Investigation of the interaction of FAT10 and VCP (p97)
Dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.)
Presented by
Ricarda Schwab
at the
Faculty of Sciences Department of Biology
Date of the oral examination: 12.10.2015
First referee: Prof. Dr. Marcus Groettrup
Second referee: Prof. Dr. Martin Scheffner
Table of Content
Table of Content ... I Summary ... III Zusammenfassung ... IV
1. Introduction ... 1
1.1 Ubiquitin ... 1
1.2 Ubiquitination ... 2
1.3 Ubiquitin chains ... 3
1.4 Deubiquitination ... 5
1.5 Ubiquitin‐like modifiers ... 6
1.5.1. ISG15... 6
1.5.2. NEDD8 ... 7
1.5.3. SUMO ... 7
1.5.4. ATG12 and ATG8 ... 8
1.5.5. FAT10 ... 8
1.6 Ubiquitin‐like and ubiquitin‐binding domains ... 13
1.7 The Proteasome ... 14
1.7.1. Proteasome Activators ... 15
1.7.2. The immunoproteasome ... 17
1.7.3. Proteasome inhibitors ... 18
1.8 Endoplasmic reticulum quality control ... 19
1.8.1. ERAD ... 19
1.8.2. Unfolded‐protein response ... 21
1.9 VCP ... 22
1.9.1. VCP cofactors ... 26
1.9.2. VCP and diseases ... 28
1.9.3. VCP inhibitors ... 28
Aim of the study ... 30
2. Results ... 31
2.1 FAT10 is conjugated to VCP ... 31
2.2 FAT10 interacts with VCP non‐covalently ... 34
2.3 FAT10 directly interacts with VCP ... 35
2.4 VCP function is not necessary for degradation of FAT10 conjugates ... 37
2.5 FAT10 does not disturb the hexamerization of VCP ... 43
2.6 FAT10 does not influence the ATPase activity of VCP ... 46
2.7 FAT10 might influence the degradation of 1AT under ER stress ... 49
3. Discussion ... 52
3.1 FAT10 is conjugated to VCP ... 52
3.2 FAT10 interacts with VCP non‐covalently ... 54
3.3 FAT10 directly interacts with VCP ... 55
3.4 VCP function is not necessary for degradation of FAT10 conjugates ... 57
3.5 FAT10 does not disturb the hexamerization of VCP ... 58
3.6 FAT10 does not influence the ATPase activity of VCP ... 59
3.7 FAT10 might influence the degradation of 1AT under ER stress ... 60
4. Materials and Methods ... 62
4.1 Antibodies ... 62
4.2 Cell culture ... 63
4.3 Immunoprecipitation ... 63
4.4 Plasmids ... 64
4.5 Primer ... 65
4.6 siRNA ... 66
4.7 Quantitative real‐time PCR ... 66
4.8 Recombinant proteins ... 66
4.9 In vitro assays ... 68
4.10 Gel filtration chromatography ... 69
5. Literature ... 70
Danksagung ... 81
Summary
In the present study the interaction of the ubiquitin‐like modifier HLA‐F adjacent transcript 10 (FAT10) with the putative substrate Valosin‐containing protein (VCP) was investigated. VCP is a hexameric ATPase associated with various activities (AAA) and extracts proteins from the Endoplasmic Reticulum (ER) membrane or from protein complexes. In this function as a segregase VCP is involved in different ubiquitin‐dependent processes reaching from the ER associated degradation (ERAD) and cell cycle regulation to membrane fusion events.
In addition to a minor covalent attachment of FAT10 to VCP, which led to the degradation of the VCP‐FAT10 conjugate by the proteasome, the more prominent non‐covalent interaction of both proteins was examined in more detail and the functional consequences arising out of it. It was shown that FAT10 and VCP interact under endogenous conditions and further in vitro experiments revealed that they interact directly. Treatment with the VCP specific inhibitor Eeyarestatin I or use of an ATPase dead VCP mutant didn’t alter the degradation rate of bulk FAT10 conjugates, whereas treatment with the inhibitor DBeQ increased the degradation rate of the FAT10 conjugates. Furthermore the effect of FAT10 on VCP function was studied.
Neither the hexamer stability nor the ATPase activity of VCP was influenced by FAT10. Only a difference in the degradation of the ERAD model substrate 1‐Antitrypsin was observed in preliminary experiments. Taken all results together it can be concluded that the consequences of the VCP‐FAT10 interaction are different to consequences arising from the interaction of VCP with ubiquitin, and that the FAT10‐VCP interaction might play a specific role in the immune system or during inflammatory processes when FAT10 is highly up‐regulated.
Zusammenfassung
In dieser Arbeit wurde die Interaktion von “HLA‐F adjacent transcript (FAT) 10” (FAT10) mit dem potenziellen Substrat „Valosin‐containing protein (VCP)“ untersucht. VCP ist eine hexamere “ATPase assoziiert mit verschiedenen Aktivitäten (AAA)” und extrahiert Proteine aus der Membran des Endoplasmatischen Retikulums (ER) oder aus Proteinkomplexen. In dieser Funktion als „Segregase“ ist VCP in viele ubiquitinabhängige Prozesse involviert, von der ER assoziierten Degradation (ERAD) und der Zellzykluskontrolle bis hin zur Membranfusion.
Zusätzlich zur geringfügigen kovalenten Verknüpfung von FAT10 und VCP, die zum Abbau des FAT10‐VCP‐Konjugats führte, wurde eine markantere nichtkovalente Interaktion beider Proteine erforscht. Es konnte gezeigt werden, dass FAT10 und VCP unter endogenen Bedingungen interagieren und in vitro Experimente zeigten, dass sie direkt miteinander interagieren. Anwendung des VCP‐spezifischen Inhibitors Eeyarestatin I oder Einsatz einer VCP‐Mutante ohne ATPase‐Aktivität veränderte die Abbaurate der gesamten FAT10‐
Konjugate nicht, hingegen die Anwendung des Inhibitors DBeQ beschleunigte den Abbau der Konjugate. Ausserdem wurde der Effekt von FAT10 auf die Funktion von VCP untersucht.
Weder die Stabilität des Hexamers, noch die ATPase‐Aktivität von VCP wurden von FAT10 beeinflusst. Lediglich ein Unterschied im Abbau des ERAD Modellsubstrats 1‐Antitrypsin konnte beobachtet werden, was jedoch als vorläufiges Ergebnis zu betrachten ist.
Zusammenfassend lassen die Resultate den Schluss zu, dass die Auswirkungen der FAT10‐VCP‐
Interaktion andere sind, als die der Interaktion von Ubiquitin mit VCP und die FAT10‐VCP‐
Interaktion eine spezielle Rolle im Immunsystem oder während entzündlicher Prozesse spielen könnte, wenn die Expression von FAT10 hochreguliert ist.
1. Introduction
Cells have evolved sophisticated control mechanisms that sense misfolded or redundant proteins in order to eliminate them and thereby maintain homeostasis. Besides autophagy the main protein degradation pathway goes via the ubiquitin‐proteasome‐system (UPS). Target proteins are marked with ubiquitin, a 76 amino acid polypeptide that is recognized by a multicatalytic complex, the proteasome. It cleaves proteins into short peptides to prevent their accumulation and thus it takes part in regulation of many cellular processes from gene transcription, signalling and protein quality control up to antigen presentation.
