Elucidating the cause of FAT10 over-expression in liver and colon carcinomas

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Elucidating the cause of FAT10 over-expression in liver and colon

carcinomas

Dissertation

zur

Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

des Fachbereiches Biologie an der

Universität Konstanz vorgelegt von

Sebastian Lukasiak

Tag der mündlichen Prüfung: 29. Juli 2009

Referent: Prof. Dr. Marcus Groettrup Referent: Prof. Dr. Martin Scheffner Referent: Prof. Dr. Alexander Bürkle

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Acknowledgments

First of all, I would like to thank Prof. Dr. Marcus Groettrup for giving me the opportunity to do this work in his lab, where I began as a diploma student. He provided me with important advice and constant support during my work.

Many thanks also go to …

Gunter Schmidtke, who was always open to give me advice, not only in biochemical terms

My fellow PhD students on the FAT10 project, especially Birte Kalveram, Christiane Pelzer, Kathrin Kluge and our “Pretty Queen” Neha Rani for a lot of fun in the lab

Marc Müller and Annette Sommershof for nice coffee breaks on the roof and beer at

“Klimperkasten”

My dear friend Khalid, a constant source of high spirits and amusement, who taught me some “nice” Hindi words

All my colleagues in the Groettrup lab, who made the time that I spend here really enjoyable!!

Gerardo for making great Caipirinha’s during fantastic lab parties.

Elmar Spies for many, many hours on our road bikes, his delicious Käßspätzle and for

“The Pump”

Peter Öhlschläger, for being an extraordinary person and friend

I am really grateful to Anja Holtz, for pleasant coffee breaks, her delicious cakes and many stimulating discussions on any topic.

I am also very thankful to Christopher, for being my friend since the first day in Konstanz!

Najbardziej chciałbym podziekować mojej rodzinie, która zawsze we mnie wierzyła i mi pomagała jak tylko mogła.

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FAT10 is a relatively new member of the ubiquitin-like protein family, since it is only expressed in mammals. FAT10 consists of two ubiquitin-like domains arranged in tandem and bears the typical di-glycine motif in its C-terminus, which is necessary for the conjugation to so far unidentified target proteins. The fat10 gene is located in the major histocompatibility complex class I (MHC I), adjacent to other genes that play a role in the immune system. Its expression was originally found to be restricted to mature B- cells and mature dendritic cells (DCs) but it has been shown that it is synergistically inducible with the proinflammatory cytokines TNF-α and IFN-γ in almost all tissues. One study reported that the fat10 promotor was negatively regulated by p53. Over expression of wild type FAT10 in some human cell lines and also from mice led to the induction of caspase-dependent apoptosis. This was not the case when a di-glycine mutant (ΔGG) was used instead.

A recent study reported that FAT10 is highly over expressed in 90% of human Hepatocellular carcinomas (HCC) and also in other gastrointestinal and gynaecological carcinoma (Lee et al. 2003). With the finding of a different study that FAT10 non- covalently interacted with the spindle assembly checkpoint protein MAD2 that prevents premature entry into anaphase, the authors concluded that FAT10 may play a role in tumour development.

One of the main aims of this thesis was to re-evaluate this startling finding more thoroughly with more HCC samples and with the use of quantitative RT-PCR since the antecedent study measured FAT10 mRNA levels using semi-quantitative northern blots.

A SYBR Green I based assay was designed and validated on cDNA samples from seven common laboratory cell lines, including cell lines derived from HCC. Experiments in this thesis show that FAT10 was up regulated by an IFN-γ and TNF-α treatment in all cell lines, suggesting that no cell line, including the ones derived from HCC, has lost its responsiveness to both cytokines. Furthermore, FAT10 mRNA levels were determined in 51 tissue samples from patients with HCC and in 15 samples derived from Colon carcinoma (CC). Additionally, levels of LMP2 mRNA, an IFN-γ and TNF-α inducible proteasome subunit, were measured in all samples. This thesis shows that significant up regulation of FAT10 was found only in 37/51 (72%) HCC samples and 8/15 (53%) CC samples. Moreover, the expression of FAT10 correlated in both cancer types in most cases with the expression of LMP2, suggesting that an ongoing immune response against

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the cancerous tissue is responsible for the up regulation of FAT10. A soft agar assay also showed that FAT10 on its own has no transforming capabilities. Therefore, this thesis shows evidence that FAT10 qualifies as a marker for an ongoing immune response, mediated by proinflammatory cytokines, in HCC and in CC.

The second aim of this thesis was to find further enzymes involved in the fatylation cascade, since only the E1 enzyme for FAT10, Uba6 has been identified so far and furthermore, to find substrates that are modified with FAT10.

The E2 enzyme USE1 (Uba6-specific E2 enzyme 1) was identified as a new interaction partner in a yeast two-hybrid screen, using a human thymus cDNA library. Furthermore, this thesis shows that USE1 is an E2 enzyme for FAT10, since FAT10 was covalently linked to USE1 in vitro and in vivo. The interaction was not present when a catalytic inactive USE1 was used instead. This covalent conjugate could be reduced with β- mercaptoethanol, suggesting a thioester linkage. SiRNA mediated knock-down of USE1 strongly reduced FAT10 conjugate formation in a cell line stably transfected with Flag- FAT10. Subsequent experiments also show that USE1 itself is the first physiological substrate of fatylation, since it still formed a conjugate under reducing conditions. USE1 can be fatylated in cis but not in trans and co-expression of NUB1L, a linker protein that facilitates the degradation of FAT10 and its conjugates, lead to proteolytic down regulation of USE1. These results suggest that USE1 can auto modify itself with FAT10 thereby negatively regulating the FAT10 conjugation pathway.

Deutsche Zusammenfassung

FAT10 ist ein relativ junges Mitglied der Familie der Ubiquitin-ähnlichen Proteine, da es nur in Säugetieren vorkommt. Es ist aus zwei Ubiquitin-ähnlichen Domänen aufgebaut, die über einen kurzen Linker miteinander verbunden sind. C-terminal weist es das typische Di-Glycin Motif auf, mit dem es an bis jetzt nicht identifizierte Zielproteine konjugiert werden kann. Das fat10 Gen ist in dem Lokus des Haupt- Histokombatibilitätskomplexes der Klasse I (MHC I) kodiert, wo auch andere Gene, die eine wichtige Rolle im Immunsystem spielen, kodiert sind. Ursprünglich wurde die Expression von FAT10 nur in reifen B-Zellen und reifen Dendritischen Zellen (DCs) entdeckt. Später hat man aber entdeckt, daß es durch die entzündungsfördernden

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Zytokine IFN-γ und TNF-α, in fast allen Geweben synergistisch induziert werden kann.

Eine andere Studie hat festgestellt, daß die Transkription von FAT10 von dem Tumorsuppressor p53 negativ reguliert wird. Weitere Studien haben gezeigt, daß die ektopische Überexpression von FAT10 in manchen humanen und auch in murinen Zellinien Caspase-abhängige Apoptose induzieren kann. Die war nicht der Fall war als man eine Di-Glycin Mutante (ΔGG) für die gleichen Experimente benutzt hat.

Eine vor kurzem veröffentlichte Studie hat gezeigt, daß FAT10 in Gewebeproben von Patienten mit Hepatozellulärem Karzinom (HCC) in 90% der Fälle stark hochreguliert war (Lee et al. 2003). Des weiteren fanden sie auch eine starke Überexpression in Proben, die aus gastrointestinalen und gynäkologischen Karzinomen stammten.

Zusammen mit den Daten aus einer früheren Veröffentlichung, die besagen, daß FAT10 nicht kovalent mit dem „Spindle assembly checkpoint“ Protein MAD2 interagiert, welches den vorzeitigen Eintritt in die Anaphase der Zellteilung verhindert, haben die Autoren dieser Studien die Vermutung aufgestellt, daß FAT10 eine Rolle in der Tumorentstehung spielen könnte.

