT10 conjugation pathway 31
3.2.4 The small ubiquitin-like modifier SUMO 32
3.3 Inhibitor of apoptosis protein (IAP) family 34 3.3.1 BRUCE represents a special BIR containing protein (BIRP) 36
3.4 Autophagy 39
3.4.1 Ambra1 (activating molecule in Becn1-regulated autophagy) 44
3.5 Transcription factors 46
3.5.1 The transcription factor AP-1 47
3.5.1.1 Structural and biochemical properties of AP-1 48 3.5.1.2 Transcriptional and post-transcriptional regulation of AP-1 50
3.5.2 The transcription factor JunB 50
Table of contents
4
Aim of this study 55
5
Materials and Methods 56
5.1 Materials 56
5.1.1 Chemicals and Materials 56
5.1.2 Reagents and reaction kits 57
5.1.3 Buffers and solutions 57
5.1.3.1 Media for cell culture 57
5.1.3.2 Stock solutions 58
5.1.3.3 Antibiotics and Inductors 58
5.1.3.4 Buffers and Solutions for Agarose Gel Electrophoresis (AGE) 58 5.1.3.5 Buffers and Solutions for SDS-Polyacrylamide Gel Electrophoresis 59
5.1.3.6 IP and lysis buffer 60
5.1.3.7 Washing and elution buffer 61
5.1.3.8 Cultivation Media and Media Additives for bacteria 62 5.1.3.9 Cultivation Media and Media Additives for yeast 63
5.1.4 Cell culture 67
5.1.4.1 Eukaryotic cell culture 67
5.1.4.2 Prokaryotic cell culture 67
5.1.4.3 Yeast 67
5.1.5 Antibodies 68
5.1.6 Enzymes and other cloning components 68
5.1.7 Recombinant proteins 69
5.1.8 Vectors used for cloning 69
5.1.9 Plasmid constructs 69
5.1.9.1 Primers 70
5.2 Methods 70
5.2.1 Plasmid DNA purification 70
5.2.2 Cloning 71
5.2.2.1 PCR (Polymerase chain reaction) 71
5.2.2.2 Site-directed mutagenesis 73
5.2.2.3 Agarose gel electrophoresis 73
5.2.2.4 Restriction digest 73
5.2.2.5 Ligation 74
5.2.2.6 Preparation of chemically competent E. coli cells 74
5.2.2.7 Transformation into E. coli TOP 10 F’ 75
5.2.3 Expression and purification of recombinant GST-TRIM11 from E. coli 75
5.2.4 Cell culture 75
5.2.5 Transient transfection 76
5.2.6 SDS-PAGE (Sodium dodecylsulphate polyacrylamide gel electrophoresis) 77
5.2.7 Immunoblotting 77
5.2.8 Immunoprecipitation (IP) 78
5.2.9 In vitro FAT10ylation assay of JunB 79
5.2.10 Growth of yeast strains 79
5.2.11 DNA isolation from yeast with glass beads 79
5.2.12 Yeast two-hybrid assay 80
5.2.13 Luciferase reporter assays 82
5.2.14 Immunofluorescence and confocal microscopy 83
6
Results 84
6.1 Yeast two-hybrid screen with UBA6 84 6.1.1 BRUCE interacts non-covalently with UBA6 and FAT10 87 6.1.2 Endogenous FAT10 co-immunoprecipitates with BRUCE 92 6.2 Yeast two-hybrid screen with TRIM11 94
6.2.1 TRIM11 interacts specifically with JunB and Ambra1 in a yeast two-hybrid screen 94 6.2.2 Interaction of JunB with TRIM11 in human cell culture 96 6.2.3 Ubiquitin and FAT10 become isopeptide linked to to JunB 98 6.2.4 Proteasome inhibition augments conjugate formation between JunB and
FAT10 102 6.2.5 JunB has no influence on the degradation rate of FAT10 105 6.2.6 Co-expression of FAT10 hardly affects the degradation of unconjugated
JunB 106 6.2.7 CHX data reveal a role for proteasome dependent degradation of the
conjugate between JunB and FAT10 108
6.2.8 TRIM11 becomes degraded via the proteasome 110 6.2.9 TRIM11 turnover in presence of FAT10 is slightly accelerated 112 6.2.10 Co-expression of TRIM11 does not change protein turnover rates of
JunB and FAT10 113
6.2.11 Conjugate formation of endogenous JunB and FAT10 115 6.2.12 Conjugate formation of JunB and FAT10 under semi-endogenous
conditions 116
Table of contents
6.2.13 In vitro auto-FAT10ylation assay 117
6.2.14 JunB is conjugated with FAT10 on lysine 237 119 6.2.15 Identification of JunB in mass spectrometry analysis 122 6.2.16 Post-translational modification: Phosphorylation of JunB on Serine 259? 124 6.2.17 FAT10 and JunB co-localize at the nuclear membrane and in the cytosol 125 6.2.18 FAT10ylation controls JunB transcriptional activities on minimal AP-1
driven reporter genes 128
6.2.19 Interaction of Ambra1 and TRIM11 in a human cell line 131 6.2.20 Ambra1 interacts non-covalently with ubiquitin and FAT10 133 6.2.21 The proteasome is involved in Ambra1 degradation 136 6.2.22 Ambra1 co-localizes with FAT10 in punctuated structures 140
7
Discussion 146
8
References 175
9
Abbreviations 202
10
Addendum 206
1 Danksagung
Mein Dank gilt Herrn Prof. Dr. Marcus Groettrup für die Bereitstellung des Themas und für die wissenschaftliche und menschliche Unterstützung. Seine Begeisterung für die Wissenschaft ist unendlich und ansteckend.
Danken möchte ich....
Frau Dr. Annette Aichem für die Betreuung am Biotechnologie-Institut Thurgau (BITg) und für die Unterstützung während der gesamten Doktorarbeitszeit.
der Konstanzer Research School Chemical Biology (KoRS-CB) und besonders meinem Promotionskommitee Frau Prof. Elke Deuerling und Herrn Prof. Valentin Wittman und allen Mitgliedern der Graduiertenschule.
Herrn Dr. Daniel Legler für seine freundliche Aufnahme am BITg und dafür, dass er stets ein offenes Ohr für mich hatte.
Karin Schäuble für unzählige Gespräche über Wissenschaft, nächtelangen Laborsessions und vieles mehr, Verena „die Göttin“ Wörtmann und Nicola Catone, die neben der Laborarbeit zu guten Freunden geworden sind.
natürlich der gesamten „FAT10“-Gruppe für eine tolle Zusammenarbeit.
insbesondere Valentina Spinnenhirn, Kathrin Kluge, Andrea Kniepert, Annegret Bitzer und Khalid „the champ“ Wasim für eine unvergessliche Zeit in Konstanz und wissenschaftliche Hilfe. Ihr seid mir sehr ans Herz gewachsen.
Hesso Farhan und Veronica Reiterer für die Hilfe am Mikroskop, für viele tolle Gespräche und kontinuierlichen Schokoladennachschub.
den guten Seelen des BITgs Josepha, Ilona, Conni und besonders Edith Uetz für die Unterstützung bei den Reporterassays, für die „Schweizerdeutsch“-Nachhilfe und für viel Spaß bei der Laborarbeit.
Gunter Schmidtke und Michi Basler, für viele hilfreiche Tipps.
meinen Bürokollegen Margit, Valentina, Francesco und Bruxe.
all meinen Kollegen des BITg und der Immuno-Gruppe des Groettrup-Labors, die ich leider aufgrund von Platzgründen nicht alle persönlich benennen kann.
meiner Familie und meinen Freunden, die mich während meiner gesamten Doktorandenzeit unterstützt hatten. Ganz besonders Andoni, für seine Liebe und Geduld.
Zusammenfassung / Summary
7
2 Zusammenfassung / Summary
2.1 Deutsch
Posttranslationale Modifikationen können die Aktivität, Funktion, Stabilität oder die intrazelluläre Lokalisation von Proteinen verändern. Posttranslationale Konjugation von einen oder mehreren Ubiquitin oder Ubiquitin-ähnlichen Proteinen zu ausgewählten Substraten, Ubiquitinierung genannt, ist eines der wichtigsten und vielfältigsten regulatorischen Mechanismen in der Biologie und erfordert das fortlaufende Zusammenspiel einer 3-Schritt Enzymkaskade. Initiierender Schritt zur Konjugation von Ubiquitin oder Ubiquitin-ähnlichen Proteinen an seine Zielproteine ist die Ausbildung eines energiereichen Thioesters an seinem C-Terminus. Diese Aktivierung erfolgt durch ein Ubiquitin-aktivierendes E1 Enzym.