In the following sections the key players of the UPS as well as associated proteins and processes are described in more detail.
1.1 Ubiquitin
The small molecule ubiquitin was initially discovered as a factor required for ATP‐dependent protein breakdown (Schlesinger, Goldstein et al. 1975, Ciechanover, Elias et al. 1980, Ciechanover, Heller et al. 1980, Wilkinson, Urban et al. 1980) and is conserved between species (Schlesinger and Goldstein 1975). In mammals ubiquitin is encoded by the four genes Uba52, Uba80, Ubb and Ubc. The first two genes encode each one fusion protein with a ribosomal subunit, the latter two encode tandem repeats in head‐to‐tail formation translated as precursors which need to be processed by specific hydrolases (Ozkaynak, Finley et al. 1984, Lund, Moats‐Staats et al. 1985, Jonnalagadda, Butt et al. 1989). This liberates a 76 amino acid polypeptide of 8.5 kilodalton (kDa) with a ‐grasp‐fold and a C‐terminal diglycine motif necessary for conjugation to substrate proteins.
Ubiquitination is ‐ besides phosphorylation ‐ the most frequent posttranslational modification and is one possibility to regulate cellular processes by degradation or changing the localisation or activity of involved proteins. Recently ubiquitin itself was found to be phosphorylated which creates an additionally mechanism to regulate ubiquitinated proteins (Kane, Lazarou et al.
2014, Kazlauskaite, Kondapalli et al. 2014, Koyano, Okatsu et al. 2014).
1.2 Ubiquitination
The conjugation of ubiquitin to its substrates is executed by a three enzyme cascade resulting
in an isopeptide bond between the carboxyl group of the C‐terminal glycine of ubiquitin and the ‐amino group of a lysine in a substrate protein. However, ubiquitin can also be attached via a linear peptide bond to the N‐terminus of a substrate or to other ubiquitins or to a serine, threonine or cysteine to build an ester or thioester.
The cascade starts with an ubiquitin‐activating enzyme (E1) (Hershko, Heller et al. 1983) binding ubiquitin and ATP to build an ubiquitin adenylate with release of pyrophosphate. This results in a thioester bond of the carboxyl group of ubiquitin to the active site cysteine of the E1 and to free AMP. A second ubiquitin is recruited to the adenylation site and the first ubiquitin is transferred to the active site cysteine of the ubiquitin‐conjugating enzyme (E2).
After the transthiolation reaction the ubiquitin ligase (E3) catalyses the transfer of ubiquitin onto the ‐amino group of a lysine in a substrate protein (Scheffner, Nuber et al. 1995).
Homologous to E6‐AP carboxyl terminus (HECT) E3 ligases take over ubiquitin to form a thioester intermediate at their active site cysteine and transfer it to the substrate (Huibregtse, Scheffner et al. 1995). Really interesting new gene (RING) E3s which don’t have an active site cysteine bring substrate and E2 into close proximity to allow a direct transfer of ubiquitin onto the substrate (Joazeiro, Wing et al. 1999). This is achieved by two zinc ions coordinated between cysteine and histidine residues in a crossbrace arrangement, the so called RING‐
finger. The most abundant E3s belong to the Cullin‐RING E3 ligase (CRL) superfamily (Feldman, Correll et al. 1997, Skowyra, Craig et al. 1997).
U‐box E3s have a similar scaffolding function without zinc ions (Aravind and Koonin 2000, Hatakeyama, Yada et al. 2001). RING‐between‐RING (RBR) E3s work like RING E3s but have an active site cysteine in one of their RING‐like domains to transfer a thioester intermediate (Aguilera, Oliveros et al. 2000). A special member of the RBR family is the linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL‐1L and the HOIL‐1L interacting protein (HOIP) linking ubiquitin molecules in a head‐to‐tail conformation to each other (Kirisako, Kamei et al.
2006).
The target of an ubiquitin molecule is specified by the consecutive action of the three
McGrath, Jentsch et al. 1991) and UBA6 (Chiu, Sun et al. 2007, Jin, Li et al. 2007, Pelzer, Kassner et al. 2007), have a selection of about 35 E2s in humans (van Wijk, de Vries et al. 2009). A supposed redundancy of the E1s is contradicted by the fact that the UBA6 specific E2 (USE) 1 accepts transfer of ubiquitin only from UBA6 (Jin, Li et al. 2007) and the knockout of UBA6 (Chiu, Sun et al. 2007) or UBA1 is lethal (McGrath, Jentsch et al. 1991). However substrate specificity is achieved at the final conjugation step and thus there are hundreds of E3s encoded in the human genome to accomplish ubiquitination of a myriad of substrates (Metzger, Pruneda et al. 2014).
1.3 Ubiquitin chains
The kind of ubiquitination determines the fate of the substrate protein. Monoubiquitination at single or multiple lysine residues is important for regulation of signalling processes or endocytosis (Haglund, Sigismund et al. 2003). Additionally to the modification of a substrate by a single molecule more ubiquitin molecules can be attached to one another and depending on the chosen lysine (K) residue within ubiquitin (K6, 11, 27, 29, 33, 48, 63) different chains can be build (Figure 1).
At least four ubiquitins linked at K48 were believed to be the principal chain type that designate the substrate protein for degradation by the proteasome (Chau, Tobias et al. 1989).
Later it was shown that K11 chains (Baboshina and Haas 1996, Xu, Duong et al. 2009) and all other chain types, except K63, contribute to degradation via the proteasome, too (Xu, Duong et al. 2009). For instance mainly K11 chains are found to associate with UBA/UBX proteins (Alexandru, Graumann et al. 2008) which interact with Valosin‐containing protein (VCP) (Hartmann‐Petersen, Wallace et al. 2004, Schuberth, Richly et al. 2004), a key component in endoplasmic reticulum associated degradation (ERAD) (Ye, Meyer et al. 2001). And interestingly the E3 ligase anaphase‐promoting complex/cyclosome (APC/C) assembles K11 chains on substrates that need to be degraded during cell cycle (Jin, Williamson et al. 2008).
K63 chains are implicated in the DNA‐damage response (Hofmann and Pickart 1999) and in signalling events leading to the activation of the transcription factor NF‐B (Deng, Wang et al.
2000) but have also been described in endocytosis of cell surface receptors (Geetha, Jiang et al. 2005, Duncan, Piper et al. 2006).
Figure 1: Lysine residues in ubiquitin. All lysine residues within ubiquitin are highlighted in red with blue nitrogen atoms and putative function of chains are indicated. The sulphur atom in Met1 is depicted in green.
The C‐terminal Gly75‐Gly76 motif involved in isopeptide bond formation is marked in red for oxygen atoms and blue for nitrogen atoms (Komander 2009).