Eines der Hauptziele dieser Doktorarbeit war es, diese überraschenden Erkenntnisse noch genauer zu evaluieren. Dazu wurde eine noch größere Anzahl an Gewebeproben mit der quantitativen RT-PCR Methode untersucht, da die frühere Studie nur semi- quantitative Northern blots benutzt hat, um die Menge der mRNA zu untersuchen. Ein SYBR Green I basierter Assay wurde während der vorliegenden Arbeit entwickelt und an cDNA Proben aus sieben humanen Zellinien validiert. Darunter waren auch vier Zellinien, die aus Leberkarzinomen stammen. Experimente mit IFN-γ und TNF-α behandelten Zellen zeigen, daß FAT10 in allen Zellinien induziert wurde. Dies zeigt, daß keine der untersuchten Zellinien, inklusive der HCC-Zellinien, die Fähigkeit verloren haben auf die eingesetzten Zytokine zu reagieren. Weiterhin wurde die Expression von FAT10 in 51 Gewebeproben untersucht, die aus Patienten mit Leberkarzinom stammen.

Um eine weitere Krebsart zu untersuchen, wurden auch 15 Proben des Kolonkarzinomes (CC) untersucht. Neben FAT10 wurde auch die mRNA-Menge an LMP2, einer IFN-γ und TNF-α induzierbaren Proteasomuntereinheit, in denselben Proben gemessen. In dieser Arbeit wurde gezeigt, daß eine Überexpression von FAT10 nur in 37/51 (72%) der HCC- und in 8/15 (53%) der CC-Proben vorlag. Überdies korrelierte die FAT10 Expression mit der LMP2 Expression in den meisten Fällen beider

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Karzinomarten, was darauf hindeutet, daß eine anhaltende Immunreaktion, die gegen den Krebs gerichtet ist, aller Wahrscheinlichkeit nach für die beobachtete Überexpression von FAT10 verantwortlich ist. Ein Soft Agar Assay zeigte außerdem, daß FAT10 an sich keine Transformation von trasfizierten Zellen verursacht hat. Deswegen eignet sich FAT10 als Marker für eine Immunantwort, die mit entzündungsfördernden Zytokinen begleitet wird, die sich gegen das krebsartige Geweben richtet.

Eine weitere Aufgabe dieser Dissertation war, weitere Enzyme zu finden die in der Kaskade der FAT10-Konjugation beteiligt sind, da bis jetzt nur das E1 Enzyme (Uba6) für FAT10 identifiziert wurde. Des weiteren sollten Substratproteine gefunden werden, die mit FAT10 modifiziert werden.

Das Ubiquitin-konjugierende E2 Enzyme USE1 (Uba6-specific E2 enzyme 1) wurde in einem yeast two-hybrid Versuch, bei dem eine cDNA-Bank aus humanem Thymus verwendet wurde, als weiterer Interaktionspartner von FAT10 identifiziert.

Anschließend wurde gezeigt, daß USE1 auch das konjugierende E2 Enzyme für FAT10 ist, da FAT10 in vitro und in vivo kovalent an USE1 konjugiert werden konnte. Dies war nicht der Fall als eine katalytisch inaktive Mutante von USE1 verwendet wurde. Dieses kovalente Konjugat konnte durch β-Mercaptoethanol reduziert werden, was auf eine Thioester-Bindung hindeutet. Der siRNA vermittelte „knock-down“ von USE1 verminderte sehr deutlich die Formation von FAT10 Konjugaten in einer Zellinie, die stabil mit Flag-FAT10 transfiziert wurde. Anschließende Experimente zeigten, daß USE1 auch das erste physiologische Substrat der Fatylierung ist, da es ein Konjugat mit FAT10 gebildet hat, das nicht reduzierbar war. USE1 wird dabei in cis und nicht in trans fatyliert. Die Koexpression von NUB1L, einem Linker-Protein das den Abbau von FAT10 und seiner Konjugate fördert, bewirkte auch den Abbau von fatyliertem USE1. Die Ergebnisse dieser Dissertation weisen auf eine Automodifikation von USE1 mit FAT10 hin, dessen Konsequenz eine Inhibition des FAT Konjugationsweges ist.

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Introduction

General Introduction

The Proteasome

The 26S proteasome is the most important protease in the cell and is located in the cytoplasm as well as in the nucleus. It is responsible for the degradation of 80-90% of all cellular proteins. It is crucial for the degradation of proteins that are damaged or misfolded/denatured but is also responsible for the degradation of regulatory proteins that control a variety of basic cellular processes such as cell cycle and division, signalling and DNA repair. Further, it is involved in processing of antigens and activation or destruction of transcription factors (Hershko et al. 1998; Ciechanover et al. 2000; Fang et al. 2004). As the principle tag for proteasomal degradation serves the covalent attachment of a highly conserved protein called ubiquitin (described below).

The 26 proteasome (Fig. 1) is a highly conserved ~2.5 MDa multi-catalytic protease that is composed of two distinct subcomplexes. The 20S core particle (CP) that contains the catalytic activity and the 19S regulatory particle (RP) that prepares substrates for entry into the CP (Pickart et al. 2004; Wolf et al. 2004). The 20S CP has a cylindrical structure formed out of four stacked heptameric rings. The two outer rings are composed of seven different α-subunits (α1-α7) and the two inner rings are composed of seven β-subunits (β1-β7). Enzymatic activity is restricted to the lumen of the cylinder (Lowe et al. 1995;

Groll et al. 1997) and is attributed to three of the seven β-subunits (β1, β2 and β5). All catalytic subunits belong to the group of threonine-proteases that can cleave C-terminal of virtually all amino acids (aa) but they display proteolytic preferences for certain aa residues. The β1-subunit shows caspase-like activity and cleaves preferentially after acidic residues, whereas β2 shows a trypsin-like activity and cleaves after basic residues and β5 cleaves after hydrophobic amino acids due to its chymotrypsin-like activity (Groll et al. 2003). Proteins destined for degradation are cleaved by the proteasome to short peptides ranging from 3 to 25 aa (Nussbaum et al. 1998; Kisselev et al. 1999), which are then further degraded by cellular aminopeptidases to single amino acids. If the resulting peptides have a length between 7-10 residues they can be transported to the endoplasmatic reticulum (ER), loaded onto MHC I molecules and

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Introduction

subsequently be presented on the cell surface to circulating cytotoxic T-lymphocytes (Coux et al. 1996; Baumeister et al. 1998).

Figure 1. Schematic representation of the 26S proteasome. The 20S core particle that contains the proteolytic activity, can be capped on both ends with a 19S regulatory particle, which is responsible for substrate recognition, removing of the ubiquitin tag, unfolding of the target protein and translocation into the 20S chamber, where proteins are degraded to short peptides (Modified from (Sullivan et al. 2003).

Both ends of the 20S CP can be associated with a 19S RPs. The 19S RP (also known as PA700) has to fulfil several important tasks such as recognition of polyubiquitylated substrates, partial unfolding of the target protein, removing the ubiquitin tag and finally opening of the 20S CP and translocation of the polypeptide into the chamber. It can be further subdivided into two subcomplexes called the “base” and the “lid” (Glickman et al.

1998). The base consists of 10 different subunits. Six of them (Rpt1-6) belong to the AAA-ATPase family and four are non-ATPase subunits (Rpn1, 2, 10 and 13). The ATPases form a hexameric ring that directly contacts the 7 α-subunits of the 20S CP.