Das aktivierte Ubiquitin wird in einer Transesterifizierungskaskade auf eines von mehreren Ubiquitin konjugierenden E2-Enzymen übertragen. Das Ubiquitin-beladene E2 und ein spezifisches Substratprotein werden dann von einer Ubiquitin Proteinligase (E3) gebunden, welche den Transfer des aktivierten Ubiquitins zwischen seiner carboxy-terminalen Hydroxylgruppe auf die ε-Amino-Seitenkette eines internen Lysinrestes des Akzeptorproteins katalysiert. Ubiquitin-ähnliche Proteine (UBLs) wie beispielsweise SUMO, NEDD8 und ISG15 werden durch eine vergleichbare E1-E2-E3 Multienzym-Kaskade an ihre Zielproteine ligiert.
Für das Ubiquitin-ähnliche Protein FAT10 ist der Konjugierungsmechanismus noch nicht vollständig erforscht.
Das IFN-γ und TNF-α induzierbare Protein FAT10 ist ein junges Mitglied der Ubiquitin- ähnlichen Proteine, welches über sein C-terminales Di-Glycin-Motiv kovalent an Zielproteine binden kann. Zudem ist es bisher das einzig identifizierte Ubiquitin-ähnliche Protein, welches Substratproteine Ubiquitin-unabhängig für den proteasomalen Abbau markieren kann. Erst vor Kurzem wurde ein FAT10 aktivierendes E1-Enzym, UBA6 und ein FAT10 konjugierendes E2 Enzym, USE1, identifiziert, welches interessanterweise zugleich das erste bekannte Substrat für FAT10 darstellt, da es in cis autoFAT10yliert wird.
Das Ziel dieser Doktorarbeit war die Charakterisierung des Konjugierungswegs von FAT10, beginnend mit der Identifizierung von UBA6 interagierenden Proteinen in einem „Yeast two- hybrid Screen“. Da zu Beginn der Doktorarbeit noch kein FAT10-spezifisches E2 Enzym bekannt war, lag der Fokus hier in der Identifikation von möglichen FAT10 E2 Enzymen.
8 In einem „Yeast two Hybrid Screen“ konnte eine direkte Interaktion von UBA6 und dem C- terminalen Ende von BRUCE gezeigt werden, welches eine vollständige hochkonservierte Ubiquitin-konjugierende Domäne enthält. Diese Interaktion wurde in dieser Arbeit mittels Co- Immunopräzipitations-Experimente mit einem Expressionskonstrukt, welches für das vollständige BRUCE-Protein kodiert, besonders hinsichtlich einer möglichen FAT10 E2 Funktion weiter charakterisiert.
Ein weiteres Ziel der Doktorarbeit lag in der Identifikation von möglichen FAT10 Substraten.
In einem „Yeast Two-Hybrid Screen“ mit einer möglichen FAT10 E3 Ligase TRIM11 als
„bait“-Protein und einer cDNA-Bank aus humanem Thymus, konnte eine spezifische Interaktion zwischen TRIM11 sowohl mit JunB, als auch mit Ambra1 nachgewiesen werden.
Die Ring-Finger E3 Ligase TRIM11 wurde zuvor in einem „Yeast Two-Hybrid Screen“ mit FAT10 und einer cDNA-Bank aus humanem Thymus identifiziert. Sowohl für JunB als auch für Ambra1 konnte eine spezifische Interaktion in HEK293 Zellen mittels Co- Immunopräzipitations-Experimenten mit TRIM11 ermittelt werden. Zudem konnte gezeigt werden, dass JunB kovalent mit Ubiquitin oder FAT10 modifiziert wird, nicht jedoch in Anwesenheit einer FAT10 Mutante, der das C-terminale Di-Glycin Motiv fehlt, welches für die Isopeptidbindung an Substratproteine verantwortlich ist. Dieses Ergebnis weist auf die Bildung eines Konjugats zwischen JunB mit beiden Ubquitin-ähnlichen Proteinen hin, was vermuten lässt, dass JunB sowohl ein Ubiquitin, als auch ein FAT10 spezifisches Substrat darstellt. Zudem konnte auch eine nicht kovalente Bindung von JunB sowohl mit Ubiquitin als auch FAT10 detektiert werden. Ferner konnte mittels „Cycloheximid-Chase“ Experimenten verdeutlicht werden, dass nicht nur die Isopeptid-gebundene Form von JunB und FAT10 für den proteasomale Abbau markiert wird, sondern auch JunB, welches nicht kovalent mit FAT10 modifiziert wurde. Proteasomale Inhibition mittels der Zugabe von MG132 führte zu einer Akkumulation des JunB-FAT10 Konjugats, welches im Falle eines FAT10 Substrats erwartet werden würde. Die Rolle von TRIM11 als FAT10 spezifische E3 Ligase mit JunB als Substrat konnte nicht eindeutig geklärt werden, da JunB in vitro in Anwesenheit von rekombinantem FAT10, UBA6 (E1), USE1 (E2) und TRIM11 (mögliche E3-Ligase) nicht FAT10yliert wurde. Überexpression von TRIM11 führte zu einer Herunterregulierung von Ubiquitin, FAT10 und JunB, als auch den JunB-Ubiquitin oder JunB-FAT10 Konjugaten auf Proteinebene. Mittels Konfokalmikroskopie konnte eine klare Co-Lokalisation von FAT10 und JunB an der nukleären Membran detektiert werden. Die Zugabe von MG132 führte zu einer Translokation von JunB in das Cytoplasma. Als vermutlich funktionale Konsequenz hatte JunB FAT10ylierung in Reporterassays eine deutlich verminderte JunB Transaktivierungs- Leistung zur Folge.
Zusammenfassung / Summary
9 Im Falle von Ambra1 konnte eine nicht-kovalente Bindung sowohl mit Ubiquitin, als auch mit FAT10 mittels Co-Immunopräzipitationsversuchen nachgewiesen werden. MG132 Zugabe führte zu einer Akkumulation von überexprimiertem Ambra1, welches darauf hinweist, dass Ambra1 proteasomal degradiert wird. Akkumulation nach proteasomaler Inhibition konnte allerdings nicht beobachtet werden, wenn Ambra1 und FAT10 co-exprimiert wurden, was darauf schließen lässt, dass die nicht-kovalente Interaktion mit FAT10 vermutlich zu einem anderen oder zusätzlichen Degradationsmechanismus führt. Experimente mit Konfokalmikroskopie verdeutlichen eine eindeutige Co-Lokalisation von FAT10 und Ambra1 in aggresomalen Strukturen, was vermuten lässt, dass die Interaktion von Ambra1 und FAT10 zu einer Translokalisation beider Proteine hin zu solchen Strukturen führt.
2.2 English
Posttranslational modifications are important means to alter a proteins’ activity, function, stability or its intracellular localization. The post-translational conjugation of one or more molecules of Ub and ubiquitin-like proteins (UBLs) to selected substrates, namely ubiquitination, is one of the most important and multifaceted regulatory mechanisms in biology and requires the sequential interaction of a 3-step enzyme cascade. The initiating step for ubiquitin or UBL conjugation to its target proteins is the formation of an energy-rich thioester at its C-terminus. This activation takes places through an ubiquitin activating E1 enzyme. The activated ubiquitin is then transferred in a trans-thioesterification cascade to one of multiple E2 conjugating enzymes. The ubiquitin-charged E2 enzyme and a specific substrate protein are then both bound by a ubiquitin protein ligase (E3), which catalyzes the transfer of the activated ubiquitin between its carboxy-terminal hydroxyl-group onto the ε- amino-side chain of an internal lysine residue of the acceptor protein. Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, SUMO, NEDD8, and ISG15 are transferred by a similar E1-E2-E3 multi-enzyme cascade to its targets. For the ubiquitin-like modifier FAT10, the enzyme cascade has not yet been characterized completely.