Other types required in NF‐B activation are linear chains (Rahighi, Ikeda et al. 2009) with the C‐terminal glycine of one ubiquitin connected to the N‐terminal methionine of the next ubiquitin. The function of other linkages is not that clear yet. It was shown for Notch signalling that K29 linked chains lead to degradation of a regulator protein via the lysosome (You and Pickart 2001), whereas the use of K29 chains in the ubiquitin fusion degradation pathway rather points to degradation by the proteasome (Johnson, Ma et al. 1995). Mixed chains have been proposed to regulate E3 ligase activity (Ben‐Saadon, Zaaroor et al. 2006) and even branched chains have been observed (Kim and Goldberg 2012) whereas their function is still elusive.
1.4 Deubiquitination
The modification of a protein by ubiquitin is a highly regulated process and is also reversible.
It’s known from the degradation machinery that there are enzymes that can cut the isopeptide linkage between the substrate protein and the attached ubiquitin (Swaminathan, Amerik et al. 1999, Verma, Aravind et al. 2002). As about 85 deubiquitinating enzymes (DUBs) are encoded in the human genome, it is obvious that there must be a greater variety in specificity and selectivity for substrates and particular types of polyubiquitin chains. Despite the differences in number and type of additional domains DUBs can be divided into five classes defined by the structure of their catalytic domain: ubiquitin‐specific protease (USP), ovarian tumour (OTU), ubiquitin C‐terminal hydrolase (UCH), Josephin (MJD) cysteine proteases and the Jab1/Mov34/Mpr1 (JAMM) metalloproteases.
The cysteine proteases need a histidine‐asparagine/aspartate to activate the cysteine for the nucleophilic attack on the peptide linkage (Storer and Menard 1994). Most of the proteases have these residues in a row but some USP (Hu, Li et al. 2002) and OTU (Edelmann, Iphofer et al. 2009) family members have to undergo conformational changes – sometimes induced by the substrate – to get the residues aligned in order to be activated. Thereby specificity for a substrate can be accomplished, e.g. OTUB1 cleaves only K48 linked ubiquitin chains (Edelmann, Iphofer et al. 2009, Wang, Yin et al. 2009) and OTULIN cleaves linear ubiquitin chains (Keusekotten, Elliott et al. 2013, Mevissen, Hospenthal et al. 2013).
Many DUBs are found in complexes with other proteins, often E2 conjugating enzymes or E3 ligases, which can regulate their activity or localisation and vice versa (Sowa, Bennett et al.
2009, Keusekotten, Elliott et al. 2013). Structural studies suggest that the binding of partner proteins induces conformational changes that keeps the catalytic residues aligned and stabilizes the DUB in an active conformation (Samara, Datta et al. 2010).
1.5 Ubiquitin‐like modifiers
Although ubiquitin is the most abundant modifier there are other family members that share not so much sequence homology but the three dimensional ubiquitin fold and that are targeted to proteins in a similar way. All ubiquitin‐like modifiers (ULMs) can be conjugated to lysines of substrate proteins via a diglycine motif at their C‐terminus which is executed by a three enzyme cascade, partially the same as for ubiquitin. The best studied ULMs are shortly described in the paragraphs below.
1.5.1. ISG15
Interferon‐stimulated gene (ISG) 15 was the first family member discovered after ubiquitin in
1979 (Farrell, Broeze et al. 1979). It is not constitutively expressed but can be induced together with its conjugation machinery by interferon (IFN) and (Nielsch, Pine et al. 1992, Yuan and Krug 2001)and lipopolysaccharide (LPS) (Hamerman, Hayashi et al. 2002). After processing of the precursor ISG15 is a 15 kDa protein consisting of two domains with 29% and 27% sequence homology to ubiquitin, respectively. The E1 enzyme UBE1L (Yuan and Krug 2001) hands over the activated ISG15 to the bispecific ubiquitin E2 UbcH8 (Zhao, Beaudenon et al. 2004). The RING E3 estrogen‐responsive finger protein (EFP) (Zou and Zhang 2006) and the E3 HECT domain and RCC1‐like domain containing protein (Herc) 5 (Dastur, Beaudenon et al. 2006)
were first found to mediate the transfer of ISG15 to its target proteins, e.g. serine protease inhibitor (serpin) 2a (Hamerman, Hayashi et al. 2002), PLC1, JAK1, STAT1 or ERK1 (Malakhov, Kim et al. 2003). ISG15 can be deconjugated by USP18 (UBP43) (Malakhov, Malakhova et al.
2002) and by USP3, 5, 13 and 14 (Catic, Fiebiger et al. 2007).
Considering its expression during infection and its secretion by monocytes and lymphocytes (Knight and Cordova 1991) ISG15 was thought to be involved in the IFN type I induced immune response, but in the first place ISG15‐/‐ mice showed no obvious phenotype (Osiak, Utermohlen et al. 2005) as well as UBE1L‐/‐ mice (Kim, Yan et al. 2006). Only later ISG15 and ISGylation could be shown to be important in some cases of viral infection (Lenschow, Lai et al. 2007), however its role in tumorigenesis is still not clear (Bektas, Noetzel et al. 2008, Feng,
1.5.2. NEDD8
Neural‐precursor‐cell‐expressed‐developmentally‐down‐regulated (NEDD) 8 (Kamitani, Kito et al. 1997) or related‐to‐ubiquitin (Rub) 1 in yeast (Liakopoulos, Doenges et al. 1998) is in respect of sequence homology the closest relative of ubiquitin with 53% identity and 72%
similarity. The NEDD8 precursor is processed to a 76 amino acid polypeptide by NEDD8‐
specific protease 1 (NEDP1, also known as DEN1) (Gan‐Erdene, Nagamalleswari et al. 2003) or ubiquitin C‐terminal hydrolase (UCH)‐L3 (Wada, Kito et al. 1998). It is conjugated by the heterodimeric E1 APPBP1‐UBA3 via the E2 Ubc12 (Osaka, Kawasaki et al. 1998) and several E3s to substrate proteins to modulate their activity. For example the neddylation of p53 inhibits its transcriptional activity (Xirodimas, Saville et al. 2004). In contrast neddylation of parkin stimulates its E3 ligase activity (Choo, Vogler et al. 2012). Likewise neddylation of cullins activates the multisubunit Cullin‐RING ubiquitin E3 ligases (CRLs) (Hori, Osaka et al. 1999), which can be deneddylated by the COP9 signalosome (CSN) (Lyapina, Cope et al. 2001).
Interestingly an attachment of NEDD8 to ubiquitin was observed in which NEDD8 terminated ubiquitin chain (Singh, Zerath et al. 2012).
1.5.3. SUMO
The small ubiquitin‐related modifier (SUMO) has four isoforms in mammals with partially overlapping functions in cell cycle progression (Dieckhoff, Bolte et al. 2004), nuclear protein import (Kirsh, Seeler et al. 2002) and protein stabilisation (Desterro, Rodriguez et al. 1998).
Sentrin/SUMO‐specific proteases (SENPs) can not only hydrolyse the precursors but can also cut the isopetide bonds to substrates (Gong and Yeh 2006). After activation of SUMO by the Aos1‐UBA2 heterodimer (Gong, Li et al. 1999), the E2 enzyme UBC9 (Schwarz, Matuschewski et al. 1998) is able to transfer SUMO directly to substrates, but this can also be mediated by the E3 enzyme RanBP2‐RanGAP1 (Werner, Flotho et al. 2012) or by protein inhibitor of activated STAT (PIAS) 1 (Kahyo, Nishida et al. 2001). SUMO can be assembled in chains (Tatham, Jaffray et al. 2001) which can be ubiquitinated by the ligase RNF4 which leads to proteasomal degradation of the substrate (Tatham, Geoffroy et al. 2008).