They are responsible for the ATP-dependent opening of the 20S CP and unfolding of substrates (Braun et al. 1999). Rpt5/S6a has been also shown to bind polyubiquitin chains and therefore it may be involved in substrate recognition (Lam et al. 2002). The function of most other subunits of the base and the lid is only poorly understood but some have been identified as ubiquitin receptors. Rpn10 and Rpn13 haven been shown to directly bind to K48 liked polyubiquitin chains through their ubiquitin interacting motif (UIM) or the pleckstrin-like receptor for ubiquitin (Pru) domain, respectively (Lam et al. 2002; Husnjak et al. 2008). The subunits Rpn1 and Rpn2 serve as indirect

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Introduction

bind to K48 polyubiquitylated cargo and dock to these 19S RP subunits through a ubiquitin-like domain located in their N-terminus. For another subunit, Rpn11, a deubiquitylating activity could be shown (Smith et al. 2006). Interestingly, Rpn13 also binds the deubiquitylation enzyme UCH37 suggesting a coupling of chain recognition and chain disassembly at the proteasome (Yao et al. 2006).

In mammals, there are three additional proteasome subunits that are induced with the pro-inflammatory cytokine interferon-γ (IFN-γ). β1i (LMP2) and β5i (LMP7) (Yang et al.

1992), which are encoded in the MHC class I region and β2i (MECL) (Groettrup et al.

1996) are incorporated into new assembling proteasomes and replace the constitutive subunits forming the so called immunoproteasome that displays an altered cleavage pattern (Gaczynska et al. 1994) thereby generating more suitable peptides that bind to MHC class I molecules (Rechsteiner et al. 2000). Furthermore, the 20S CP can associate with a new regulatory particle called PA28 (also known as 11S regulator), which interestingly is also inducible with IFN-γ. The genes for the new regulator are also encoded in the MHC class I region adjacent to the other inducible subunits LMP2 and LMP7 (Dubiel et al. 1992; Ma et al. 1992). This regulatory particle has no ATPase activity and therefore, it can promote the degradation of only short peptides but not of complete proteins. It binds to the outer α-ring of the 20S CP and induces a conformational change that opens the 20S CP gate. Together with the inducible β-subunits of the proteasome, PA28 generates different peptides with higher affinities that bind more stable to the peptide binding groove in the MHC I molecule (Groettrup et al. 2001; Goldberg et al.

2002). Recently, a novel catalytic β-subunit, called β5t, has been discovered in the thymus of mice (Murata et al. 2007) and later also in the human thymus. Human β5t is exclusively expressed in cortical thymic epithelial cells and also to some extent in cortical dendritic cells (DCs) (Tomaru et al. 2009). It has been shown that β5t reduced the chymotrypsin-like activity of the “thymoproteasome”, which is considered to be important for generating peptides with high affinity binding to MHC class I molecules, but β5t deficient mice showed defects in the development of CD8⁺ T lymphocytes, suggesting a key role of thymic β5t expression for the positive selection during thymic development of a MHC class I restricted CD8⁺ T cell repertoire (Murata et al. 2007).

Besides complete degradation of substrates, the 26S proteasome is involved in processing of certain proteins. For example, p50 a subunit of the heterodimeric NF-κB transcription factor, is generated by a partial cleavage and removal of the C-terminal

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Introduction

domain of the p105 precursor (Lin et al. 1996). The complete degradation in this case is prevented by an N-terminal Rel homology domain (RHD) (Lin et al. 2003). Two yeast transcription factors (SPT23 and MGA2) that are yeast relatives of NF-κB are also expressed as inactive precursors that require proteasome-dependent processing to yield active components (Hoppe et al. 2000).

Ubiquitin

Ubiquitin is a highly conserved protein of 76 aa expressed in all eukaryotic species. The sequence differs only in 3 aa between S. cerevisiae and the human protein (Vijay-Kumar et al. 1987). Post-translational modification with ubiquitin regulates many diverse processes, including cell cycle progression, receptor signalling, immune responses and transcription to name few. Ubiquitin is encoded by a family of genes and the products are always fusion proteins, either as ubiquitin-oligomeres or ubiquitin fused to a ribosomal protein. In both cases, enzymes called C-terminal hydrolases recognise the di- glycine motif at the C-terminus along with the ubiquitin domain and release ubiquitin monomers (Jentsch et al. 1991). The di-glycine motif is used to conjugate ubiquitin to a lysine of a substrate protein. Ubiquitin itself has 7 conserved lysines within its sequence that can theoretically be used to generate polyubiquitin chains but only chains linked via K48, K63 and to a much lesser extent K29 are readily detected in vivo (Haglund et al.

2005). The process of ubiquitin conjugation is termed ubiquitylation (Fig. 2), which is highly regulated and requires a sequential cascade of three different enzymes. In the first step a ubiquitin activating enzyme (E1) adenylates the C-terminal glycine of ubiquitin in an ATP-dependent manner. Then the activated ubiquitin is transferred to the catalytic cysteine within the E1, forming a thioester bond. The E1 enzyme then binds to one of many ubiquitin-conjugation enzymes (E2) and transfers the activated ubiquitin to the catalytic cysteine of the E2. In the last step of this cascade a ubiquitin ligase (E3), which confers substrate specificity, catalyses the isopeptide linkage of ubiquitin to an ε- amino group of a lysine in target proteins (Hershko et al. 1998). In some cases an additional chain elongation factor is needed, termed E4 (Koegl et al. 1999). However, conjugation to non-lysine residues was reported for MyoD (Breitschopf et al. 1998) and p21 (Bloom et al. 2003), which can be tagged for degraded via ubiquitylation of the N- terminus. One group could also show that a viral E3 ubiquitin ligase is able to attach ubiquitin to a cytoplasmic cysteine residue of MHC I molecules (Cadwell et al. 2005).

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Introduction

Figure 2. The ubiquitin conjugation pathway. In the first step, free ubiquitin (Ub) is ATP-dependently activated by an E1 enzyme, and then it is transferred to an Ub-conjugation enzyme (E2). In the final step, Ub is transferred to a lysine of a substrate protein through the action of an Ub-ligase or E3. There are two major E3 enzyme families: the HECT-type and the RING-finger E3s. Ring-type E3s, which are often multi-subunit complexes, do not have enzyme activity. They act as facilitators of ubiquitylation by physically linking substrate binding proteins and Ub-activated E2s together. In the case of HECT domain E3s, Ub is passed on to the catalytic site cysteine of an E3. The ligase binds the substrate and attaches the Ub moiety directly to the target protein (modified from Di Fiore et al. 2003).

The best characterised modification with ubiquitin (Fig. 3) is the polyubiquitylation of a substrate protein with a K48 linked ubiquitin chain. It has been shown that four K48 linked ubiquitins attached to a lysine within a protein are the minimal signal that leads to proteasomal degradation of the tagged protein (Chau et al. 1989) and this turned out to be the most prevalent function of the ubiquitylation system. It is very little known about the function of K29 linked polyubiquitin chains but it could be shown that in some cases this modification also leads to proteasomal degradation (Johnson et al. 1995;

Glickman et al. 2002). However, all other variations of ubiquitylation have different meanings and are not involved in protein degradation. For example, K63 linked chains function rather as protein-protein interaction platforms and are involved in processes diverse as the Rad6-dependent DNA repair (Spence et al. 1995; Hofmann et al. 1999), regulation of ribosome function (Spence et al. 2000), endocytosis (Haglund et al. 2003)

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Introduction

or the activation of NF-κB (Chen et al. 1996; Schwartz et al. 2009). Alongside, proteins can be modified with only one ubiquitin (monoubiquitylation) at one time or with several separate ubiquitins (poly-monoubiquitylation) on different lysines. This is sufficient to change the activity of a protein or to create a binding site for adaptor proteins. Ligand binding to the epidermal growth factor receptor leads to monoubiquitylation of the receptor and this drives its endocytosis and thus terminates signalling (Mosesson et al. 2003). It has been shown that monoubiquitylation is also involved in processes such as viral budding, DNA repair, vesicular sorting and transcriptional regulation of histone activity (Hicke 2001; Di Fiore et al. 2003).

Figure 3. Different forms of Ub modification. Attachment of one Ub moiety (monoubiquitylation but also multi- monoubiquitylation) to a protein is associated with various processes such as endocytosis, membrane trafficking and histone regulation. Whereas, modification with K48 and also K29 linked polyubiquitin chains leads to degradation of the substrate by the proteasome. K63 linked polyubiquitin chains rather serve as platforms for additional factors that regulate processes such as signalling and DNA repair.

