Ubiquitin like modifier (ULM): an overview

Ubiquitin-like modifiers in turn function as their name already suggests in “ubiquitin-like”

manner, what means, that they exert their function in being covalently attached to substrate molecules (see Table 1)

Introduction

25

Table 1: Ubiquitin like modifiers.

Modified and extended from (Hochstrasser, 2009).

ULM Identity with

ubiquitin [%] Enzymes Substrates Comments and Functions

NEDD8 58

SUMO encoded by 3-4 genes in vertebrates, depending on the species.

Control of protein stability, function and localization, antagonist to ubiquitin,

FUB1 38 NI NI Derived from a ribosomal protein

precursor proteins MoaD and ThiS. Contains a β-grasp fold. Most ancient ULM. Role in tRNA modification. Urm1 modification in the response of cells to oxidative damage.

Important for the prevention of ER stress-induced apoptosis.

NI, not identified.

26 3.2.2 The ubiquitin like modifier FAT10

The fat10 gene was discovered in 1996 by chromosomal sequencing of the human major histocompatibility complex (MHC) class I locus (Fan et al., 1996) close to the HLA-F locus, leading to the designation of HLA-F adjacent transcript 10 (FAT10) (Liu et al., 1999). The region of human chromosome 6 encoding the MHC complex contains a diverse set of genes, including genes whose function can be directly related to immune function such as the MHC class I and II gene products, and genes encoding for members of the complement cascade, TNF-a and -ß, the transporter associated with antigen processing (TAP), and the LMP2 and 7 components of the proteasome (Pichon et al., 1996).

FAT10, consisting of 165 amino acids is an ~18 kDa ULM which comprises two ubiquitin-like domains in a head to-tail formation, connected by a short linker peptide. These domains form the same three-dimensional core structure - the β-grasp fold – like Ub, revealing a common ancestry for the modification systems. Similar to Ub, it possesses a free C-terminal di-glycine motif required for covalent conjugation to USE1 (Aichem et al., 2010), p53 (Li et al., 2011) and huntingtin (Nagashima et al., 2011) and to several so far unknown target proteins (Chiu et al., 2007; Raasi et al., 2001). There exists a high degree of sequence similarity between murine and human FAT10 at mRNA and protein levels.

Due to its analogy to a tandem fusion of two Ubs, it was originally called “ubiquitin D” or

“diubiquitin”. The N-terminal and C-terminal ubiquitin-like domains of FAT10 are more closely related to Ub than to each other and show 29 % and 36 % sequence identity to Ub, respectively. Four of the lysine residues involved in poly-ubiquitin-chain formation – corresponding to K27, K33 and most notably K48 and K63 – are conserved in both ubiquitin-like domains of FAT10 (Figure 6). Atypical to Ub, FAT10 contains 4 cysteine residues in its sequence (Bates et al., 1997).

Like Ub, FAT10 is activated by UBA6 (Chiu et al., 2007; Groettrup et al., 2008; Pelzer et al., 2007) and can be transferred to the E2 enzyme USE1 (Aichem et al., 2010). FAT10 is only expressed in vertebrates, i.e. it is evolutionary one of the youngest members of the ULM family. The importance of the regulation of FAT10 expression has been highlighted by different observations.

Initially, up-regulation of FAT10 expression was shown to be restricted to mature dendritic cells (DCs) and B-cells (Bates et al. 1997). Unlike ubiquitin, which is expressed constitutively, constitutive FAT10 mRNA expression at tissue level seems to be limited to organs of the immune system. This was confirmed by Northern blot analysis, in situ hybridization as well as quantitative real-time PCR (qRT-PCR) in organs of the immune system like spleen, gut, lymphnodes and especially thymus (Liu et al., 1999; Lukasiak et al., 2008).

Introduction

27

Figure 6: Sequence comparison and ribbon diagram of Ubiquitin and the predicted tertiary FAT10 model structure

(a) FAT10 is composed of two ubiquitin-like domains (UBLs) which are more closely related to ubiquitin than to each other. Both, the N- and C-terminal UBL show the typical β-grasp fold and displays 29 % and 36 % sequence identity to ubiquitin, respectively (Groettrup et al., 2008). (b) FAT10 encompasses two ubiquitin-like domains (UBLs), in which the C-terminal di-glycine motif is conserved in the second domain of FAT10. In addition, four of the lysines involved in poly-ubiquitin-chain formation – corresponding to K27, K33, K48 and K63 – are conserved.