1.5.4. ATG12 and ATG8
Autophagy‐related gene (ATG) 12 was the first ULM identified to be involved in autophagy (Mizushima, Noda et al. 1998). It’s already translated with a free diglycine motif and can directly be activated by the E1 ATG7 and conjugated by the E2 ATG10 to its substrate ATG5.
This ATG12‐ATG5 conjugate recruits factors required for phagophore elongation and closure, like the coiled‐coil protein ATG16. Together they build a complex which has E3‐like activity for ATG8‐Phosphatidylethanolamine (PE) (Fujita, Itoh et al. 2008).
ATG8 (LC3 in humans) is an exception in the ULM family as it targets PE and is not conjugated to proteins (Ichimura, Kirisako et al. 2000). After processing by the cysteine protease ATG4, ATG8 is activated by ATG7, transferred to its E2 ATG3 and finally conjugated to the amino group of PE incorporated into the phagophore (Kirisako, Ichimura et al. 2000).
1.5.5. FAT10
In 1996 Fan et al. found seven new genes in the human major histocompatibility complex (MHC) class I region at the human leukocyte antigen (HLA)‐F locus on chromosome 6, one of them showing high similarity to a doubled ubiquitin (Fan, Cai et al. 1996). Thus the 18 kDa protein was named diubiquitin or ubiquitin D. Later the nomenclature HLA‐F adjacent transcript (FAT) 10 was asserted.
FAT10 was first found to be expressed in mature dendritic cells and mature B‐cells (Bates,
Ravel et al. 1997), but then it was discovered that FAT10 is strongly upregulated in all cell types after stimulation with the proinflammatory cytokines Interferon (IFN) and Tumour necrosis factor (TNF) (Liu, Pan et al. 1999, Raasi, Schmidtke et al. 1999). Further studies revealed that under non‐inflammatory conditions endogenous FAT10 expression is highest in spleen, thymus, gut and lymph nodes of mice (Lukasiak, Schiller et al. 2008), suggesting a role of FAT10 in the immune system. The mild phenotype of FAT10 knockout mice under laboratory conditions merely can be challenged by lipopolysaccharide (LPS) to induce endotoxin hypersensitivity (Canaan, Yu et al. 2006). This hints to an involvement in specialized immune reactions as FAT10 ‐ in contrast to other parts of the adaptive immune system ‐ is found only
The two domains of FAT10 have 29% and 36% sequence homology to ubiquitin, respectively, but only 20% homology to each other (Bates, Ravel et al. 1997). Although the structure is not yet completely solved (Theng, Wang et al. 2014), both domains are expected to have the typical ubiquitin fold with a five amino acid linker in between which is assumed to allow flexible positioning to each other (Figure 2). Corresponding to the amino acid sequence of ubiquitin human FAT10 harbours four lysines (K27, 33, 48 and 63) at the same positions whereas in mice only lysine 48 is conserved. Different to ubiquitin FAT10 contains two cysteines per domain but it exhibits the C‐terminal diglycine motif as a common feature of all ULMs (Bates, Ravel et al. 1997). Most of them ‐ as well as ubiquitin itself ‐ are produced as precursors and need to be processed. However FAT10 is translated with a free diglycine motif and can be directly attached to lysines in substrate proteins in a ubiquitin‐like manner (Raasi, Schmidtke et al. 2001) by an enzyme cascade sharing even the same enzymes with ubiquitin.
Figure 2: Ribbon model of ubiquitin (left) and FAT10 (right). Both domains of FAT10 display the typical ‐grasp fold of ubiquitin, witha central ‐helix (turquoise) surrounded by ‐sheets (purple) (Groettrup, Pelzer et al.
2008).
FAT10 is activated by the E1 enzyme UBA6 which was found to prefer ubiquitin for thioester formation (Chiu, Sun et al. 2007, Pelzer, Kassner et al. 2007). Nevertheless Gavin et al.
measured a higher binding affinity of FAT10 to UBA6 in vitro (Gavin, Chen et al. 2012). It is assumed that massive expression of FAT10 during infections changes the ratio to ubiquitin increasing the probability for FAT10 to be taken by UBA6 for activation (Chiu, Sun et al. 2007, Gavin, Chen et al. 2012). The E2 conjugating enzyme UBA6 specific enzyme (USE) 1 is bispecific
for ubiquitin and FAT10, too (Aichem, Pelzer et al. 2010), and takes over – as its name indicates
‐ both ULMs only from UBA6. However, FAT10 can’t be activated and conjugated by the UBA6 dependent E2s Ubc5 and Ubc13 or by the second ubiquitin E1 (UBE1) and corresponding E2s (Chiu, Sun et al. 2007). Knockdown of either UBA6 or USE1 massively reduce FAT10 conjugates proposing that these are the main if not only E1 and E2 enzymes for FAT10, respectively (Chiu, Sun et al. 2007, Aichem, Pelzer et al. 2010).
As there are no E3 ligases for FAT10 published yet, one might speculate that in vitro the E1 UBA6 and the E2 USE1 are sufficient for conjugation to substrate proteins likewise it was published for SUMO (Tatham, Jaffray et al. 2001, Werner, Flotho et al. 2012). Recently it could be demonstrated that at least in vitro even the E1 UBA6 is sufficient for conjugation of FAT10 to its substrate protein UBE1 (Bialas, Groettrup et al. 2015), which might be due to high protein concentrations and thus close proximity of all involved proteins. Nevertheless the need for an E3 ligase to allow substrate specific FAT10ylation in vivo can’t be ruled out and several attempts have been done to identify putative candidates, which are still under investigation (J. Bialas, unpublished data).
The incomplete knowledge about the FAT10‐conjugation enzyme cascade arises the question about deconjugating enzymes. Expression of linear ubiquitin‐GFP in cells leads to rapid cleavage of the fusion proteins, whereas FAT10‐GFP stays intact (Hipp, Kalveram et al. 2005).
But keeping in mind that FAT10 does not need to be processed to liberate its diglycine motif it seems to be likely that there is no enzyme cleaving linear FAT10 fusions. On the other hand endogenous FAT10 conjugates are degraded at the same rate as free FAT10 which is very short lived (Hipp, Kalveram et al. 2005), why it might be possible that there are no deconjugating enzymes for FAT10 at all.
The first substrate identified was the E2 enzyme USE1 which auto‐FAT10ylates itself leading to its degradation by the proteasome (Aichem, Pelzer et al. 2010, Aichem, Catone et al. 2014).