Substrate specificity in the ubiquitin system is conferred by E3 ubiquitin ligases. Based on shared structural motifs and with the help of sophisticated sequence comparison methods, it appears that the human genome contains up to 1000 E3s (Schwartz et al.

2009), making it the biggest class of enzymes encoded in our genome. E3 ligases are subdivided into two major families, the HECT domain (homologous to E6-AP carboxy terminus) and the RING finger domain ligases (Pickart 2001; Weissman 2001).

In the ubiquitylation cascade with a HECT domain E3, the activated ubiquitin moiety is transferred from the E2 to a conserved catalytic cysteine in the E3 enzyme, which then in turn directly catalyses the transfer of ubiquitin to target proteins (Huibregtse et al.

1995). The first member of this group E6-AP, was identified as a protein, that in a complex together with the human papilloma virus (HPV 16) protein E6, promoted degradation of the tumour suppressor p53 (Scheffner et al. 1993).

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Introduction

The larger family of E3 belongs to the RING (really interesting new gene) finger domain E3 ligases, which do not function as enzymes per se, but rather activate and bring an E2 and a substrate in close proximity to promote ubiquitylation (Vodermaier 2004). The RING finger is a special form of the Zinc finger and is defined by eight conserved cysteines and histidines that together coordinate two zinc ions (Borden et al. 1996). The RING E3s exist as monomers, with the ability to bind substrates and an E2 on the same polypeptide (e.g. Parkin and Cbl) and as multisubunit complexes such as the SCF (Skp/Cullin/F-box) or the APC (anaphase promoting complex or cyclosome). In the case of a SCF complex, the RING finger domain is located in a separate protein (e.g. ROC1) and different F-box proteins confer the substrate specificity and selectivity (Kipreos et al.

2000). Recognition of substrates is often regulated by phosphorylation (Deshaies 1999) or in the case of the Hypoxia-inducible factor (HIF-1α), by hydroxylation (Kaelin 2007).

Other small subfamilies with variations of the RING finger are the U-box (Jiang et al.

2001) and the PHD domain-containing E3 (Coscoy et al. 2003).

Ubiquitylation is tightly regulated and additionally to proteins that recognise ubiquitin and attach it to substrate proteins, over 90 putative deubiquitylating enzymes (DUBs) are encoded in the human genome, which can reverse this process and add a further layer of complexity to the system (Nijman et al. 2005). Deubiquitylating enzymes mediate the removal and processing of ubiquitin and are crucial regulatory proteins.

This is underscored with several reports, for example, mutation in CYLD (cylindromatosis), a DUB that negatively regulates TNF-α and Toll-like receptor mediated signalling, leads to a benign skin cancer (Bignell et al. 2000). Several human pathogenic viruses such as Coronaviruses (SARS), Adenoviruses and Herpesviruses encode their own DUBs or hijack host DUBs to prevent ubiquitin-dependent degradation of viral proteins (Edelmann et al. 2008). There are two classes of DUBs, the cysteine and the metallo proteases, although most DUBs belong to the group of cysteine proteases.

The cysteine DUBs can be further divided: the ubiquitin C-terminal hydrolases (UCHs) with only four members, which were originally identified to process ubiquitin precursors and ubiquitin specific processing proteases (USPs) that remove ubiquitin from substrates and disassemble polyubiquitin chains to reuse ubiquitin before proteasomal degradation of target proteins. Some of the DUBs show specificity for either K48 or K63 linked chains (Nijman et al. 2005).

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Introduction

For over 20 years only one E1 enzyme (UBE1) was known that activates ubiquitin. UBE1 deletion is lethal (McGrath et al. 1991) but mutations in the single copy of ube1, which is encoded on the X chromosome, generated few temperature sensitive (ts) cell lines, that allowed further characterisation of UBE1 function in the ubiquitylation system (Ciechanover et al. 1984; Finley et al. 1984; Kulka et al. 1988; Zacksenhaus et al. 1990).

Experiments in the ts85 cell line showed a reduction of polyubiquitylation to < 15%. The remaining ubiquitin activation was attributed to an incomplete UBE1 inactivation at the restrictive temperature rather than to a second ubiquitin activating enzyme (Groettrup et al. 2008). Only recently, three independent groups reported the discovery of a second ubiquitin activating enzyme which was named UBA6 (Chiu et al. 2007; Jin et al. 2007;

Pelzer et al. 2007). Deletion of either E1 enzyme is embryonically lethal, indicating little redundancy in the ubiquitylation system. Both have distinct E2 charging activities in vitro where UBE1 could charge all E2 enzymes examined but one. USE1 (UBA6 specific E2 enzyme 1) was found to be exclusively charged by UBA6 (Jin et al. 2007). The same study also reported that nine different E2 enzymes could only be charged in vitro by UBE1 and not by UBA6.

While there are only two identified ubiquitin E1 enzymes in the mammalian genome, over 30 E2 enzymes with a conserved core domain (UBC) have been identified. Some members of the E2 family possess N- or C-terminal extensions that may be involved in subcellular localisation or specific E2-E3 interactions (von Arnim 2001). Most E2 enzymes are rather small with a molecular weight between 11-40 kDa (Jentsch et al.

1990) except for BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), a huge 528 kDa protein located in the peripheral membrane of the trans-Golgi network (TGN) which bears a single BIR domain most strongly related to survivin (Hauser et al. 1998) and a C-terminal UBC domain. A group could show that BRUCE can function as an unusual chimeric E2/E3 ubiquitin ligase (Bartke et al. 2004).

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Introduction

Ubiquitin-like proteins

The human genome encodes additional proteins that are related to ubiquitin and share either sequence or structure homology but have otherwise different biological functions.

They can be divided into two distinctive groups, the so called ubiquitin-like modifiers (UBLs) or the ubiquitin domain proteins (UBDs) (Fig. 4). Members of the first group are covalently attached to target proteins in a manner analogous to ubiquitylation. Members that belong to the second group are larger proteins that contain a ubiquitin-like domain within their sequence which is mostly responsible for protein-protein interactions (Jentsch et al. 2000; Schwartz et al. 2003).

Figure 4. Two different groups of Ubiquitin-like proteins. Members of the first group are related to ubiquitin and possess the di-glycine motif needed for conjugation and function as modifiers. Whereas, proteins from the second group are mostly large proteins that contain a Ubiquitin-like domain, which is an integral part of their structure. They are not processed nor conjugated to other proteins (Modified from Jentsch et al. 2000).

Ubiquitin-like modifiers

UBLs have a di-glycine motif at the C-terminus which is a cardinal feature for the whole family of UBLs. In most cases, except for ATG12, URM1 and FAT10, all members are expressed as precursor proteins that need to be processed with specific C-terminal hydrolases to gain a free di-glycine motif. Most of the UBLs have a high sequence homology to ubiquitin, except for ATG8, ATG12 and URM1 but basically, they all share the same three dimensional structure, called the β-grasp or ubiquitin fold.

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Introduction

SUMO (Small ubiquitin-like modifier)

SUMO is poorly related to ubiquitin with a sequence identity of only 18%, but it contains nearly an identical structural fold. Only one SUMO protein is expressed in lower eukaryotes such as yeast, nematodes and insects. There are three different SUMO proteins expressed in mammals (Hay 2005) and up to eight different versions can be found in the genome of plants (Kurepa et al. 2003). SUMO-2 and SUMO-3 are almost identical in sequence and differ only by three N-terminal residues. They are only about 50% identical to SUMO-1 and are functionally different (Saitoh et al. 2000; Tatham et al.

2001). The first identified SUMO substrate was the GTPase activating protein RanGAP1, which was modified with SUMO-1 (Matunis et al. 1996). Virtually, all SUMO-1 is engaged in conjugates, whereas, there is a larger pool of unconjugated SUMO-2/3. This more abundant pool seems to be utilised upon different cellular stresses (Saitoh et al. 2000).