The IFN-γ and TNF-α inducible modifier FAT10 is a young member of ubiquitin-like proteins, which can be conjugated to target proteins via its C-terminal diglycine motif. Moreover, it is to date the only identified ubiquitin-like protein, which can assign substrate proteins, in an ubiquitin-independent manner, for proteasomal degradation.
Recently, the FAT10 activating enzyme (E1) UBA6 and a FAT10 conjugating enzyme (E2), namely USE1 was identified, which interestingly, was at the same time the first known substrate for FAT10, as it was auto-FAT10ylated in cis.
10 The aim of this thesis was the characterization of the FAT10 conjugation pathway, starting with the identification of UBA6 interacting proteins in a yeast two-hybrid screen.
Given that at the beginning of the doctoral thesis no FAT10 conjugating E2 enzymes were known so far, the focus here was the identification of potential FAT10 E2 enzymes. In a yeast two-hybrid approach, a direct interaction of UBA6 and the C-terminal end of BRUCE, containing the entire highly conserved ubiquitin conjugating domain, could be shown. This interaction was further characterized with a construct encoding for full length BRUCE via co- immunoprecipitation experiments, especially in terms of exhibiting a putative FAT10 E2 function.
A further aim of this doctoral thesis was the identification of putative FAT10 substrates. In a yeast two-hybrid screen with the putative FAT10 E3 ligase TRIM11 and a cDNA-library from human thymus, a specific interaction between TRIM11 and JunB as well as with Ambra1 could be observed. The RING finger containing E3 ligase TRIM11 was previously identified in a yeast two-hybrid screen with FAT10 and a cDNA-library from human thymus. For both JunB and Ambra1, a specific interaction with TRIM11 could be verified in co- immunoprecipitation assays. Moreover, it could be demonstrated that JunB becomes covalently linked to either ubiquitin or FAT10, but not in the presence of a FAT10 mutant, lacking the di-glycine motif, which is required for isopeptide linkages to substrate proteins.
This result points to a conjugate formation between JunB with the ubiquitin-like modifier ubiquitin and FAT10, which indicates, that JunB is a ubiquitin as well as FAT10 specific substrate. Moreover, a non-covalent linkage of JunB with either ubiquitin or FAT10 could be detected. Furthermore, cycloheximide experiments revealed evidence that not only the isopeptide linked form of JunB and FAT10 became assigned for proteasomal degradation but also JunB, which was non-covalently modified with FAT10. Proteasome inhibition with MG132 led to an accumulation of the JunB-FAT10 conjugate, which would be expected for a FAT10 substrate. The role of TRIM11 as a FAT10 specific E3 ligase could not be solved definitely, due to the fact that JunB, in presence of recombinant FAT10, UBA6 (E1), USE1 (E2) and TRIM11 (putative FAT10 E3) did not become FAT10ylated in vitro. TRIM11 overexpression resulted in a decreased protein level of ubiquitin, FAT10, JunB and also the JunB-ubiquitin and JunB-FAT10 conjugates.
A clear co-localization of FAT10 and JunB at the nuclear membrane could be detected by means of confocal microscopy. MG132 treatment caused the translocation of JunB into the cytosol. As a presumable functional consequence, JunB FAT10ylation led to a reduced JunB trans-activating capacity in reporter assays.
Zusammenfassung / Summary
11 In case of Ambra1, a non-covalent interaction with ubiquitin and FAT10 could be verified in co-immunoprecipitation experiments. Addition of MG132 led to an accumulation of over- expressed Ambra1, indicating that Ambra1 becomes degraded by the proteasome. However, no accumulation after proteasome inhibition was observable, when Ambra1 was ectopically co-expressed with FAT10, suggesting that the non-covalent interaction with FAT10 led to a different degradation mechanism other than the proteasome. Experiments with confocal microscopy illustrate an unambiguous co-localization of FAT10 and Ambra1 in punctuated structures, suggesting that the interaction of Ambra1 and FAT10 led to translocation of both proteins into punctuated structures.
12
3 Introduction
3.1 The Ubiquitin conjugation system
Ubiquitin (Ub) is a highly conserved protein of 76 amino acids (aa), encoded on multiple genes, which was originally isolated by Goldstein and co-workers in the search for hormones derived from the thymus (Goldstein et al., 1975). It is a heat-stable protein found throughout the cells of eukaryotes that folds into a compact globular structure, a so-called ‘β-grasp fold’, wherein five β -sheets pack around a central α-helix.
Although prokaryotes do not possess an Ub homolog, several prokaryotic proteins adopt a β- grasp fold, including MoaD and ThiS (Iyer et al., 2006). There are several mechanistic parallels between the ATP-dependent activation of Ub and MoaD/ThiS, although they differ broadly in function (Iyer et al., 2006; Miranda et al., 2011).
Ub is either found solely distributed in the cell body or mostly covalently attached to substrate proteins. Modifications of substrate proteins with Ub alter their functions, locations or target them for destruction by the 26S proteasome (Kirkin and Dikic, 2007).
The post-translational conjugation of one or more molecules of Ub and ubiquitin-like proteins (UBLs) to selected substrates, namely ubiquitination, is one of the most important and multifaceted regulatory mechanisms in biology (Hershko and Ciechanover, 1998; Jentsch, 1992; Pickart, 2004). It plays an integral role in a wide variety of functions in eukaryotic cells including signal transduction, transcription, heterochromatin formation, genome stability, protein trafficking, cell division, morphogenesis, DNA repair, endocytosis, apoptosis, autophagy and proteasome-mediated proteolysis (Hershko and Ciechanover, 1998;
Hochstrasser, 2009; Jentsch, 1992; Pickart, 2004). Proteasomal degradation was not only the first consequence of ubiquitination to be identified, but is still recognized today as the most prevalent function of ubiquitin modification. The ultimate mechanisms that cells use to ensure the quality of intracellular proteins are on one hand the selective destruction of misfolded or damaged polypeptides to prevent the accumulation of non-functional, potentially toxic proteins (Goldberg, 2003). On the other hand, there is the participation in regulatory mechanisms by selectively destroying key molecules, like transcription factors (Hammond- Martel et al., 2011) or cell cycle regulators by activating cell cycle dependent kinases (Cdks), for instance (Glotzer et al., 1991; Reed, 2003). Another crucial function is the processing of antigens for later presentation on MHCI (Rock and Goldberg, 1999).
Besides, several studies provided evidences that Ub conjugation is involved in the recognition and elimination of intracellular bacteria (Fujita and Yoshimori, 2011; Steele- Mortimer, 2011).
Introduction
13 Prior to conjugation, Ub is expressed as a pro-protein with a C-terminal extension and needs to be proteolytically processed by UBL specific proteases (ULPs) or deubiquitinating enzymes (DUBs), to expose the C-terminal di-glycine motif before yielding functional, monomeric Ub (Jentsch, 1992).
Ubiquitination usually results in the formation of an isopeptide bond between the C-terminus of Ub (G76) and the ε-amino group of a substrate lysine residue. Substrate proteins can be modified by Ub in different ways. Conjugation of a single Ub to a single lysine residue (mono- ubiquitination) is a regulatory modification involved in diverse processes including endocytosis, endosomal sorting, histone regulation, transcription, virus budding and nuclear export (Haglund and Dikic, 2005; Mukhopadhyay and Riezman, 2007). Multiple mono- ubiquitination occurs, when several lysine residues of a substrate are modified by single Ub molecules. Here, a role in receptor internalization and endocytosis has been described (Mosesson et al., 2003).
The ability of Ub to form isopeptide linked polymers is crucial for the versatility of the Ub system. Ub itself contains seven internal lysine residues (K6, K11, K27, K29, K33, K48, and K63) which can be potentially used as acceptors for the attachment of other Ub molecules, allowing the formation of different types of Ub chains (poly-ubiquitination). In addition, linear poly-Ub can be linked by amide bonds formed between the C-terminal glycine residue of Ub and the N-terminal methionine residue of a following Ub (Kirisako et al., 2006).