(c) Sequence alignment of the N- and C-terminal parts of FAT10 with ubiquitin (Ub). Conserved lysine residues are highlighted in yellow.

Moreover, an induction of FAT10 could be observed under particular conditions, such as inflammation, and has to be removed efficiently, when its inducing signals are turned off.

However, FAT10 can be synergistically induced in many tissues by the proinflammatory cytokines IFN-γ and TNF-α (Liu et al., 1999; Raasi et al., 1999). Induction of the FAT10 mRNA was independent of protein neosynthesis but partially dependent on proteasome activity as treatment with proteasome inhibitors prevented induction of FAT10 with TNF-α, but not IFN-γ (Raasi et al., 1999).

Although its function has not been fully elucidated, FAT10 has been implicated to play important roles in various cellular processes, for instance cancer, antigen presentation, cytokine response, apoptosis and mitosis. Studies in a murine fibroblast cell line revealed that induced expression of FAT10 resulted in massive caspase dependent cell death within 24 to 48 hours. Assumedly, the induction of apoptosis was dependent on the conjugation of FAT10 to so far unidentified target proteins (Raasi et al., 2001).

28 Interestingly, fat10 is one of the most highly upregulated genes in HIV-infected renal tubular epithelial cells (RTECs). Down-regulation of FAT10 expression was shown to reduce apoptosis in RTECs infected by human immunodeficiency virus (HIV), suggesting a novel role for FAT10 in epithelial apoptosis (Ross et al., 2006).

Moreover, FAT10 is a critical mediator of Viral Protein R (Vpr) induced apoptosis in human and murine RTECs, whereby the vpr gene plays an important role in FAT10 up-regulation.

These proteins interact non-covalently and co-localize to mitochondria (Snyder et al., 2009).

In seeming contradiction, up-regulation of FAT10 expression could be observed in several carcinomas, most notably in hepatocellular carcinoma (HCC) and in gastrointestinal and gynecological cancers (Lee et al., 2003; Lukasiak et al., 2008).

In 2008, Oliva et al. identified FAT10 as a potential marker for liver preneoplasia, as it was highly overexpressed in a model of Mallory-Denk body containing chronic liver diseases, which are thought to progress to hepatocellular carcinoma (Oliva et al., 2008). Both suggested an active involvement of FAT10 in tumorigenesis based on its non covalent interaction with the spindle assembly checkpoint protein mitotic arrest deficiency 2 (MAD2), as previously shown in a yeast two hybrid assay (Liu et al., 1999).

FAT10 is thought to displace MAD2 from the kinetochore during prometaphase, associated with incomplete chromosomal segregation and increased mitotic non-disjunction, resulting in a generation of cells that contain aberrant chromosome numbers, which is commonly observed in several cancers but no direct evidence was demonstrated (Ren et al., 2006).

This finding strengthens the hypothesis that FAT10 plays a role in the regulation of genomic stability. FAT10 expression at transcript level undergoes cell cycle–specific changes, with the highest expression during the S phase and a low expression during G2/M phase (Lim et al., 2006). This finding further supported the hypothesis that FAT10 disturbs correct chromosomal segregation and is thus cell cycle regulated.

Moreover, a presumable role for FAT10 in carcinogenesis has been proposed, as the presence of wild-type p53, which is known to play an important role in cell-cycle regulation, negatively regulates FAT10 mRNA expression and promoter activity and prevents reaching high FAT10 levels in the cell (Zhang et al., 2006), whereas mutant p53 provokes a contrary effect. Very recently Li et al. revealed evidence, that p53 becomes FAT10ylated and p53 transcriptional activity was found to be substantially enhanced in FAT10-overexpressing cells (Li et al., 2011).

However, a further study identified that FAT10 possesses no transforming capability and increased FAT10 expression in several tumors is due to the up-regulation of proinflammatory cytokines (Lukasiak et al., 2008). So far, there is still a matter of debate whether it functions as a tumor suppressor or rather an oncogene.

Introduction

29 Hipp et al. reported in 2004, that degradation of FAT10 and its conjugates is accelerated in vitro and in vivo via its non-covalent interaction with the UBL-UBA domain protein NEDD8 ultimate buster 1-long (NUB1L), which binds to the proteasome through its UBL domain (Hipp et al., 2004; Schmidtke et al., 2006). The UBA domains of NUB1L are required for binding but not for accelerated degradation of FAT10 by the proteasome.