An interesting feature of SUMO-2/3 is that they are the only known ubiquitin-like modifiers apart from ubiquitin that can build poly-UBL chains (Tatham et al. 2001).

The SUMO conjugation cascade consists of a heterodimeric E1 enzyme SAE1 and SAE2 (Aos1 and Uba3 in yeast, respectively), the E2 enzyme Ubc9 and several E3 enzymes (Hochstrasser 2001; Melchior et al. 2003). A unique feature of the E2 enzyme Ubc9 is its ability to directly recognise target proteins through the minimal sumoylation motif ΨKXE (Ψ represents a hydrophobic aa, X represents any aa) present in many known SUMO substrates (Rodriguez et al. 2001; Sampson et al. 2001; Bernier-Villamor et al.

2002). SUMO proteins are expressed as precursors that have to be processed to reveal the di-glycine motif. This is accomplished by SUMO specific proteases. Seven genes in humans have been found that have C-terminal hydrolase activity and/or isopeptidase activity to specifically remove conjugated SUMO from its substrates (Melchior et al.

2003).

Sumoylation seems to have quite different effects on the target proteins. Rather than ubiquitylation that leads in many cases to protein degradation, attachment of SUMO changes the subcellular localisation of certain target proteins. In the case of RanGAP1, which is cytoplasmic, modification with SUMO-1 targets the protein to the nuclear core complex where it is involved in nuclear import/export (Matunis et al. 1996).

Sumoylation on the same lysine can also have the opposite effect of ubiquitylation and stabilise the target protein, as has been shown for the NF-κB inhibitor IκBα. Sumoylation of lysine 21 prevents ubiquitylation and proteasomal degradation and hence the

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Introduction

activation of NF-κB (Desterro et al. 1998). PCNA (Proliferating cell nuclear antigen) acts as a processivity factor for DNA Polymerase δ during DNA replication. In response to DNA damage, PCNA can be monoubiquitylated on lysine 164 (Hoege et al. 2002).

Monoubiquitylation on this lysine activates translesion synthesis (TLS), which is synthesis across the damaged site. TLS is error-prone because incorrect nucleotides can be incorporated (Friedberg et al. 2005). Sumoylation on lysine 164 attracts the anti- recombinogenic helicase Srs2 that may aid replication by inhibiting unwanted and deleterious recombination during DNA synthesis (Haracska et al. 2004; Papouli et al.

2005; Pfander et al. 2005). Interestingly, PCNA can also be ubiquitylated on lysine 164 with K63 chains, this results in activation of an error-free DNA repair mechanism (Leach et al. 2005). Other targets of sumoylation include certain transcription factors, such as c- Jun and p53 but while sumoylation of c-Jun inhibits its activity, over expression of SUMO-1 activated transcriptional activity of wild type p53 (Rodriguez et al. 1999; Muller et al. 2000).

Neural precursor cell-expressed developmentally down-regulated 8 (NEDD8)

NEED8 (or RUB1 in yeast and plants) has the highest identity (60%) and similarity (80%) to ubiquitin of all ubiquitin-like modifiers (Kumar et al. 1993). Like ubiquitin and most other UBL modifiers it is necessary to cleave off several aa from the C-terminus to gain a free di-glycine motif before conjugation. This is accomplished by the ubiquitin C- terminal hydrolase UCH-L3 (Wada et al. 1998) and more specifically by DEN1 (Gan- Erdene et al. 2003; Mendoza et al. 2003; Wu et al. 2003). The E1 enzyme for NEDD8 consists of a heterodimer of APP-BP1 and UBA3. After activation NEDD8 is then transferred to its E2 enzyme UBC12 (Liakopoulos et al. 1998). A recent paper describes a new E2 enzyme called UBE2F hat expands the NEDD8 conjugation pathway. This new E2 enzyme which is a relative of UBC12 seems to specifically transfer NEDD8 to CUL5- RBX2 (Huang et al. 2009), while UBC12 neddylates other cullins that are associated with RBX1 (Schulman et al. 2009).

Originally, NEDD8 was found to be conjugated to Cdc53 a component of the SCF complex (Skp1-Cdc53/CUL1-Fbox protein), a RING E3 ubiquitin ligase in Saccharomyces cerevisiae (Lammer et al. 1998; Liakopoulos et al. 1998). Subsequently, it was shown that with the exception of APC2, each member of the cullin family is modified by NEDD8

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Introduction

(Hori et al. 1999; Pan et al. 2004). Cullins act as scaffolding platform for the largest group of RING-based E3 ligases. They share a conserved neddylation site which is defined by the aa sequence IVRIMKMR with few variations among members of the cullin family (Hori et al. 1999). Neddylated cullins, together with their RING finger partner ROC1/Rbx1 and different F-box proteins, that confer substrate specificity, form a complex that supports polyubiquitin chain assembly onto target proteins (Pan et al.

2004). NEDD8 attachment to the CUL1, e.g. seems to assist in the recruitment of an RING E2 to the SCF complex, thereby promoting the SCF-mediated ubiquitylation of several substrates including IκBα (Read et al. 2000; Kawakami et al. 2001) or p27 (Morimoto et al. 2000; Podust et al. 2000). The same was also found for CUL2, that neddylation was required for efficient ubiquitylation of HIF-α by the pVHL E3 ligase (Ohh et al. 2002).

The NEDD8 conjugation pathway plays an important role in the control of many different cellular pathways. It is essential for cell viability in S. pombe (Osaka et al. 2000), for embryogenesis in Drosophila (Ou et al. 2002) and for cell cycle progression and morphogenesis in mice (Tateishi et al. 2001). The activity of SCF complexes is regulated by NEDD8 specific proteases such as the COP9 signalosome (CSN), which contains a subunit (CSN5) with metallo-protease activity that specifically removes NEDD8 from CUL1 (Cope et al. 2002).

Recently, p53 was identified as a new target of neddylation. Mdm2, which is an E3 ubiquitin ligase responsible for the continuous turnover of p53, can also transfer NEDD8 to p53 and also to itself. Mdm2 mediated conjugation of NEDD8 to p53 inhibits its transcriptional activity (Xirodimas et al. 2004). Furthermore, ribosomal proteins have been found to be novel NEDD8 substrates and modification of a subset of ribosomal proteins with NEDD8 provided higher protein stability (Xirodimas et al. 2008). It is now evident, that there are more NEDD8 modified substrates and this expands the diverse functions of neddylation to transcriptional regulation, membrane trafficking and ribosomal protein stability (Xirodimas 2008). Since NEDD8 plays a central role in cell cycle progression and growth it has been implicated in tumourigenesis, making it an interesting clinical target for the pharmaceutical industry. Recently, an inhibitor of the NEDD8 E1 enzyme APP-BP1/Uba3 has been shown to promote apoptotic cell death in human tumour cells and suppressed growth of human tumour xenografts in mice (Soucy et al. 2009).

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Introduction

ISG15 (Interferon stimulated gene 15)

ISG15 (or Ubiquitin cross reactive protein, owing to its ability to cross react with some ubiquitin specific antibodies) is the oldest known member of the ubiquitin-like modifier family (Farrell et al. 1979) but till recently it was only little known about this protein partly due to the lack of homologues in lower eukaryotes. It is one of the most up regulated genes that are induced with IFN-α/β (Blomstrom et al. 1986), LPS (Li et al.