This auto‐modification relies on the active site cysteine of USE1 which transfers the activated FAT10 onto its lysine323, however when it’s mutated another lysine is chosen (Aichem, Catone et al. 2014). Interestingly also the ubiquitin E1 enzyme UBA1 (also UBE1) is a substrate of FAT10 and becomes degraded by the proteasome (Rani, Aichem et al. 2012, Bialas, Groettrup et al. 2015), which might be a mechanism to bias the conjugation machinery
Involvement of FAT10 in other degradation mechanisms can be assumed e.g. because of the modification of the autophagy adapter protein p62 (Aichem, Kalveram et al. 2012). It is multi‐
monoFAT10ylated meaning modified with one FAT10 at several lysines simultaneously which marks p62 for degradation via the proteasome. Additionally p62 interacts also non‐covalently with FAT10 (Aichem, Kalveram et al. 2012), but a function for the interaction couldn’t be
established yet.
NEDD8 ultimate buster 1 long (NUB1L; see also in section 1.6), which is inducible by IFN (Kito, Yeh et al. 2001), was shown to interact non‐covalently with the N‐terminal domain of FAT10 accelerating its degradation. As NUB1L interacts also with the proteasome subunit Rpn10 it was proposed to transfer FAT10 to Rpn10 in order to facilitate the degradation of FAT10 (Hipp, Raasi et al. 2004, Tanji, Tanaka et al. 2005, Rani, Aichem et al. 2012).
FAT10 itself was reported to be modified by ubiquitination (Hipp, Kalveram et al. 2005, Buchsbaum, Bercovich et al. 2012) and by at least slight acetylation (Kalveram, Schmidtke et al. 2008). As it was shown that degradation of FAT10ylated substrates is independent of further ubiquitination (Hipp, Kalveram et al. 2005, Schmidtke, Kalveram et al. 2009), the significance of these modifications for the function of FAT10 is still under debate.
Despite the characterization of the degradation of some substrates the role of FAT10 in biological processes is poorly understood. Before substrate proteins or even E1 and E2 enzymes were identified, the spindle assembly checkpoint protein mitotic arrest deficiency (MAD) 2 was found as an non‐covalent interaction partner of FAT10 (Liu, Pan et al. 1999).
Because it’s expressed in B‐cells and dendritic cells the authors speculated that FAT10 modulates cell growth during development and activation. Further investigation by Ren et al.
showed delocalization of MAD2 from the kinetochore upon FAT10 overexpression, leading to missegregation of chromosomes (Ren, Kan et al. 2006), which could be inhibited by siRNA knockdown of FAT10 at simultaneous TNF treatment (Ren, Wang et al. 2011). As FAT10 expression was claimed to be upregulated in hepatocellular, colorectal, ovarian and uterus carcinomas it is implied to promote tumorigenesis (Lee, Ren et al. 2003). The tumour suppressor p53 was first linked to FAT10 as a negative regulator of expression and a loss of p53 leading to transcriptional upregulation of FAT10 contributing to tumorigenesis (Zhang, Jeang et al. 2006). Additionally, FAT10 was described to upregulate transcriptional activity of
p53 and modify inactive p53 (Li, Santockyte et al. 2011). Choi et al. proposed a synergistic activation of FAT10 expression by NF‐B and STAT3 which leads to p53 repression (Choi, Kim et al. 2014). Further FAT10 was suggested, besides mutant p53, to be used as prognostic marker for gastric cancer (Ji, Jin et al. 2009).
Contradictory to the statements mentioned before cytokine‐induced expression of FAT10 could be a response to pro‐inflammatory stimuli of the surrounding tissue to fight the tumour.
The simultaneous upregulation of the immunoproteasome subunit LMP2, changing the subset of MHC class I ligands (see also section 1.7.2) argues for this (Lukasiak, Schiller et al. 2008).
Whether FAT10 expression promotes or suppresses induction of apoptosis is discussed very oppositional. Some groups could demonstrate that FAT10 overexpression induces apoptosis in mouse fibroblasts, HeLa cells and renal tubular epithelial cells (Liu, Pan et al. 1999, Raasi, Schmidtke et al. 2001, Ross, Wosnitzer et al. 2006) and that FAT10 knockdown protects from apoptosis (Ross, Wosnitzer et al. 2006). In a recent study FAT10 was found to interfere with the NF‐B pathway by modifying leucine‐rich repeat Fli‐I‐interacting protein (LRRFIP) 2 (Buchsbaum, Bercovich et al. 2012). FAT10‐modified LRRFIP can’t be recruited to the LPS‐
sensing Toll‐like receptor (TLR) 4 anymore and therefore NF‐B mediated transcription of apoptosis inhibitors is prevented. In contrast, Ren et al. observed a protective function of FAT10 expression from TNF induced apoptosis in the colon cancer cell line HCT116 (Ren, Kan et al. 2006).
Various other interaction partners were found which point to different tasks of FAT10. The histone deacetylase (HDAC) 6 mediates the transport of polyubiquitinated proteins to aggresomes upon proteasome inhibition. It acts as a linker between ubiquitin chains and dynein which brings substrates to these inclusion bodies for degradation via autophagy. Under these conditions FAT10 interacts non‐covalently with HDAC6 and localizes to aggresomes, too (Kalveram, Schmidtke et al. 2008).
A possible role for FAT10 in eye development was suggested recently. The aryl hydrocarbon receptor‐interacting protein‐like 1 (AIPL1) which is mutated in Leber’s congenital amaurosis (LCA) was shown to interact with NUB1 (van der Spuy and Cheetham 2004) and free as well as conjugated FAT10 build a ternary complex (Bett, Kanuga et al. 2012). The authors claimed that
is abolished by pathogenic mutations in AIPL1. Additionally AIPL1 associates with UBA6 which implicates further regulation of FAT10ylation (Bett, Kanuga et al. 2012).
As FAT10 seems to be involved in various processes and its role there is heavily discussed, a lot of investigation is needed to shed light on its actual impact on these pathways.
1.6 Ubiquitin‐like and ubiquitin‐binding domains
Additionally to ubiquitin‐like modifiers there are proteins containing one or more integral ubiquitin‐like folds which are not conjugated to substrate proteins. The ubiquitin‐like (UBL) domain is a common feature present in proteins like proteasome shuttle factors, E3 ligases, DUBs or even kinases. A slightly modified ubiquitin analogue is the ubiquitin‐regulatory x (UBX) domain of VCP cofactors (reviewed in (Schuberth and Buchberger 2008); see also section 1.9.1). In contrast the Phox and Bem1 (PB1) domain containing proteins are functionally unrelated.
Besides the domains having the ubiquitin‐like structure there are domains possessing a more or less high affinity to bind to ubiquitin itself or to ubiquitin family members. Most of these ubiquitin‐binding (UBD) domains share a similar ‐helix‐based structure and bind to the same hydrophobic patch around isoleucine 44 in ubiquitin such as the ubiquitin‐associated (UBA) domain, ubiquitin‐associating (UAS), ubiquitin‐interacting motif (UIM), double‐sided ubiquitin‐interacting motif (DUIM), motif interacting with ubiquitin (MIU), the coupling of ubiquitin conjugation to ER degradation (CUE) domain or the von Willebrand factor type A (VWA) domain. Other UBDs like Npl4 zinc finger (NZF) or polyubiquitin‐associated zinc finger (PAZ), the ubiquitin‐conjugating (UBC) domain, ubiquitin‐conjugating enzyme variant (UEV), GRAM‐like ubiquitin‐binding in Eap45 (GLUE), Jab1/MPN and PLAA family ubiquitin binding (PFU) domains differ in structure and binding site preference (reviewed in (Grabbe and Dikic 2009)).