Substrates with four or more lysine 48 (K48) ~ glycine 76 (G76) linked Ub moieties are usually targeted to the 26S proteasome for degradation, whereas Ub is not degraded along with the substrate but removed and subsequently recycled (Hanna and Finley, 2007;
Hershko and Ciechanover, 1998). However, recent publications reported that other than K48 linked ubiquitin chains are also accepted by the proteasome (Jacobson et al., 2009; Xu et al., 2009).
Chains linked through lysine 63 have a role in endocytosis and NF-kB signaling (Deng et al., 2000; Galan and Haguenauer-Tsapis, 1997; Ikeda and Dikic, 2008). In contrast, K11-linked chains are involved in endoplasmic reticulum associated degradation (ERAD) and mitosis whereas K29-linked chains may participate in Ub fusion degradation (Johnson et al., 1995).
Ubiquitination can in some cases also occur on substrate serine, threonine or cysteine residues (Vosper et al., 2009; Wang et al., 2007).
Moreover, beside modification of internal amino acid residues, Ub conjugation to the N- terminal residue of substrates has been described for several proteins (Breitschopf et al., 1998; Ciechanover and Ben-Saadon, 2004). The impact of Ub conjugation on its substrates is tightly controlled by a variety of specific enzymes which in turn designate the fate of ubiquitinated proteins.
14 Elaborate ATP-dependent conjugation systems covalently and reversibly attach Ub and UBLs to their target proteins (overview in Figure 1).
This process of conjugation and deconjugation is carried out by a stringent enzymatic cascade consisting of a three-step mechanism, whereby in general, the different UBLs have their own discrete E1–E2–E3 cascades and have distinct effects on their targets (Hershko, 1983; Schulman and Harper, 2009).
Figure 1: General conjugation pathway of ubiquitin and ubiquitin-like proteins (UBLs)
Ubiquitin and some ubiquitin-like proteins (UBLs) are processed by either deubiquitinating enzymes (DUBs) or UBL-specific proteases (ULPs) to expose a C-terminal glycine. Conjugation to substrates is an ATP dependent process, facilitated with the help of an enzyme cascade composed of at least three different enzymes. The ubiquitin activating enzyme E1 binds first ATP and then the Ub/UBL which leads to the formation of an Ub- adenylate that serves as a donor of Ub to the E1 active site cysteine. A subsequent trans-thiolation reaction transfers the Ub/UBL to a conserved cysteine residue on the ubiquitin conjugating E2 enzyme. In the final step of the cascade, the Ub/UBL is transferred from the E2 to an ε-amino group of lysine residues on protein substrates.
This final step is usually mediated by E3 ligases that may function in one of two distinct ways: The HECT-like E3 ligases transfer the Ub/UBL from E2 to an internal cysteine through a further trans-thiolation step before transferring it to the target, whereas the RING (U-Box) and A20 finger-type E3 ligases seem to mediate a direct transfer of the UBL to the substrate within the target lysine residues. Figure taken from (Kerscher et al., 2006).
Ub is first linked to an ubiquitin activating enzyme (E1) and becomes activated in an ATP dependent manner (see 3.1.1). Thereupon, the activated Ub is transferred to the ubiquitin conjugating enzyme (E2), where Ub become trans-thiolated to its own conserved active site cysteine (see 3.1.3).
Introduction
15 From an E2 enzyme Ub is transferred to the ε-amino group of a lysine residue either within the target protein or the growing poly-Ub chain, thus forming an isopeptide bond.
This transfer is often assisted by a ubiquitin E3 ligase (see 3.1.4). Ubiquitin E3 ligases recognize substrates for ubiquitination, and are considered to be crucial for determining ubiquitination specificity.
Ubiquitin specific proteases known as deubiquitinating enzymes (DUBs) can remove covalently attached Ub from proteins, thereby controlling substrate activity and/or abundance (Ventii and Wilkinson, 2008).
In some circumstances, an E4 enzyme can act as auxiliary factor to catalyze multi-ubiquitin chain assembly in collaboration with E1, E2 and E3 (Hoppe, 2005; Koegl et al., 1999).
The process of dynamically modifying proteins with Ub and other UBLs creates reversible switches between different functional states of a substrate protein, allowing fine-tuned control of numerous cellular pathways.
3.1.1 E1 ubiquitin activating enzymes
Ubiquitin-activating enzyme E1 (UBA1, UBE1) is the archetype for a family of enzymes, which catalyze the ATP-coupled activation of Ub and other UBLs required for their subsequent conjugation to cellular targets. The general physical and structural features of the E1 family are well conserved. E1s can be classified on the basis of the domain structure.
The so-called canonical E1s include UBE1, NEDD8-activating enzyme (NAE), SUMO- activating enzyme (SAE), UBA6 and UBA7 owing to their related domain structures and enzymatic mechanisms, and the non-canonical E1s include Atg7, UBA4 and UBA5 (Schulman and Harper, 2009).
In the first step, ATP and UBL bind together to form a UBL–acyl adenylate intermediate, releasing inorganic pyrophosphate. The C-terminus of free UBL is adenylated by an ubiquitin activating (E1) enzyme, leaving the Ub–AMP adduct bound to the enzyme. The UBL–AMP then reacts with the E1 active-site thiol to form an E1~UBL thioester.
Subsequently, a second ATP and UBL bind the enzyme as in the first step to form a ternary complex that contains two UBL molecules bound to the E1 (Haas and Rose, 1982; Haas et al., 1982). Charging of an E1 with Ub or a UBL triggers conformational changes in the E1, which exposes a negatively charged groove within a Ub fold to allow the formation of a proper E1~E2 complex (Lee and Schindelin, 2008). This form of E1 is competent for transthiolation of the thioester-bound UBL to a pathway-specific E2 and is required for the downstream function of UBL conjugation (Figure 2).
16
Figure 2: Enzymatic mechanism of the ubiquitin activation and conjugation cycle.
Ub(A) represents ubiquitin that is associated non-covalently at the adenylation active site, and Ub(T) represents ubiquitin that is covalently linked to the catalytic Cys of an E1 enzyme through a thioester bond. Step 1 shows adenylate formation, step 2 shows thioester formation, step 3 shows double ubiquitin loading of E1 and step 4 shows ubiquitin transfer to E2. Step 2 is repeated on the E1 Ub(A)~adenylate generated in step 4 to continue the cycle. Figure taken from (Schulman and Harper, 2009).
The initial activation of Ub was for decades believed for decades to be accomplished solely by a single enzyme designated ubiquitin activating enzyme 1 (UBE1) (Ciechanover et al., 1981; Haas et al., 1982). The surprising discovery that Ub can be stimulated by two different essential E1 enzymes, namely ubiquitin-activating enzyme E1 (UBE1) and ubiquitin-like modifier activating enzyme 6 (UBA6) (Chiu et al., 2007; Pelzer et al., 2007) and that UBA6 can activate two different UBLs (Ub and FAT10) (Chiu et al., 2007) illustrates, that a unilateral assignment of an E1 enzyme to a select UBL is no longer valid, and raises the question how these modifiers compete for activation.
3.1.2 The ubiquitin-like modifier activating enzyme 6 (UBA6)
The activating enzyme UBA6 was 2007 identified as novel E1 enzyme, which can be charged by ubiquitin as well as FAT10 (Chiu et al., 2007; Pelzer et al., 2007). Human UBA6 and UBE1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s.
UBE1 is phylogenetically more closely related to UBA7 (the E1 for ISG15) (Jin et al., 2007), whereas UBE1 and UBA6 show only about ~40 % sequence identity.
UBA6 is uniquely responsible for transferring ubiquitin (Jin et al., 2007) as well as FAT10 to a UBA6-specific E2 enzyme USE1 (Aichem et al., 2010). UBA6 and USE1 are found from humans to zebrafish, as well as sea urchin, and are ubiquitously expressed, but they are absent from worms, flies, plants and yeast, which indicates a selective role in certain multicellular organisms.
Introduction
17 Deletion of the mouse uba6 gene results in embryonic lethality (Chiu et al., 2007) and can block the conjugation of FAT10 to unknown proteins (Aichem et al., 2010). However, mice that lack FAT10 are viable (Canaan et al., 2006), suggesting that the essential functions of UBA6 are not linked to FAT10 activation.
3.1.3 E2 conjugating enzymes
Ubiquitin-conjugating enzymes (E2s) are responsible for transferring UBLs to substrate proteins. Activated UBLs are subsequently transferred to an E2 enzyme, where UBLs become trans-thiolated to its own conserved active site cysteine.