2001) and during a viral infection (Haas et al. 1987). ISG15 consists of two ubiquitin-like domains arranged in tandem, similar to FAT10 (Haas et al. 1987). It is expressed as a precursor with a C-terminal extension that masks its di-glycine motif; therefore, it has to be processed before conjugation to its substrates. Conjugation to target proteins is akin to the ubiquitin system and comprises of an ISG15 specific E1 enzyme called UBE1L (Yuan et al. 2001), two E2 ubiquitin enzymes UBCH6 (Takeuchi et al. 2005) and UBCH8 (Kim et al. 2004; Zhao et al. 2004) that also act in the ISG15 pathway. RNAi experiments in HeLa cells suggest that UBCH8 acts as the principal E2 enzyme in the ISG15 pathway (Kim et al. 2004). Finally, two E3 ubiquitin ligases HERC5 (Wong et al. 2006) and TRIM25 (Zou et al. 2006) have been identified to conjugate ISG15 to target proteins.

Adequately, all enzymes involved in the conjugation pathway that have been identified so far are also inducible with type I interferons like ISG15 itself. ISGylation is a reversible process analogous to ubiquitylation and several enzymes that catalyse the removal of ISG15 from its substrates have been identified. UBP43 (also called USP18) specifically removes ISG15 from target proteins and is also inducible with IFN-α/β (Malakhov et al. 2002). Since the tail region of ISG15 and ubiquitin is identical, one group identified several promiscuous deubiquitinating enzymes (USP2, USP5, USP13 and USP14) that are capable of removing ISG15 and ubiquitin from substrate proteins (Catic et al. 2007).

Up to now 158 putative ISG15 substrates have been identified (Giannakopoulos et al.

2005; Zhao et al. 2005; Takeuchi et al. 2008). Many of them are important players in the IFN response against viral infections, such as the key signalling components JAK1, STAT1, ERK1 and Phospholipase Cγ1, pattern recognition receptor RIG-I (retinoic-acid- inducible gene I) and very important anti viral effector proteins PKR, RNaseL, HuP56 and MxA(Malakhov et al. 2003; Zhao et al. 2005). Concordant with its important role in an anti viral response are the reports that ISG15 deficient mice are more susceptible to infections with several different viruses including influenza A and B viruses, Sindbis

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Introduction

virus (SV), and both herpes simplex virus 1 (HSV-1) and murine γ–herpesvirus (Lenschow et al. 2005; Osiak et al. 2005; Lenschow et al. 2007). Further evidence comes from studies that identified the influenza B virus protein NS1 that targets different stages of the ISGylation pathway (Yuan et al. 2001; Chang et al. 2008). At last, viral proteases from SARS coronavirus, crimean-congo hemorrhagic fever virus, equine arteritis virus, porcine respiratory and reproductive syndrome virus, and SV have been identified to mediate deISGylation (Arguello et al. 2007; Frias-Staheli et al. 2007;

Lindner et al. 2007).

In contrast to every other ubiquitin-like modifier, ISG15 is secreted by human monocytes and lymphocytes treated with IFN-β (Knight et al. 1991; D'Cunha et al. 1996) in large amounts and acts itself as an interferon that stimulates IFN-γ production in CD3⁺ cells (Recht et al. 1991) and promotes proliferation of CD56⁺ natural killer cells (D'Cunha et al. 1996).

An intriguing finding in the ISG15 conjugation pathway is that its E1 enzyme UBE1L was found to be absent in almost all small cell lung cancers and also from renal cell carcinomas, which led to the hypothesis that UBE1L acts as a potential tumour suppressor (Carritt et al. 1992; Kok et al. 1993; Pitterle et al. 1998). Recently, it was found that transfection of UBE1L into lung cancer cells reduced cyclin D1 protein levels and conferred growth suppression (Feng et al. 2008).

FAT10

The ubiquitin-like modifier FAT10 (HLA-F-locus adjacent transcript 10) was discovered when the human Major Histocompatibility Complex I locus was sequenced (Fan et al.

1996). The protein is composed of 165aa and has a molecular mass of about 18 kDa.

Akin to ISG15, which is another ubiquitin-like modifier, it consists of two ubiquitin-like domains separated by a short linker of 5 aa, for which reason it was originally denominated “diubiquitin” (Fig. 5). The N-terminal and C-terminal domain of FAT10 are more closely related to ubiquitin than to each other and have a sequence identity of 29%

and 36% compared to ubiquitin itself, respectively. The C-terminus bears the typical di- glycine motif (Bates et al. 1997) that is necessary for the covalent conjugation to so far unknown target proteins (Raasi et al. 2001; Chiu et al. 2007). Four of the lysine residues responsible for poly-ubiquitin chain formation, most notably K48 and K63, are conserved in both UBL domains but currently there is no experimental evidence for

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Introduction

FAT10 chain formation. Atypical to ubiquitin, FAT10 contains 4 cysteine residues in its sequence (Bates et al. 1997).

Figure 5. Comparison of the tertiary structure of Ubiquitin and the predicted model of FAT10. Both UBL domains of FAT10 show the typical β-grasp of ubiquitin fold (Modified from Groettrup et al. 2008).

The fat10 gene of humans and mice is encoded on chromosome 6 and chromosome 17, respectively, in the MHC class I locus, adjacent to other genes which play an important role in the immune system such as TAP (Transporter associated with antigen processing) and the interferon-γ inducible proteasome subunits LMP2 and LMP7 (Fan et al. 1996; Bates et al. 1997). Initially, expression of FAT10 was shown to be restricted to mature dendritic cells and mature B-cells (Bates et al. 1997) and mRNA was detected in spleen and thymus by Northern blot and in situ hybridisation (Liu et al. 1999). Its expression pattern and genomic localisation led to the assumption that FAT10 might play a role in antigen presentation in professional antigen presenting cells. However, FAT10 induction did not affect cell surface expression of MHC class I molecules or class I restricted antigen presentation (Raasi et al. 2001). Subsequently, two independent groups have shown that FAT10 can be synergistically induced with the proinflammatory cytokines IFN-γ and TNF-α, but not IFN-α/β, in various cell lines derived from different tissue origins (Liu et al. 1999; Raasi et al. 1999). Recently, a group could show that the fat10 promoter bears a p53-binding site and that FAT10 expression is negatively regulated by a factor of 2 by p53 (Zhang et al. 2006).

Currently, there are only two pieces of data that connect FAT10 with a function in the immune system. One study found that FAT10 inhibited Hepatitis B virus gene expression in a human hepatoblastoma cell line after IFN-γ treatment (Xiong et al. 2003). The second hint comes from FAT10 deficient mice which are viable and display no obvious phenotype except that they show hypersensitivity to a sublethal dose of lipopolysaccharides (LPS) (Canaan et al. 2006), but the function and mechanism of its

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Introduction

action in the immune system remains to be determined with much more work focused on FAT10 deficient mice.

FAT10 is synthesised with a free di-glycine motif at its C-terminus, without the need for processing before conjugation to its target proteins. Activation of FAT10 is accomplished by its E1 enzyme UBA6, which interestingly, is a second E1 enzyme for ubiquitin apart from UBE1 (Chiu et al. 2007), but no E2 nor E3 enzymes are known yet for the

“fatylation”-cascade. Like ubiquitin and other ubiquitin-like modifiers, it could be shown that FAT10 is covalently transferred to so far unidentified target proteins (Raasi et al.

2001) and that it tags these proteins for proteasomal degradation as efficiently as ubiquitin since N-terminal fusions with the long-lived protein GFP where degraded with similar rates as compared to a fusion with ubiquitin (Hipp et al. 2004). It could also be shown that this process is ubiquitin independent since a target protein covalently modified with a lysine-less FAT10 mutant was degraded at the same rate (Hipp et al.

2005). Furthermore, in the same study the authors showed that FAT10 is degraded along with its target protein rather than being recycled and reused like it is the case for ubiquitin. This finding suggests that there are probably no FAT10 specific proteases and once a protein is tagged with FAT10 its fate is sealed.

In yeast two-hybrid screens two non-covalent interaction partners of FAT10 could be identified. The first one is called Nedd8 ultimate buster 1 long (NUB1L). It was shown that when NUB1L is co-expressed along with FAT10, it binds to FAT10 and accelerates the degradation rate of FAT10 and its conjugates (Hipp et al. 2004).