This finding led to the assumption that NUB1L might not only act as a linker between the 26S proteasome and ULMs, but also as a facilitator of proteasomal degradation (Schmidtke et al., 2006). The degradation of FAT10 and its conjugates was initially described to be independent of Ub. Fusion of FAT10 to the N-termini of very long-lived proteins, like green fluorescent protein (GFP) for instance, enhanced their degradation rate as potently as fusion with Ub did. Therefore, it was suggested that FAT10 is the first ULM which provides a signal for proteasomal degradation of other proteins as an alternative route for Ub mediated protein degradation (Hipp et al., 2005).

Further, FAT10 degradation occurred normally in E1 temperature-sensitive mutants, however it should be emphasized that Ub conjugation in this mutant is largely deficient at the restrictive temperature but a small share of poly-ubiquitin conjugate formation remained, which can lead to FAT10 ubiquitination (Hipp et al., 2005).

Because no evidence for the deconjugation of FAT10 from its substrates has been obtained, it was believed that FAT10 is probably degraded, along with its substrates, in a manner similar to that seen with Ub-modified substrates when deconjugation is inhibited (Hanna and Finley, 2007).

Contradictory, a very recent article assumed that FAT10 degradation by the proteasome requires its prior ubiquitination (Buchsbaum et al., 2011), based on the observation that a non-ubiquitinable lysine-less form of FAT10 is rapidly aggregated and precipitated in a insoluble fraction which is probably not sensible to the proteasome, whereas the WT protein appears to be less susceptible to aggregation. Moreover, FAT10 stabilization could be observed by using cells expressing non-polymerizable Ub and in cells harboring a thermo-labile mutation in the ubiquitin-activating enzyme, E1. The discrepancy to the previous article (Hipp et al., 2005) could origin in the different experimental setups to inactivate the E1 enzyme (Buchsbaum et al., 2011). Their own statement could be confuted by an experiment where they showed, that degradation of FAT10-GFP occurs in the presence of a nonpolymerizable mutant of ubiquitin which can be explained by the fact that the interaction of FAT10 with the proteasome is sufficient to promote, at least partially, the degradation of a downstream fused protein. This is in line with previous findings, showing that FAT10 can constitute a degradation signal without further ubiquitination (Hipp et al., 2005).

.

30 Along with this, some interaction partners of FAT10 have been determined to link FAT10 with aggregate formation. Kalveram et al. reported in 2008 that a cytoplasmic protein, histone deacetylase 6 (HDAC6), can interact non-covalently with FAT10 under proteasome inhibition, leading to the localization of FAT10 in aggresomes (Kalveram et al., 2008). This could provide an alternative route to ensure sequestration and subsequent removal of FAT10-conjugated proteins if FAT10 fails to subject its target proteins to proteasomal degradation.

FAT10 also co-localizes with the catalytic immunoproteasome subunits LMP2 and LMP7 and its expression increases in liver cells forming Mallory-Denk bodies due to accumulation and aggregation of ubiquitinated cytokeratins (Bardag-Gorce et al., 2010).

Many late-onset neurodegenerative diseases are associated with the formation of intracellular aggregates by misfolded or toxic proteins, revealing a high importance in the degradation pathways acting on such aggregate-prone cytosolic proteins including the ubiquitin-proteasome system and macroautophagy (Ross and Pickart, 2004; Williams et al., 2006). A recent report described that FAT10 molecules were covalently attached to the soluble fraction of the aggregate prone protein huntingtin and FAT10-modified huntingtin is prone to degradation by the proteasome. Moreover, completely aggregated huntingtin lacks any FAT10 and FAT10 knockdown enhanced aggregate formation. These data let suggest that FAT10 plays a role in stabilizing soluble huntingtin by facilitating the interaction with the proteasome (Nagashima et al., 2011).

Several evidences for the involvement of FAT10 in the immune system are given. One hint for its immunological relevance is that the fat10 gene is encoded in the MHC locus, for instance (Fan et al., 1996). Moreover, FAT10 is constitutively expressed in immune cells and in organs of the immune system like spleen, gut, lymphnodes and especially the thymus (Lukasiak et al., 2008). Further, it is synergistically inducible in many tissues with the proinflammatory cytokines IFN-γ and TNF-α (Liu et al., 1999; Raasi et al., 1999).

Interestingly, FAT10 can inhibit hepatitis B virus expression in a hepatoblastoma cell line after IFN-γ treatment (Xiong et al., 2003) and fat10 gene targeted mice demonstrated a high level of sensitivity toward lipopolysaccharide challenge and their lymphocytes are more susceptible to spontaneous apoptotic death (Canaan et al., 2006).