As mentioned above UBL and UBD domains can interact with each other and often they are comprised even in the same protein. Many of them, as most of the UBL‐UBA domain proteins, participate in the process of protein degradation but their overall domain structure and the specific event as well as other processes they are involved in are diverse (reviewed in (Su and Lau 2009)). For example, NEDD8 ultimate buster‐1 long (NUB1L) binds FAT10 via its UBA
domains and via its UBL domain it binds to the von Willebrand factor type A (VWA) domain of the 26S proteasome subunit Rpn10 and additionally to Rpn1 ((Rani, Aichem et al. 2012); see also section 1.7.1) to accelerate the degradation of FAT10‐coupled substrate proteins. The cofactors of VCP inclosing a UBX domain also have a UBA domain give rise to the UBA‐UBX domain protein family, but other combinations are possible, too, (Schuberth and Buchberger 2008).
1.7 The Proteasome
The ubiquitin‐proteasome‐system (UPS) is the major mechanism of eukaryotic cells to degrade proteins in order to maintain protein homeostasis. The centre of the pathway is the 26S proteasome, a multi‐subunit ATP‐dependent complex degrading ubiquitinated proteins not only in the cytoplasm but also in the nucleus. It consists of a 20S core particle (CP) cleaving its substrates endocatalytically and 19S regulatory particles (RP, PA700) which can associate at both sites to control the entry of the substrates (Figure 3). The 20S proteasome is found in Bacteria, Archaea and Eukarya and its overall structure is well conserved. It consists of four stacked heptameric rings forming a barrel‐like shape with a central pore divided into three chambers (Lowe, Stock et al. 1995, Groll, Ditzel et al. 1997). In eukaryotes the outer rings are composed of seven different ‐subunits, the inner rings of seven different ‐subunits including the three catalytically active ones with N‐terminal threonine residues as active‐site
nucleophiles. Those subunits favour different amino acids as signal for hydrolysis of peptide bonds. 1 (PSMB6, Y, ) comprises a caspase‐like activity cleaving after acidic amino acids, 2 (PSMB7, Z, MC14) has a trypsin‐like activity preferring basic amino acids and 5 (PSMB5, X, MB1) bares a chymotrypsin‐like activity choosing hydrophobic residues.
Proteasomes are the main proteases supplying ligands for major histocompatibility complex (MHC) class I molecules as they produce peptides of eight to ten amino acids in length with preferentially hydrophobic or basic amino acids at the C‐terminus as anchor residues particularly fitting into the cleft of the MHC molecules.
The transcription factor nuclear factor erythroid 2‐related factor (Nrf) 1 is a type II
the proteasome. Upon impaired proteasome function Nrf1 is only partially processed and thereby activated. It increases the transcription of all proteasome subunits and PA200 as well as VCP and its cofactors Ufd1, Npl4 and p47 (see section 1.9) to compensate the reduced degradation capacity (Sha and Goldberg 2014).
Figure 3: Scheme of the 26S proteasome. The 20S core particle consists of four heptameric rings stacked onto each other. The outer rings are built of ‐subunits and the inner rings are comprised of ‐subunits including the catalytically active ones 1, 2 and 5. The 19S regulatory particle contains the AAA subunits Rpt1‐6 which build together with Rpn1, 2 and 13 the base for the assembly of the subunits Rpn3, 5‐9, 11, 12 and 15 which form a lid. The 19S particle regulates the entry of substrates into the catalytic chamber of the core particle.
1.7.1. Proteasome Activators
The ‐subunits of the 20S CP confine the entry of the substrate into the pore by building a gate which can be opened through conformational changes caused by binding of the 19S RP or the other proteasome activators (PA) 11S RP (PA28) or Blm10 (PA200). This gains the access of substrates which need to be degraded and restricts uncontrolled proteolysis (reviewed in (Forster, Unverdorben et al. 2013)).
The base of the 19S RP is composed of regulatory‐particle triple‐A proteins (Rpt) 1‐6 forming a ring and the regulatory particle non‐ATPase proteins (Rpn) 1, 2 and 13. The subunits Rpn 3, 5‐9, 11, 12 and 15 form a lid which is connected to the base via Rpn10 (Figure 3). The C‐termini
of the Rpt subunits extend into pockets between the ‐subunits of the 20S CP and the N‐
termini of the‐subunits are arranged at the inside of the RP ring (reviewed in (Kunjappu and Hochstrasser 2014)). Ubiquitin chains attached to substrates are recognized by the receptor subunits Rpn10 and Rpn13 so that the substrate can be grasped by the Rpt subunits to translocate the protein through the ring which is ATP‐dependent and thereby unfold it. In the meantime the deubiquitinating enzyme Rpn11 removes the ubiquitin chain from the substrate which then can enter the cylinder of the 20S CP and reach the middle chamber where the cleavage of the substrate occurs (Verma, Aravind et al. 2002). The ubiquitin chain can be further processed and the single moieties can be reused for conjugation.
The other two proteasome activators PA28 and PA200 bind to the ‐ring of the 20S CP the same way as the 19S RP in order to open the gate but in an ATP‐independent manner. The activators can also combine with the 19S RP to build a hybrid proteasome (Hendil, Khan et al.
1998, Cascio, Call et al. 2002). PA200 is a 250 kDa monomer which was indicated to activate the proteasome during in DNA repair and spermatogenesis by stimulating the caspase‐like activity of the 20S CP (reviewed in (Savulescu and Glickman 2011)). PA28is a subtype of PA28 mainly expressed in the nucleus forming a homoheptameric ring. It was shown to selectively stimulate the trypsin‐like activity without affecting the other two activities and to promote
ubiquitin‐independent degradation of some substrates involved in cell cycle progression and induction of apoptosis. The other subtype of PA28 is the heteroheptamer PA28 3 and 4
subunits, whose expression is induced by the cytokine IFN, assemble to form a ring which associates with the 20S proteasome in the cytosol. PA28 is involved in the MHC I restricted antigen presentation as it enhances processing of small peptides resulting in MHC I ligands (reviewed in (Vigneron and Van den Eynde 2014)). This relates to double‐cleavage of substrates presumably by retention of the substrates in the catalytic chamber (Dick, Ruppert et al. 1996). Accordingly it was demonstrated that PA28 promotes presentation of virus antigens leading to an increased recognition by T‐cells (Groettrup, Ruppert et al. 1995).
1.7.2. The immunoproteasome
As response to an infection the proinflammatory cytokine IFN is produced which in turn triggers a lot of reactions to fight pathogens. One of those downstream effects is the induction of proteasome subunits which substitute the catalytically active subunits in newly assembled proteasomes.
In the so called immunoproteasome the subunits 1 and 5 are replaced by low‐molecular mass peptide 2 (LMP2 or 1i) and LMP7 (5i), respectively, which are encoded in the MHC
class II locus (Brown, Driscoll et al. 1991, Glynne, Powis et al. 1991, Kelly, Powis et al. 1991, Ortiz‐Navarrete, Seelig et al. 1991). 2 is exchanged by multicatalytic endopeptidase complex like‐1 (MECL‐1 or 2i), which is not encoded in the MHC (Boes, Hengel et al. 1994, Groettrup, Kraft et al. 1996, Hisamatsu, Shimbara et al. 1996).