E2s often function with a single or limited number of E3 ligases, although in some cases no E3 is required. Thus, E2s function as key mediators of Ub chain assembly. These enzymes are able to govern the switch from Ub chain initiation to elongation, regulate the processivity of chain formation and establish the topology of assembled chains, thereby determining the consequences of ubiquitination for the modified proteins (Ye and Rape, 2009).
Together, these factors determine the fate of ubiquitinated substrate proteins depending on whether they are mono or poly-ubiquitinated and on the site(s) to which Ub is conjugated. E2 family members possess a highly conserved core ubiquitin-conjugating (UBC) domain, consisting of approximately 150 amino acids (aa) containing the catalytic cysteine (Cys) residue which resides in a shallow groove (Wenzel et al., 2011). Ubiquitin E2 variant (UEV) proteins also have a UBC domain but lack an active site Cys residue (Hurley et al., 2006).
After being charged with a UBL, E2s engage E3s to catalyse ubiquitin transfer to the ε-amino group of a lysine residue either within the target protein or the growing poly-Ub chain, thus forming an isopeptide bond, whereby a single E2 can interact with several different E3s.
3.1.4 E3 Ligases
E3-mediated attachment of Ub/UBL to substrates is highly regulated in response to cellular cues, and can modulate a target protein’s half-life, localization, interactions with protein or DNA partners and many other functions. Substrate selectivity of the Ub proteasome system relies primarily on the specificity of hundreds of E3 ubiquitin–protein ligases in the human genome, which mediate the transfer of activated Ub/UBL from an E2 enzyme to substrates (Varshavsky, 1997). Moreover, the E3 enzyme, in combination with the E2, is also important for determining the topology of the poly-Ub chain.
18 Coupled to various cellular signaling events, ubiquitin E3 ligases ensure that the ubiquitination process is temporally controlled and tightly regulated with a high degree of substrate specificity which enables their function as key regulators in many cellular pathways (Ciechanover, 2003)
There are two major types of E3s in eukaryotes, defined by the presence of either a homologous to E6-AP C-terminus (HECT) (see 3.1.4.1) or a really interesting new gene (RING) domain (see 3.1.4.2), which are characterized by distinct Ub conjugation mechanisms (Fang and Weissman, 2004).
3.1.4.1 HECT E3 ligases
HECT E3 ligases contain an approximately 350–amino acid long C-terminal region homologous to that of E6-associated protein (E6-AP), with a conserved active-site cysteine residue near the C-terminus, through which HECT domain E3 ligases form thioester intermediates with Ub before transferring it to the target protein (Huibregtse et al., 1995;
Scheffner et al., 1995; Schwarz et al., 1998). N-terminal regions are highly variable and may be involved in substrate recognition (Hershko and Ciechanover, 1998).
The HECT domain protein E6-AP is known for its role in binding the E6 protein of oncogenic human papilloma viruses and was first identified to be involved in the rapid degradation of p53 via the Ub dependent proteolytic pathway (Scheffner et al., 1994). In addition to ubiquitinate proteins for degradation by the 26S proteasome, HECT-E3 enzymes regulate the trafficking of many receptors, channels, transporters and viral proteins (Rotin and Kumar, 2009).
3.1.4.2 RING finger E3 ligases
The really interesting new gene (RING) family, which includes the related U-box, B-box, leukocyte-association protein (LAP) domain and plant homeodomain (PHD) containing proteins, is conserved from yeast to humans. This family represents with over 600 members, the most abundant class of E3 ligases which mediates protein ubiquitination. Some of the ubiquitin ligases are single subunit ubiquitin ligases, such as Mdm2, containing the RING finger and the substrate-binding site in the same molecule (Marine and Lozano, 2010), while the majority of the RING E3s are multi-subunit complexes. Well investigated representatives are the cullin RING finger ubiquitin ligases (CRL) (Liu and Nussinov, 2009) and the high molecular mass anaphase promoting complex (APC) (Peters, 2002).
Introduction
19 The RING domain was originally described by Freemont and colleagues as a novel cysteine- rich sequence motif (Freemont et al., 1991). RING domain proteins compared to HECT E3s, do not have catalytic activity themselves but rather act as scaffolding proteins which facilitate the interaction between an E2 and target proteins (Joazeiro and Weissman, 2000).
They bind the E2~Ub thioester together with the substrate, which brings them in close proximity to each other, often conveyed by conformational changes, and thereby permit the Ub transfer from the E2 directly to the target proteins (Deshaies and Joazeiro, 2009; Jentsch, 1992; Kerscher et al., 2006).
In general, an ε-amino group of a Lys residue in the associated substrate attacks the thioester of the transiently associated charged E2, making an isopeptide bond with Ub or UBLs. The discharged E2 then dissociates from the E3, allowing a second charged E2 to interact with the E3 to facilitate a second round of Ub/UBL transfer, either by attack of a Lys residue in ubiquitin itself or by attack of a different Lys in the substrate.
Multiple E2 cycles of E1-mediated Ub/UBL loading and subsequent unloading through a range of mechanisms lead to poly-ubiquitination of the substrate (Figure 3).
Figure 3: Reaction cycle of a RING E3.
RING E3s are bisubstrate enzymes that catalyze the conversion of the reactants E2~Ub and substrate to the products E2 and substrate-Ub. Un-liganded E3 (a) binds substrate and E2~Ub to form the Michaelis complex (b).
It is generally assumed that the two substrates do not need to bind in a predetermined order. (c) Ubiquitin is transferred from E2~Ub to substrate to yield the products, E2 and substrate-Ub. (d) For further ubiquitination to occur, E2 must dissociate to allow a fresh molecule of E2~Ub to bind (e). E2 cannot be recharged on E3 because E1 and E3 use overlapping surfaces to bind E2. The newly recruited E2~Ub transfers its cargo to yield di- ubiquitinated substrate (f). From this scheme, it is evident that the relative rates of substrate-Ub dissociation and E2~Ub recruitment/E2 dissociation can have a major impact on the number of ubiquitins that a substrate receives every time it binds to an E3. Figure taken from (Deshaies and Joazeiro, 2009).
20 The ~70 aa residue RING finger domains bind two zinc ions in a unique "cross-brace"
arrangement through a defined motif of eight highly conserved cysteine and histidine residues as depicted in Figure 4 (b) and (c).
Unlike zinc fingers, the zinc coordination sites in a RING “finger” are interleaved and this arrangement endows the RING domain with a globular conformation, characterized by a central alpha-helix and loops of variable-length separated by several small beta-strands, suitable for protein-protein as well as protein-DNA interactions (see Figure 4 (a)) (Borden and Freemont, 1996). Structures of different RING E3s have shown, that the RING domains interact directly with E2s (Passmore and Barford, 2004).
Figure 4: The RING finger domain
(a) A ribbon diagram, based on a model of the RING finger of CNOT4 bound to the E2 UBCH5B (blue; catalytic cysteine of E2 in yellow), the amino and carboxyl termini are indicated, respectively. (b) A schematic model of a RING finger domain. (c) RING-like sequence variants (coordinating residues are numbered in red, X indicates intervening amino acids followed by spacing in numbers). Figure taken from (Lipkowitz and Weissman, 2011).
Another structurally related domain which confers E3 ligase activity is the U-box domain, which does not contain any zinc coordinating residues but is still able to recruit E2 enzymes (Hatakeyama et al., 2001). In comparison, B-box domains of the TRIM subfamily of RING E3 ligases can bind one zinc atom, respectively, and adopt a similar structure reminiscent of the RING domain but are not capable to recruit E2s (Tao et al., 2008).
Introduction
21 3.1.5 The tripartite motif (TRIM) protein family
The largest family of higher order RING finger-containing proteins are the tripartite motif (TRIM) or RING/B-box/Coiled Coil (RBCC) proteins (Borden, 1998; Reddy et al., 1992).
TRIM proteins are characterized by the presence of the tripartite motif consisting of a RING domain, one or two B-box motifs followed by a coiled-coil region, which frequently mediates hetero- or homodimerization and a variable C-terminus (Borden, 1998; Henry et al., 1998;
Reddy et al., 1992; Rhodes et al., 2005). Notably, this arrangement is conserved throughout evolution, further supporting its functional relevance (Reymond et al., 2001).