The second non-covalent interaction partner is Histone deacetylase 6 (HDAC6), which is a cytosolic histone deacetylase that deacetylates α-tubulin (Hubbert et al. 2002;

Matsuyama et al. 2002; Zhang et al. 2003), HSP90 (Bali et al. 2005) and cortactin (Zhang et al. 2007). It contains two catalytic domains responsible for its deacetylase activity, a dynein binding domain (Kawaguchi et al. 2003) and a C-terminal BUZ-domain that is able to bind polyubiquitin chains as well as monomeric ubiquitin (Seigneurin-Berny et al. 2001; Hook et al. 2002). Kawaguchi et al. (2003) could show that under conditions of misfolded protein stress or when the proteasome is inhibited, HDAC6 associates with the microtubule network via its dynein binding domain and shuttles polyubiquitylated proteins, bound with its BUZ-domain, to the aggresome. In that way, HDAC6 connects the ubiquitin pathway with the autophagy system. Kalveram et al. (2008) showed that HDAC6 binds FAT10 in vivo only under conditions of proteasome impairment and

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Introduction

shuttles FAT10 and fatylated proteins to the aggresome. Interestingly, FAT10 is the only ubiquitin-like modifier except ubiquitin itself that targets proteins directly for proteasomal degradation and it uses the same rescue mechanism like ubiquitin when the proteasome is overwhelmed.

Another intriguing property of FAT10 is that its ectopic over expression is not compatible with cell survival since two independent groups reported that generated stable FAT10 expressing clones in HeLa cells suffered from continuous cell death and poor proliferation and clones that survived the selection lost their FAT10 expression (Liu et al. 1999; Raasi et al. 1999). Studies on a mouse fibroblast cell line expressing FAT10 in a tetracycline inducible manner showed that the over expression of wild type FAT10 induced caspase dependent cell death within 24 to 48h. This process was FAT10 specific since over expression of a di-glycine mutant of FAT10 did not induce apoptosis (Raasi et al. 2001). A different study found that in vitro infection of human renal tubular epithelial cells (RTEC) with HIV-1 induced FAT10 and that endogenous FAT10 expression promoted apoptosis in RTECs (Ross et al. 2006).

An apparent contradiction to the above mentioned cell death promoting properties of FAT10 is the report of Lee et al (2003). This group found, using Northern blots, that FAT10 is over expressed in 90% of patients with hepatocellular carcinomas (HCC), over 80% of colon carcinomas (CC) and also highly up regulated in other gastrointestinal and gynaecological cancers. This group found also no up regulation of other genes implicated in an immunological response against the tumour. Therefore, they proposed that FAT10 may somehow be involved in tumourigenesis of these malignancies. Consistent with this hypothesis were two reports that FAT10 interacted non-covalently with the spindle checkpoint assembly protein MAD2 during mitosis (Liu et al. 1999; Ren et al. 2006).

MAD2 binds to unattached kinetochores and prevents premature entry into anaphase (Li et al. 1996). It is proposed that FAT10 displaces MAD2 from kinetochores and this results in an abbreviated mitotic phase which contributes to chromosome instability and abnormal chromosome numbers (Ren et al. 2006). Another group identified FAT10 as an epigenetic marker for liver preneoplasia in a drug-primed mouse model of HCC but contrary to the data of Ren et al. (2006) they found no signs of aneuploidy at any stage, including tumour formation over a time period of 14 months (Oliva et al. 2008).

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Introduction

Ubiquitin domain proteins

Attachment of ubiquitin changes the molecular landscape of the substrate protein and therefore influences the protein-protein interaction with other proteins that act downstream of this event. Proteins with a ubiquitin domain (UDPs) represent a structurally and functionally heterogeneous group that is not conjugated to other proteins. Instead, they non-covalently bind to monoubiquitin or polyubiquitin chains attached to substrate proteins. The preference for certain chains or monoubiquitin makes sense in vivo. For example, for ubiquitin receptors that link polyubiquitylated substrates to the proteasome, K48 liked chains are fundamental (Elsasser et al. 2005).

For UDPs that are involved in the endocytic pathway, monoubiquitin recognition is essential since endocytic cargo and the responsible machinery is mostly monoubiquitylated, but still little is known about the molecular mechanism how the domains discriminate between the different signals (Hicke et al. 2003).

Proteins that belong to this family often possess a ubiquitin-like domain in their N- terminus and one to several ubiquitin domains located in their C-terminal part. They mostly function as mediators of protein-protein interactions through their binding to ubiquitin. The interaction of an Ubiquitin domain protein with ubiquitin is in most cases mediated by the contact with the conserved residue Ile44 within the hydrophobic patch of ubiquitin (Hicke et al. 2005).

Interestingly, some of the UDPs bind directly to the 19S regulatory particle of the 26S proteasome via their UBL domain (Hartmann-Petersen et al. 2004). Many of the UDP proteins contain other characterised modules that belong to at least 16 different ubiquitin-binding domains, e.g. UBA (Ubiquitin associated domain), UIM (Ubiquitin interacting motif), UBZ (Ubiquitin-binding zinc finger) or PAZ (polyubiquitin-associated zinc finger) to name few (reviewed in Hurley et al. (2006)). Some of these proteins seem to function as soluble ubiquitin receptors and facilitate proteasomal degradation.

Examples of well studied UBL-UBA proteins are Rad23/hHHR23 and Dsk2/hPLIC. Both proteins interact with the proteasome via their UBL domain and they bind polyubiquitylated substrate proteins via their UBA domain(s) and function most probably as shuttle carriers to transport proteins tagged for proteasomal degradation (Hartmann-Petersen et al. 2003). This could be shown with deletion experiments, mostly carried out in yeast. Deletion of either one of these proteins led to accumulation of polyubiquitylated proteins and impaired degradation of certain substrates (Chen et al.

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Introduction

2002; Rao et al. 2002). In the case of Rad23 it has been shown that its UBA domains preferentially bind to K48 linked polyubiquitin chains (Raasi et al. 2005) and this event can also lead to inhibition of proteasomal degradation of substrates by sequestering the ubiquitin tag (Raasi et al. 2003). These experiments also showed that there is high redundancy in this system since only the combined deletion of Rad23, Dsk2 and the proteasomal subunit Rpn10, which serves as a ubiquitin receptor directly associated with the proteasome, was lethal or displayed a severe phenotype in yeast (Wilkinson et al. 2001; Saeki et al. 2002).

BAG1 (Bcl-2-associated athanogene) serves as a nucleotide exchange factor for cytosolic Hsc/Hsp70 thereby triggering substrate unloading from the chaperone. Additionally, it contains a N-terminal UBL domain. It functions as a physical link between the molecular chaperone HSP70, the E3 ligase CHIP and the proteasome, thereby linking protein folding and proteolysis (Luders et al. 2000; Alberti et al. 2003).

NUB1

NUB1 was originally identified in yeast two-hybrid screens to interact with NEDD8. It belongs to the UBL-UBA protein family and was shown to reduce the levels of NEDD8 and its conjugates in pulse-chase experiments by targeting NEDD8 to proteasomal degradation, which could be totally blocked with proteasome inhibitors, and was therefore named “NEDD8 ultimate buster-1” (Kamitani et al. 2001; Kito et al. 2001).