Because of the altered cleavage specificities of the immunoproteasome, meaning a lower caspase‐like activity and higher chymotrypsin‐like activity, a different subset of MHC I ligands is produced (Gaczynska, Rock et al. 1994). Ligands with hydrophobic amino acids at their C‐
terminus have a higher affinity to the cleft of the MHC I molecules. Thus these peptides are preferentially presented on the cell surface which leads to the activation of a different T‐cell repertoire (Fehling, Swat et al. 1994, Van Kaer, Ashton‐Rickardt et al. 1994, Chen, Norbury et al. 2001, Basler, Youhnovski et al. 2004). As the peptides are often derived from viral or tumour proteins a more efficient immune response is initiated. In return primarily the constitutive proteasome is capable to produce antigenic peptides resulting from some proteins (Chapiro, Claverol et al. 2006). In experiments using knockout (KO) mice and inhibitors of the inducible subunits it became clear that not only the cleavage specificity but sometimes just their presence is needed for proper antigen presentation by protecting peptides from cleavage by the constitutive proteasome (Basler, Lauer et al. 2012). Not only the generation of antigens for presentation but also the production of inflammatory cytokines is changed. This is an important observation for the therapy of autoimmune diseases, which can be ameliorated by treatment with specific immunoproteasome inhibitors (Muchamuel, Basler et al. 2009, Basler, Dajee et al. 2010, Basler, Mundt et al. 2014).
As mentioned above the immunoproteasome subunits are only incorporated into newly assembled proteasomes interdependently. Therefore mainly immunoproteasomes containing
all three inducible subunits are built (Groettrup, Standera et al. 1997, Griffin, Nandi et al.
1998). But there are exceptions leading to intermediate proteasomes containing 1, 2 and
5i (Dahlmann, Ruppert et al. 2000, Guillaume, Chapiro et al. 2010). This might give rise to a greater variety of peptides for presentation on MHC I molecules.
In normal body cells the presence of immunoproteasomes is tightly regulated as they have a much shorter half‐life than constitutive proteasomes (Heink, Ludwig et al. 2005), whereas in cells of the lymphoid tissues such as thymus, spleen and lymph nodes immunoproteasomes are permanently expressed (Stohwasser, Standera et al. 1997). In cortical thymic epithelial
cells a third type of proteasome is expressed, the thymoproteasome with the catalytic subunit
5t, which is important for positive selection of T‐cells (Murata, Sasaki et al. 2007).
1.7.3. Proteasome inhibitors
In order to study the role of the proteasome in various cellular processes previous known protease inhibitors were adopted. Depending on the structure of the peptide aldehyde leupeptin, inhibitors with higher potency and increased selectivity towards the 20S proteasome were developed, e.g. MG115 and MG132 (Rock, Gramm et al. 1994). To increase selectivity further other classes of molecules, like epoxyketones, e.g. the natural compound epoxomycin, or boronic ester derivatives, were refined. Belonging to the latter, the compound MG‐341 (PS‐341 and later Bortezomib) was found to be a reversible inhibitor of the 20S proteasome. Under the trade name Velcade® it is intensively applied for treatment of Multiple Myeloma. Because of reduced resistance and relapse rates, also Carfilzomib (Kyprolis®) is now used as treatment. However, because of the massive side effects of the available drugs, there is still need for design of more selective compounds (reviewed in (Dou and Zonder 2014)).
The immunoproteasome was identified as valuable target for treatment of haematological malignancies and autoimmune diseases. For example the LMP7 selective inhibitor ONX‐0914 (PR‐957) was successfully implemented in mouse models for treatment of experimental arthritis (Muchamuel, Basler et al. 2009), experimental colitis (Basler, Dajee et al. 2010), and experimental autoimmune encephalomyelitis (EAE)(Basler, Mundt et al. 2014).
1.8 Endoplasmic reticulum quality control
Proteins that need to be secreted or inserted into membranes are cotranslationally transported into the Endoplasmic Reticulum (ER) where their folding and assembly is monitored by the Endoplasmic reticulum quality control (ERQC). The signal recognition particle (SRP) binds signal sequences in newly synthesized polypeptides in the cytosol and
thereby arrests translation (Walter and Blobel 1981). The SRP is recognized by a receptor in the ER membrane which brings it to the translocon, a complex consisting of Sec61, and (Deshaies, Sanders et al. 1991). After its release the ribosome starts again translation of the polypeptide through the translocon pore into the ER lumen where it is bound by chaperones to assist folding. Subsequently the signal sequence of the polypeptide is cleaved by signal peptidases (Jackson and Blobel 1977) and the preformed oligosaccharide consisting of three glucose, nine mannose and two N‐acetylglucosamin residues (Glc3Man9GlcNAc2) is added to an asparagine. This oligosaccharide is directly trimmed by glucosidase I and II to GlcMan9GlcNAc2 so that the lectin‐like chaperones calnexin (CNX) and calreticulin (CRT) can interact with the polypeptide to prevent aggregation and promote proper folding (Rodan, Simons et al. 1996). The CNX/CRT interacting oxidoreductase ERp57 catalyzes the formation of disulphide bonds between the cysteines of the protein which is released upon removal of the last glucose by glucosidase II. The UDP‐glucose/glycoprotein glucosyl transferase (UGGT) controls the folding state of the protein and adds glucose again to repeat the assisted folding.
Correctly folded proteins are transported to their destination (Araki and Nagata 2011).
1.8.1. ERAD
When proteins ultimately can’t be folded correctly they have to be removed from the ER to prevent their accumulation. This process is called ER‐associated degradation (ERAD) and at least in yeast it can be divided into ERAD‐C (cytosol), ERAD‐M (membrane) and ERAD‐L (lumen) depending on the localisation of the substrate and therefore on the involved enzymes (Carvalho, Goder et al. 2006). In mammals these pathways are defined by complexes containing the ubiquitin E3 ligases Ring finger protein (RNF) 5 (or RMA1), glycoprotein (gp) 78 or HMG‐CoA reductase degradation (Hrd) 1 which share some cofactors and partially target
the same substrates (Morito, Hirao et al. 2008). During repeated folding cycles the probability of the glycosylated proteins to be exposed to ER‐mannosidase I increases. Trimmed oligosaccharides are recognized by ER degradation‐enhancing ‐mannosidase like (EDEM) proteins which can cut off even more mannosidase residues. They hand over the substrate proteins to the chaperone Binding immunoglobulin Protein (BiP, GRP78) which retains the substrate until retrotranslocation into the cytosol in an unfolded state. The reduced glycosylation increases hydrophobicity of the proteins which is recognized by the lectins OS‐9 and XTB‐3B. Abnormal non‐glycosylated proteins are also directly recognized by BiP and co‐
chaperones of the DnaJ family without the help of EDEM proteins. BiP, XTB‐3B and OS‐9 interact via SEL1L with the transmembrane HRD1 ubiquitin E3 ligase complex. Derlin‐1, which is associated with Hrd1 or gp78, interacts with Sec61 and promotes the transport of the substrate through the translocon into the cytosol. On the cytoplasmic site of the ligase complex the associated E1 UBA1 and the E2 UBC6e (Ube2j1) start the enzyme cascade resulting in the ubiquitination of the substrate mostly at serines or threonines. UBXD8 and UBXD2 are membrane bound cofactors which recruit the Valosin‐containing protein (VCP, p97) to the ligase complex. VCP is an ATPase involved in the release of the translocated ERAD substrates in order to make them accessible to degradation by the proteasome. Although VCP can also interact with Hrd1 and gp78 directly, it needs its cofactors Ufd1 and Npl4 to deliver the ubiquitinated substrates for processing. Additionally the deubiquitinating enzymes Ataxin‐
3, VCIP135 and YOD1 associate with VCP to modify ubiquitination of either substrates or parts of the ERAD machinery (reviewed in (Araki and Nagata 2011, Christianson and Ye 2014). When the substrates are translocated into the cytosol they are handed over to ubiquitin receptors like Rad23 or Dsk2 which bind to the proteasome to deliver the substrate for degradation (Medicherla, Kostova et al. 2004). There are some characteristic proteins like the T‐cell receptor ‐chain (TCR), ‐1 antitrypsin (1AT) or the cystic fibrosis transmembrane conductance regulator (CFTR) which can be used as model substrates to study ERAD‐related processes (reviewed in (Baek, Cheng et al. 2013)).