TRIM proteins are involved in a plethora of cellular processes such as development and cell growth, apoptosis, cell-cycle regulation and viral response. Consistently, their alteration results in many diverse pathological conditions (Meroni and Diez-Roux, 2005).
To date, very little is known about the biological and molecular mechanisms mediated by the TRIM proteins.
Over the past few years, several TRIM proteins have been reported to control gene expression through regulation of the transcriptional activity of numerous sequence-specific transcription factors. These proteins include the transcriptional Intermediary Factor 1 (TIF1) regulators, Trim19 or promyelocytic leukemia tumor suppressor (PML), the RET finger protein (RFP) and TRIM45, for instance (Kim et al., 1996; Le Douarin et al., 1995;
Moosmann et al., 1996; Quignon et al., 1998; Zhong et al., 2000). The N-terminal region of PML harbours the typical tripartite motif which is essential for PML nuclear body formation in vivo (Jensen et al., 2001). Overexpression of TRIM45 inhibits the transcriptional activities of the transcription factors ElK-1 and AP-1, suggesting that TRIM45 acts as transcriptional repressor in MAPK signaling pathway.
Among the plethora of cellular functions controlled by TRIM proteins, recent studies have demonstrated that TRIM proteins regulate signaling pathways which lead to type I interferon (IFN) induction in response to viral infection. IFNs, a group of secreted cytokines, are the main mediators of innate immunity against viral infection, by up-regulating the expression of many antiviral effectors within cells (Randall and Goodbourn, 2008). Moreover, expression of various TRIM genes is up-regulated in response to IFNs, suggesting the involvement of TRIM proteins in regulating host antiviral activities (Carthagena et al., 2009). Various TRIM proteins, for instance TRIM11, TRIM22, TRIM5α, TRIM19, TRIM25 or PML possess potential roles to trigger either autocrine or paracrine cell defense mechanisms and are possibly involved in innate immunity (Chelbi-Alix et al., 1998; Choi et al., 2006; Gack et al., 2007; Lin et al., 2004; Uchil et al., 2008). Furthermore, other TRIMs have been demonstrated to regulate signaling molecules downstream of pattern recognition receptors (PRRs) (McNab et al., 2011).
22 Almost all TRIM family proteins possess a RING finger domain at their N-terminus. E3 ligase activity and function in ubiquitination processes mediated through the RING finger has been confirmed for some members of the TRIM family, including MID1/TRIM18, Efp/TRIM25, TRIM32 and TRIM22 (Duan et al., 2008; Frosk et al., 2002; Gack et al., 2007; Meroni and Diez-Roux, 2005; Trockenbacher et al., 2001). Very recently it has been shown, that some members of the TRIM superfamily possess SUMO E3 ligase activity, dependent on the TRIM motif, suggesting it to be the first widespread SUMO E3 motif (Chu and Yang, 2011). These TRIM proteins bind both the SUMO-conjugating enzyme Ubc9 and substrates and strongly enhance transfer of SUMOs from Ubc9 to these substrates like for instance tumor suppressor p53 and its principal antagonist Mdm2 or the transcription factor c-Jun.
Furthermore, TRIM E3 activity may be an important contributor to SUMOylation specificity and the versatile functions of TRIM proteins (Chu and Yang, 2011).
As a number of other RING finger proteins either homo- or heterodimerize, dimerization may facilitate optimal E3 ligase activity of TRIMs. There exist variants that retain all of the TRIM domains except the RING finger. In some cases these form heterodimers with RING finger- containing TRIMs via their coiled-coil domains. TRIMs without RING fingers may help to modulate substrate interactions, or serve as substrates themselves. Although the mechanisms how many TRIM proteins operate still remain to be deciphered, the highly conserved modular structure let suggest, that a common biochemical function may underlie their assorted cellular roles.
3.1.6 TRIM11
TRIM11 (52,8 kDa) is a member of the TRIM family proteins containing an N-terminal RING- finger which is a type of zinc finger motif, a B-box type 1, a B-box type 2, a coiled-coil region and a C-terminal PRY/SPRY domain, which constitute the B30.2 domain (Figure 5).
Homooligomers can be formed through its coiled-coil domain (Reymond et al., 2001; Woo et al., 2006). This protein localizes to the nucleus and the cytoplasm (Ishikawa et al., 2006).
Figure 5: Schematic model of TRIM11.
TRIM11 is composed of an N-terminal Ring finger domain, two B-Boxes, followed directly by a leucine coiled coil domain and a Pry and Spry domain which together constitute the B30.2 domain.
Introduction
23 TRIM11 is known to interact with humanin, an inhibitor of Alzheimer-like neuronal insults via its B30.2/SPRY domain and thereby to destabilize this protein. It is proposed to regulate intracellular levels of humanin by acting as an E3 ligase and thus inducing Ub mediated humanin degradation in neural tissues (Niikura et al., 2003). Moreover, humanin interacts with the apoptotic factor Bax and induces antiapoptotic effects by inhibiting the translocation of Bax to the mitochondria (Guo et al., 2003; Zapala et al., 2010). Thus, TRIM11 may upregulate apoptosis by controlling humanin stability.
Another interaction partner for TRIM11 is the activator-recruited cofactor 105 kDa component (ARC105) which mediates chromatin-directed transcription activation and plays a crucial regulatory role for transforming growth factor β (TGF-β) signaling. Co-expression of TRIM11 increased ARC105 degradation which could be rescued through proteasome inhibition.
Further, TRIM11 suppressed ARC105-mediated transcriptional activation induced by TGF-ß which suggests that TRIM11, together with the ubiquitin-proteasome pathway, regulates ARC105 function in TGF-ß signaling (Ishikawa et al., 2006).
Besides, TRIM11 was able to interact in a yeast two hybrid screen with the homeodomain transcription factor Paired-like homeobox 2b (Phox2b), which is one of the key determinants involved in the development of noradrenergic (NA) neurons in both, the central nervous system (CNS) and the peripheral nervous system (PNS) (Hong et al., 2008).
Furthermore, TRIM11 could interact with the transcription factor Paired box gene 6 (Pax6), a protein which is involved in the development and regulation of eyes and other sensory organs, brain, certain neural and epidermal tissues as well as other homologous structures.
The interaction with TRIM11 mediates Pax6 degradation via the ubiquitin-proteasome system. However, abrogation of endogenous TRIM11 expression in the developing cortex increases the level of insoluble forms of Pax6 and enhances apoptosis. This indicates that an auto-regulatory feedback loop between TRIM11 and Pax6 maintains a balance between Pax6 and TRIM11 protein levels in cortical progenitors, indicating an essential role for the Pax6-dependent neurogenesis (Tuoc and Stoykova, 2008).
Moreover, members of the TRIM family of E3 ligases have been shown to interfere with retroviral lifecycle and to exhibit antiviral activities (see 3.1.5). Overexpression of TRIM11 suppressed infectivity of human immunodeficiency virus 1 (HIV-1), by suppressing viral gene expression. This antiviral activity has been shown to depend on a functional TRIM11 E3 ubiquitin-protein ligase domain. Downregulation of TRIM11 enhanced virus release, suggesting that this protein contributes to the endogenous restriction of retroviruses in cells.
24 Notably, while TRIM11 inhibited HIV entry, enhancing effects were observed for murine leukemia virus (MLV). Moreover, TRIM11 plays a role to regulate TRIM5 turnover, a protein which promotes innate immune signaling, via the proteasome pathway, thus counteracting the TRIM5-mediated cross-species restriction of retroviral infection at early stages of the retroviral life cycle (Uchil et al., 2008).
In our laboratory a specific interaction of TRIM11 and the UBL FAT10 could be shown in a yeast two hybrid screen (A. Aichem, personal communication). Further, TRIM11 could interact in vivo with the FAT10 specific E2 enzyme USE1, and siRNA mediated TRIM11 downregulation decreased FAT10 conjugates, indicating that TRIM11 may function as a FAT10 specific RING finger E3 ligase.
3.2 Ubiquitin-like proteins (UBLs)
All eukaryotic cells contain several additional proteins related to ubiquitin, called ubiquitin-like proteins (UBL) with either sequence or structure similarities which can be further subdivided into two separate classes.