Originally, NUB1 was described to have two UBA domains and a N-terminal UBL domain but a splice variant was found that is 14 aa longer. This insertion creates an additional UBA domain between the two existing ones. This splice variant was named NUB1long or NUB1L (Tanaka et al. 2003). In addition, NUB1 was shown to interact with the ubiquitin precursor UbC1 that is expressed as a fusion protein of 9 ubiquitin moieties. It could be shown that the UBA domains of NUB1 interacted with α-peptide bond linked polyubiquitin but not with isopeptide bond linked polyubiquitin (Tanaka et al. 2004). A different study that investigated the binding preference of isolated UBA domains from 30 different UDPs could show that the UBA domains of NUB1 are unable to interact with polyubiquitin chains or monoubiquitin (Raasi et al. 2005). NUB1 is reported to be inducible by IFN-β and IFN-γ (Kito et al. 2001) although this finding has been only shown in HeLa cells. Its subcellular expression is mostly in the nucleus, owing to the nuclear localisation signal (NLS) present in the sequence (Kito et al. 2001). In vitro

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Introduction

studies showed that NUB1 directly binds to Rpn10, a subunit of the proteasome, but surprisingly this study stated that this interaction was not mediated via its UBL domain but rather through a C-terminal region located between the residues 536 to 584 (Tanji et al. 2005). A different study could show that in vivo binding of NUB1 to the proteasome was totally dependent on the presence of its UBL domain since a deletion mutant lacking only the N-terminal UBL domain was unable to bind to the proteasome (Schmidtke et al.

2006).

In yeast two-hybrid screens, NUB1L was identified as a new non-covalent interaction partner of FAT10, which could be verified in vitro and in vivo. Experiments with ubiquitin and also with SUMO and NEDD8 showed no interaction despite previous reports that NUB1 can specifically bind NEDD8, using the same conditions. Even a ten fold excess of NEDD8 over FAT10 in the reaction was unable to compete for NUB1 binding (Hipp et al. 2004). NUB1L binds FAT10 in vivo through its three UBA domains.

With its UBL domain it can bind to the proteasome and promote the degradation of FAT10, but binding of FAT10 by NUB1L is not necessary for this accelerated degradation since a deletion mutant of NUB1L, lacking all UBA domains still accelerated FAT10 degradation. Thus, NUB1L appears not only to function as a shuttling factor for monomeric FAT10 and fatylated target proteins but also as an facilitator of FAT10 degradation (Schmidtke et al. 2006). Further evidence for the outstanding importance of NUB1L in the degradation of FAT10 comes from in vitro studies with purified FAT10, NUB1L and 26S proteasome. In this system FAT10 could be degraded by the 26S proteasome only in the presence of NUB1L. The degradation rate was independent of ubiquitylation and fully dependent on the amount of purified NUB1L in the system. Also shRNA dependent knock down of NUB1L in HeLa cells greatly reduced the degradation rate, determined by pulse-chase experiments, of a FAT10-DHFR fusion protein, further highlighting the importance of NUB1L in the degradation of FAT10 and its conjugates (Schmidtke et al. 2009).

Recently, NUB1 has been implicated with two different neurodegenerative pathologies.

One of the few identified interaction partners of NUB1 is AIPL1 (Aryl hydrocarbon receptor-interacting protein-like 1)(Akey et al. 2002), which expression in adults is restricted to rod photoreceptors in the human retina. Mutations in AIPL1 cause an early onset of the blinding disease Leber congenital amaurosis (LCA), which is the most severe retinal dystrophy causing blindness or severe visual impairment before the age of 1 year

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Introduction

(den Hollander et al. 2008). AIPL1 functions as a co-chaperone that interacts with HSP70 and HSP90 and is part of a chaperone heterocomplex that is able to modulate the translocation of NUB1 from the nucleus to the cytoplasm but the function of this interaction is still unclear (van der Spuy et al. 2004). The other link comes from a study that identified synphilin-1 as a further interaction partner of NUB1 (Tanji et al. 2006).

Synphilin-1 interacts with α-synuclein, which is predominantly expressed in neurons, and is thought to be involved in the pathogenesis of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), collectively referred to as α-synucleinopathies, where both proteins are found in cytoplasmic inclusions (Wakabayashi et al. 1998;

Engelender et al. 1999; Wakabayashi et al. 2000). It could be shown that over expression of NUB1 suppressed the formation of synphilin-1 positive inclusions, presumably by targeting the protein for proteasomal degradation (Tanji et al. 2006). The same group used immunostained sections of the brain from patients that suffered from PD and other α-synucleinopathies and showed that NUB1 co-localised with synphilin-1 and accumulated in inclusion bodies only in patients with α-synucleinopathies but not in inclusions found in other neurodegenerative disorders like Alzheimer’s disease or Pick's disease (Tanji et al. 2007).

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a primary cancer of the liver. It is one of the most frequent visceral malignancies worldwide and the fourth leading cause of cancer related death, with estimated ~600000 fatal casualties each year (Parkin et al. 2001). Men are generally more affected than women, with male:female ratios between 2:1 to 4:1 (El- Serag et al. 2007). In almost all cases, HCC develops from chronic hepatitis or cirrhosis, both regarded as preneoplastic stages (Laurent-Puig et al. 2006). These conditions are accompanied by an invasion with immune cells and the loss of many hepatocytes, since hepatocytes are very susceptible to the induction of apoptosis, which is mediated by the proinflammatory cytokine TNF-α (Czaja et al. 1995; Wang et al. 2006). This liver injury is associated with the deposition of connective tissue leading to progressive loss of liver function. The aetiology of HCC is well defined compared with other types of cancer. The main causative agents are hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol abuse and food that is contaminated with aflatoxin B1 (AFB), a mycotoxin produced by fungi from the Aspergillus genus. Chronic infection with HBV and/or HCV is the strongest risk

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Introduction

factor for HCC (Blumberg et al. 1985; Yeh et al. 1989; London et al. 1995; Bosch et al.

1999) but the incidence rate and also the onset of cancer varies widely worldwide. The highest rates are found in sub-Saharan Africa and eastern Asia, where foodstuffs are often contaminated with AFB and the hepatotrophic HBV and HCV are endemic. HCC is often diagnosed in these regions in earlier age ranging from the 20s to 50s. In North America and Western Europe, where HCC is a rare type of cancer, alcohol abuse is the most frequent cause of cirrhosis and tumours found in the liver are often not primary HCC. In many cases they are due to metastasis of cancers formed in different organs, mostly the colon. Therefore, most HCC cases are diagnosed in patients that are >60 years old. Treatment and prognosis of HCC depend on various factors but especially on tumour size and stage. The only treatment with curative potential is surgery (partial hepatectomy or transplantation) but only a minority of patients is amenable for this option because HCC is diagnosed in many cases in a late stage and surgery is only possible when the tumour occurs as a clear entity and not as poorly defined multiple spots that often show infiltrative growth or when the tumour localises to major blood vessels. Because of the aggressive growth and the lack of effective treatment options, the 5-year survival rate of patients with HCC is below 9% (Sherman 2005).

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Chapter 1

Quantitative analysis of gene expression relative to 18S rRNA in carcinoma samples using the LightCycler

^®

instrument and a SYBR GreenI based assay: determining FAT10 mRNA levels in hepatocellular carcinoma

Sebastian Lukasiak, Kai Breuhahn, Claudia Schiller, Gunter Schmidtke and Marcus Groettrup

Summary

Due to the fact that mutations and up- or down-regulation of genes can lead to the development of cancer, quantitative comparison of relative gene expression in healthy and cancerous tissue can gain valuable insights into tumourigenesis. While the semi- quantitative DNA microarrays are being used to identify differentially expressed genes on a genomic scale, real-time RT-PCR provides a power-full tool for quantitative measurement of gene expression. It presently is the most sensitive method available.

Here we describe in detail a SYBR GreenI based assay using the LightCycler^® instrument to measure the levels of mRNA for the ubiquitin-like protein FAT10 relative to 18S rRNA in human hepatocellular carcinoma tissue. This method can be easily adapted to any tissue (human or mouse, rat etc.) and any gene.

Key Words: relative gene expression; quantitative RT-PCR; SYBR GreenI; co-application reverse transcription (Co-RT); normalisation to 18S rRNA; highly pure RNA from tissue.

From: Methods in Molecular Biology, Vol. 429: Molecular Beacons: Signalling Nucleic Acid Probes, Methods and Protocols Edited by: A. Marx and O. Seitz © Humana Press Inc., Totowa, NJ

Elucidating the cause of FAT10 over-expression in liver and colon carcinomas