1.8.2. Unfolded‐protein response
In case of accumulation of proteins that don’t fold properly because of mutations or stress conditions and thus can’t be exported from the ER, the unfolded‐protein response (UPR) takes place to reduce ER stress. Three principal branches of the UPR have been defined by the ER‐
resident signalling components as inositol requiring enzyme (IRE) 1, doublestranded RNA‐
activated protein kinase (PKR)‐like ER kinase (PERK) and activating transcription factor (ATF) 6 pathway. They employ different mechanisms for reducing the amount of unfolded proteins by translational control or production of helpful proteins in order to restore protein homeostasis and normal ER function (reviewed in (Walter and Ron 2011)).
ATF6 is a transmembrane protein with a large ER‐luminal domain bound to the chaperone BiP.
Upon accumulation of unfolded proteins ATF6 is released from BiP and transported from the ER to the Golgi. ATF6 is cut by the Golgi‐resident site‐1 protease (S1P) and subsequently by S2P next to the membrane which releases the N‐terminal part of ATF6 into the cytosol. This N‐terminus acts as a transcription factor for BiP, glucose‐regulated protein (GRP) 94 and other ER‐resident chaperones (Walter and Ron 2011).
During ER stress the transmembrane kinase PERK oligomerizes, phosphorylates itself and inhibits the translation initiation factor eIF2 by phosphorylation. Translation of most proteins is stopped to reduce the burden of protein folding in the ER. However, some mRNAs are particularly translated when eIF2 is inactive, e.g. the transcription factor ATF4 which targets the transcription factor C/EBP homologous protein (CHOP) and growth arrest and DNA
damage‐inducible (GADD) 34. CHOP induces expression of genes involved in apoptosis, whereas GADD34 is a subunit of the protein phosphatase PP1C which dephosphorylates eIF2
thus restoring its normal activity (Walter and Ron 2011).
IRE1 is a transmembrane protein with kinase and endoribonuclease activity. Unfolded proteins lead to oligomerization and autophosphorylation of IRE1. This activates its nuclease activity towards the mRNA of the X‐box binding protein (XBP) 1 excising one intron. The translated protein XBP‐1s acts as a transcription factor for chaperones and other ERAD proteins as well as for proteins involved in lipid synthesis (Walter and Ron 2011).
1.9 VCP
Cell cycle‐deficient (Cdc) 48 was first found in yeast mutants to play a role in cell cycle progression (Moir, Stewart et al. 1982). Time after time a lot of processes were described where Cdc48 is involved in or it’s even essential for. In mammals the protein was named Valosin‐containing protein (VCP), as it enclosed the sequence of a peptide named valosin (Koller and Brownstein 1987), or p97 because of its molecular weight. In Xenopus it was found to build an oligomer with ATPase activity (reviewed in (Baek, Cheng et al. 2013)).
Now it is known that VCP is one of the most abundant proteins in eukaryotic cells. It was classified as an ATPase associated with various cellular activities (AAA) which is a protein family with a common structure and similar function as unwinding, disassembly, unfolding or extraction of substrate proteins. Like all type II AAA proteins each subunit of VCP consists of two ATPase domains D1 and D2 and a cofactor‐binding N‐terminal N domain as well as an unstructured C‐terminus (Figure 4). The subunits assemble into a homo‐hexameric barrel‐
shape with a central pore built by the ATPase domains and the N domains arranged at the outside of the D1 ring. Both ATPase domains contain the three motifs Walker A for binding, Walker B for hydrolysis and a second region of homology (SRH) needed for efficient hydrolysis of ATP. As the D1 domain has a higher affinity for ADP, D2 displays higher ATPase activity. The energy of ATP hydrolysis causes major conformational changes of the whole complex which can be used for transferring mechanical forces to unfold or segregate substrate proteins (Pye, Dreveny et al. 2006, Briggs, Baldwin et al. 2008). The precise procedure of extracting a substrate from membranes or protein complexes, whether it is unfolded at the VCP surface by movements of the N domains or if it’s threaded through the channel or only partially inserted, is not known (reviewed in (Meyer and Weihl 2014).
Figure 4: Structure of VCP. Each subunit consists of an N‐terminal domain (green), the two AAA ATPase domains D1 (cyan) and D2 (blue), and a C‐terminal tail (grey). D1 and D2 form two stacked hexameric rings.
The N‐domain is positioned at the outside of the D1 ring. ATP hydrolysis in D2 induces conformational changes in VCP that is used to transfer forces onto target substrates to promote their unfolding (Meyer, Bug et al.
2012).
Commonly one condition for the action of VCP is the ubiquitination of its substrates. It was described to bind polyubiquitin chains directly via the N domain (Dai and Li 2001, Ye, Meyer et al. 2003), but mostly via cofactors that exhibit stronger affinity for ubiquitin. These substrates are often facilitated for degradation by the proteasome thus VCP is part of the ubiquitin‐proteasome‐system (UPS). But there are substrates that are degraded independently from VCP which might be determined by localization, structure or solubility of the substrate. Moreover, some substrates not marked for degradation are targeted for segregation from their interaction partners by VCP. It associates with either the heterodimer Ubiquitin‐fusion degradation (Ufd) 1‐ Nuclear pore localization (Npl) 4 or with the cofactor p47 or UBXD1 to so called core complexes which are recruited by further cofactors to specify localization and action of the complex (see also section 1.9.1). Ubiquitin ligases and DUBs that bind directly or indirectly to VCP can modify ubiquitination patterns and thereby change the destiny of substrates (Baek, Cheng et al. 2013).
In the ubiquitin‐fusion degradation (UFD) pathway the consecutive action of enzymes associated with VCP leads to the degradation of substrate proteins by the proteasome.
Proteins involved in this pathway were discovered by screening for genes essential for degradation of substrates artificially fused to ubiquitin. In vivo substrates are ubiquitinated by