The first group of UBLs are a heterogeneous group of ubiquitin domain proteins (UBDs), which bear a ubiquitin-like domain embedded in their sequences and thereby enable the binding of mono- or poly-ubiquitin chains in a non-covalent manner (Di Fiore et al., 2003;
Jentsch and Pyrowolakis, 2000; Kerscher et al., 2006; Schnell and Hicke, 2003; Welchman et al., 2005). Their involvement has been described for various aspects of cellular physiology including protein degradation, receptor trafficking, DNA repair, autophagy and apoptosis (Ikeda et al., 2010). For instance, the first UBD to be published was the proteasome subunit S5A/RPN10 (Young et al., 1998).
The second group presents the ubiquitin-like modifiers (ULMs). These proteins can be covalently attached to target proteins in a similar manner like Ub. To date, there are 17 known ULMs from nine phylogenetically distinct classes (NEDD8, SUMO, ISG15, FUB1, FAT10, Atg8, Atg12, Urm1, and UFM1) that have been identified to conjugate to substrates in a manner analogous to Ub.
3.2.1 Ubiquitin like modifier (ULM): an overview
Ubiquitin-like modifiers in turn function as their name already suggests in “ubiquitin-like”
manner, what means, that they exert their function in being covalently attached to substrate molecules (see Table 1)
Introduction
25
Table 1: Ubiquitin like modifiers.
Modified and extended from (Hochstrasser, 2009).
ULM Identity with
ubiquitin [%] Enzymes Substrates Comments and Functions
NEDD8 58
E1: UBA3-Ula1 heterodimer E2: Ubc12 E3: many
Cullins, p53, Mdm2, synphilin-1
Positive regulator of ubiquitin E3s;
activation and destabilization of SCF;
transcriptional regulation of p53
SUMO-1 18
E1: Aos-1-Uba1 heterodimer E2: Ubc9 E3: RanBP2, Pc2, PIAS
c-Jun, IκB, p53, Mdm2, STAT-1, PML, RanGAP1, RanBP2, PCNA, topoisomeraseII
SUMO encoded by 3-4 genes in vertebrates, depending on the species.
Control of protein stability, function and localization, antagonist to ubiquitin, overlap with SUMO-2/3
SUMO-2/3 16
E1: Aos-1-Uba1 heterodimer E2: Ubc9
RanGAP1, topoisomeraseII
Transcription regulation, cycle progression
ISG15 29, 27
E1: UBE1L E2:UbcH8 E3: Herc5, Efp
PLCγ1, JAK1, STAT1, ERK1/2, serpin 2a
Positive regulator of IFN-related immune response, potentially involved in cell growth and differentiation
FUB1 38 NI NI Derived from a ribosomal protein
precursor
FAT10 29, 36
E1: UBA6 E2: USE1 E3: NI
p53, USE1, huntingtin
Cell cycle checkpoint for spindle assembly, ubiquitin- independent degradation
Atg8 10 E1: Atg7
E2: Atg3
Phosphatidyl- ethanolamine
Three known isoforms in humans.
Contains a β-grasp fold.
Autophagy, cytoplasm-to-vacuole targeting
Atg12 17 E1: Atg7
E2: Atg10 Atg5
20 % identical to Atg8.
Autophagy, cytoplasm-to-vacuole targeting
Urm1
10 (Emboss needle alignment)
E1: UBA4
Ahp1 (in yeast) MOCS3, ATPBD3, CTU2, CAS
Related to the small-sulphur-carrying proteins MoaD and ThiS. Contains a β- grasp fold. Most ancient ULM. Role in tRNA modification. Urm1 modification in the response of cells to oxidative damage.
UFM1
6 (Emboss needle alignment)
E1: UBA5 E2: UFC1 E3: UFL1
C20orf116
Contains a β-grasp fold.
Important for the prevention of ER stress-induced apoptosis.
NI, not identified.
26 3.2.2 The ubiquitin like modifier FAT10
The fat10 gene was discovered in 1996 by chromosomal sequencing of the human major histocompatibility complex (MHC) class I locus (Fan et al., 1996) close to the HLA-F locus, leading to the designation of HLA-F adjacent transcript 10 (FAT10) (Liu et al., 1999). The region of human chromosome 6 encoding the MHC complex contains a diverse set of genes, including genes whose function can be directly related to immune function such as the MHC class I and II gene products, and genes encoding for members of the complement cascade, TNF-a and -ß, the transporter associated with antigen processing (TAP), and the LMP2 and 7 components of the proteasome (Pichon et al., 1996).
FAT10, consisting of 165 amino acids is an ~18 kDa ULM which comprises two ubiquitin-like domains in a head to-tail formation, connected by a short linker peptide. These domains form the same three-dimensional core structure - the β-grasp fold – like Ub, revealing a common ancestry for the modification systems. Similar to Ub, it possesses a free C-terminal di-glycine motif required for covalent conjugation to USE1 (Aichem et al., 2010), p53 (Li et al., 2011) and huntingtin (Nagashima et al., 2011) and to several so far unknown target proteins (Chiu et al., 2007; Raasi et al., 2001). There exists a high degree of sequence similarity between murine and human FAT10 at mRNA and protein levels.
Due to its analogy to a tandem fusion of two Ubs, it was originally called “ubiquitin D” or
“diubiquitin”. The N-terminal and C-terminal ubiquitin-like domains of FAT10 are more closely related to Ub than to each other and show 29 % and 36 % sequence identity to Ub, respectively. Four of the lysine residues involved in poly-ubiquitin-chain formation – corresponding to K27, K33 and most notably K48 and K63 – are conserved in both ubiquitin- like domains of FAT10 (Figure 6). Atypical to Ub, FAT10 contains 4 cysteine residues in its sequence (Bates et al., 1997).
Like Ub, FAT10 is activated by UBA6 (Chiu et al., 2007; Groettrup et al., 2008; Pelzer et al., 2007) and can be transferred to the E2 enzyme USE1 (Aichem et al., 2010). FAT10 is only expressed in vertebrates, i.e. it is evolutionary one of the youngest members of the ULM family. The importance of the regulation of FAT10 expression has been highlighted by different observations.
Initially, up-regulation of FAT10 expression was shown to be restricted to mature dendritic cells (DCs) and B-cells (Bates et al. 1997). Unlike ubiquitin, which is expressed constitutively, constitutive FAT10 mRNA expression at tissue level seems to be limited to organs of the immune system. This was confirmed by Northern blot analysis, in situ hybridization as well as quantitative real-time PCR (qRT-PCR) in organs of the immune system like spleen, gut, lymphnodes and especially thymus (Liu et al., 1999; Lukasiak et al., 2008).
Introduction
27
Figure 6: Sequence comparison and ribbon diagram of Ubiquitin and the predicted tertiary FAT10 model structure
(a) FAT10 is composed of two ubiquitin-like domains (UBLs) which are more closely related to ubiquitin than to each other. Both, the N- and C-terminal UBL show the typical β-grasp fold and displays 29 % and 36 % sequence identity to ubiquitin, respectively (Groettrup et al., 2008). (b) FAT10 encompasses two ubiquitin-like domains (UBLs), in which the C-terminal di-glycine motif is conserved in the second domain of FAT10. In addition, four of the lysines involved in poly-ubiquitin-chain formation – corresponding to K27, K33, K48 and K63 – are conserved.
(c) Sequence alignment of the N- and C-terminal parts of FAT10 with ubiquitin (Ub). Conserved lysine residues are highlighted in yellow.
Moreover, an induction of FAT10 could be observed under particular conditions, such as inflammation, and has to be removed efficiently, when its inducing signals are turned off.
However, FAT10 can be synergistically induced in many tissues by the proinflammatory cytokines IFN-γ and TNF-α (Liu et al., 1999; Raasi et al., 1999). Induction of the FAT10 mRNA was independent of protein neosynthesis but partially dependent on proteasome activity as treatment with proteasome inhibitors prevented induction of FAT10 with TNF-α, but not IFN-γ (Raasi et al., 1999).
Although its function has not been fully elucidated, FAT10 has been implicated to play important roles in various cellular processes, for instance cancer, antigen presentation, cytokine response, apoptosis and mitosis. Studies in a murine fibroblast cell line revealed that induced expression of FAT10 resulted in massive caspase dependent cell death within 24 to 48 hours. Assumedly, the induction of apoptosis was dependent on the conjugation of FAT10 to so far unidentified target proteins (Raasi et al., 2001).
28 Interestingly, fat10 is one of the most highly upregulated genes in HIV-infected renal tubular epithelial cells (RTECs). Down-regulation of FAT10 expression was shown to reduce apoptosis in RTECs infected by human immunodeficiency virus (HIV), suggesting a novel role for FAT10 in epithelial apoptosis (Ross et al., 2006).
Moreover, FAT10 is a critical mediator of Viral Protein R (Vpr) induced apoptosis in human and murine RTECs, whereby the vpr gene plays an important role in FAT10 up-regulation.
These proteins interact non-covalently and co-localize to mitochondria (Snyder et al., 2009).
In seeming contradiction, up-regulation of FAT10 expression could be observed in several carcinomas, most notably in hepatocellular carcinoma (HCC) and in gastrointestinal and gynecological cancers (Lee et al., 2003; Lukasiak et al., 2008).
In 2008, Oliva et al. identified FAT10 as a potential marker for liver preneoplasia, as it was highly overexpressed in a model of Mallory-Denk body containing chronic liver diseases, which are thought to progress to hepatocellular carcinoma (Oliva et al., 2008). Both suggested an active involvement of FAT10 in tumorigenesis based on its non covalent interaction with the spindle assembly checkpoint protein mitotic arrest deficiency 2 (MAD2), as previously shown in a yeast two hybrid assay (Liu et al., 1999).
FAT10 is thought to displace MAD2 from the kinetochore during prometaphase, associated with incomplete chromosomal segregation and increased mitotic non-disjunction, resulting in a generation of cells that contain aberrant chromosome numbers, which is commonly observed in several cancers but no direct evidence was demonstrated (Ren et al., 2006).
This finding strengthens the hypothesis that FAT10 plays a role in the regulation of genomic stability. FAT10 expression at transcript level undergoes cell cycle–specific changes, with the highest expression during the S phase and a low expression during G2/M phase (Lim et al., 2006). This finding further supported the hypothesis that FAT10 disturbs correct chromosomal segregation and is thus cell cycle regulated.
Moreover, a presumable role for FAT10 in carcinogenesis has been proposed, as the presence of wild-type p53, which is known to play an important role in cell-cycle regulation, negatively regulates FAT10 mRNA expression and promoter activity and prevents reaching high FAT10 levels in the cell (Zhang et al., 2006), whereas mutant p53 provokes a contrary effect. Very recently Li et al. revealed evidence, that p53 becomes FAT10ylated and p53 transcriptional activity was found to be substantially enhanced in FAT10-overexpressing cells (Li et al., 2011).
However, a further study identified that FAT10 possesses no transforming capability and increased FAT10 expression in several tumors is due to the up-regulation of proinflammatory cytokines (Lukasiak et al., 2008). So far, there is still a matter of debate whether it functions as a tumor suppressor or rather an oncogene.
Introduction
29 Hipp et al. reported in 2004, that degradation of FAT10 and its conjugates is accelerated in vitro and in vivo via its non-covalent interaction with the UBL-UBA domain protein NEDD8 ultimate buster 1-long (NUB1L), which binds to the proteasome through its UBL domain (Hipp et al., 2004; Schmidtke et al., 2006). The UBA domains of NUB1L are required for binding but not for accelerated degradation of FAT10 by the proteasome.
This finding led to the assumption that NUB1L might not only act as a linker between the 26S proteasome and ULMs, but also as a facilitator of proteasomal degradation (Schmidtke et al., 2006). The degradation of FAT10 and its conjugates was initially described to be independent of Ub. Fusion of FAT10 to the N-termini of very long-lived proteins, like green fluorescent protein (GFP) for instance, enhanced their degradation rate as potently as fusion with Ub did. Therefore, it was suggested that FAT10 is the first ULM which provides a signal for proteasomal degradation of other proteins as an alternative route for Ub mediated protein degradation (Hipp et al., 2005).
Further, FAT10 degradation occurred normally in E1 temperature-sensitive mutants, however it should be emphasized that Ub conjugation in this mutant is largely deficient at the restrictive temperature but a small share of poly-ubiquitin conjugate formation remained, which can lead to FAT10 ubiquitination (Hipp et al., 2005).
Because no evidence for the deconjugation of FAT10 from its substrates has been obtained, it was believed that FAT10 is probably degraded, along with its substrates, in a manner similar to that seen with Ub-modified substrates when deconjugation is inhibited (Hanna and Finley, 2007).
Contradictory, a very recent article assumed that FAT10 degradation by the proteasome requires its prior ubiquitination (Buchsbaum et al., 2011), based on the observation that a non-ubiquitinable lysine-less form of FAT10 is rapidly aggregated and precipitated in a insoluble fraction which is probably not sensible to the proteasome, whereas the WT protein appears to be less susceptible to aggregation. Moreover, FAT10 stabilization could be observed by using cells expressing non-polymerizable Ub and in cells harboring a thermo- labile mutation in the ubiquitin-activating enzyme, E1. The discrepancy to the previous article (Hipp et al., 2005) could origin in the different experimental setups to inactivate the E1 enzyme (Buchsbaum et al., 2011). Their own statement could be confuted by an experiment where they showed, that degradation of FAT10-GFP occurs in the presence of a nonpolymerizable mutant of ubiquitin which can be explained by the fact that the interaction of FAT10 with the proteasome is sufficient to promote, at least partially, the degradation of a downstream fused protein. This is in line with previous findings, showing that FAT10 can constitute a degradation signal without further ubiquitination (Hipp et al., 2005).
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30 Along with this, some interaction partners of FAT10 have been determined to link FAT10 with aggregate formation. Kalveram et al. reported in 2008 that a cytoplasmic protein, histone deacetylase 6 (HDAC6), can interact non-covalently with FAT10 under proteasome inhibition, leading to the localization of FAT10 in aggresomes (Kalveram et al., 2008). This could provide an alternative route to ensure sequestration and subsequent removal of FAT10- conjugated proteins if FAT10 fails to subject its target proteins to proteasomal degradation.
FAT10 also co-localizes with the catalytic immunoproteasome subunits LMP2 and LMP7 and its expression increases in liver cells forming Mallory-Denk bodies due to accumulation and aggregation of ubiquitinated cytokeratins (Bardag-Gorce et al., 2010).
Many late-onset neurodegenerative diseases are associated with the formation of intracellular aggregates by misfolded or toxic proteins, revealing a high importance in the degradation pathways acting on such aggregate-prone cytosolic proteins including the ubiquitin-proteasome system and macroautophagy (Ross and Pickart, 2004; Williams et al., 2006). A recent report described that FAT10 molecules were covalently attached to the soluble fraction of the aggregate prone protein huntingtin and FAT10-modified huntingtin is prone to degradation by the proteasome. Moreover, completely aggregated huntingtin lacks any FAT10 and FAT10 knockdown enhanced aggregate formation. These data let suggest that FAT10 plays a role in stabilizing soluble huntingtin by facilitating the interaction with the proteasome (Nagashima et al., 2011).
Several evidences for the involvement of FAT10 in the immune system are given. One hint for its immunological relevance is that the fat10 gene is encoded in the MHC locus, for instance (Fan et al., 1996). Moreover, FAT10 is constitutively expressed in immune cells and in organs of the immune system like spleen, gut, lymphnodes and especially the thymus (Lukasiak et al., 2008). Further, it is synergistically inducible in many tissues with the proinflammatory cytokines IFN-γ and TNF-α (Liu et al., 1999; Raasi et al., 1999).
Interestingly, FAT10 can inhibit hepatitis B virus expression in a hepatoblastoma cell line after IFN-γ treatment (Xiong et al., 2003) and fat10 gene targeted mice demonstrated a high level of sensitivity toward lipopolysaccharide challenge and their lymphocytes are more susceptible to spontaneous apoptotic death (Canaan et al., 2006).
These findings indicate that FAT10 may function as a survival factor, but the function and mechanism of its action in the immune system still remains poorly understood and still need to be investigated.