These findings indicate that FAT10 may function as a survival factor, but the function and mechanism of its action in the immune system still remains poorly understood and still need to be investigated.

Introduction

31 3.2.3 FAT10 conjugation pathway

Besides Ub, several UBLs have been discovered; they all harbor structural homology to Ub, and many of them can also be conjugated to target proteins in order to modify their fate (Hochstrasser, 2009; Welchman et al., 2005). However, little is known about the regulation of UBLs, and in particular about the role of ubiquitination to control their levels. The initial activation of Ub was for decades believed to be accomplished solely by a single enzyme designated ubiquitin activating enzyme 1 (UBE1) (Ciechanover et al., 1981; Haas et al., 1982). Surprisingly, in 2007, a second E1 type enzyme, ubiquitin-like modifier activating enzyme 6 (UBA6) was identified which can activate Ub as well as the ULM FAT10 and depletion of UBA6 can block the conjugation of FAT10 to so far unknown proteins (Chiu et al., 2007; Pelzer et al., 2007). This finding raises the interesting question how this E1 enzyme can discriminate, whether an E2 enzyme needs to be charged with activated Ub or FAT10 (Groettrup et al., 2008).

Activated FAT10 can be conjugated by the UBA6 specific E2 enzyme (USE1), which was recently identified in a yeast two-hybrid screen. Therefore, UBA6 as well as USE1 are examples that enzyme sharing between ULMs is much more common than initially believed.

USE1 is not only the first described E2 enzyme in the FAT10 conjugation pathway but also the first physiological substrate of FAT10 conjugation, as it was efficiently auto-FAT10ylated in cis but not in trans (Aichem et al., 2010).

Co-expression of NUB1L, a linker protein that facilitates the degradation of FAT10 and its conjugates, led to proteolytic down regulation of USE1 which suggest that USE1 can auto-modify itself with FAT10 and thus negatively regulates the FAT10 conjugation pathway (A.

Aichem, manuscript in preparation).

To date, no E3 enzyme for FAT10 has been identified. In our laboratory a specific interaction of FAT10 and the RING finger E3 ligase TRIM11 could be shown in a yeast two hybrid screen (A. Aichem, unpublished), indicating that this protein may be involved in the FAT10 conjugation pathway. This finding could further be strengthened by the fact that TRIM11 interacts in vivo with the FAT10 specific E2 enzyme USE1, and siRNA-mediated knockdown of TRIM11 reduced the amount of FAT10 conjugates in HEK293 cells, indicating that TRIM11 may function as a FAT10 specific RING finger E3 ligase (A. Aichem, unpublished).

32 3.2.4 The small ubiquitin-like modifier SUMO

The small ubiquitin-like modifier protein (SUMO) was initially identified in 1996, almost two decades later than Ub, in the yeast Saccharomyces cerevisiae as a suppressor of mutations in the centromere protein MIF2 and designated suppressor of MIF2 mutations-3 (Smt3p) (Meluh and Koshland, 1995).

Almost coincidentally, the mammalian homolog of the yeast protein was discovered and referred to as SUMO-1 (small ubiquitin-like modifier 1), UBL1 (ubiquitin-like 1), PIC1 (promyelocytic leukemia protein (PML)-interacting protein 1), sentrin or GMP1 (GTPase-activating protein (GAP)-modifying protein 1) (Boddy et al., 1996; Mahajan et al., 1997;

Matunis et al., 1996; Okura et al., 1996). The highly conserved SUMO is ubiquitously expressed in all eukaryotic cells but is absent in bacteria and archaea.

To date, four SUMO isoforms SUMO-1/Smt3H3, SUMO-2/SmtH2, SUMO-3/Smt3H1 and SUMO-4/Smt3H4 have been described in mammals. SUMO1-3 is found to be expressed in all tissues at all developmental stages, whereas expression of SUMO-4 appears to be restricted in kidney and various immune tissues, especially lymphnodes and spleen (Bohren et al., 2004; Guo et al., 2004; Seeler and Dejean, 2003). Only three of these (SUMO-1, SUMO-2 and SUMO-3) are processed in vivo to bear the C-terminal diglycine motif required for post-translational conjugation. SUMO-2 and SUMO-3 are nearly identical and are assumed to be largely redundant in their functions. SUMOs share only ~18 % sequence identity with Ub, although structure analysis by nuclear magnetic resonance (NMR) revealed that both share a common three dimensional structure, which is characterized by a tightly packed globular fold with five anti-parallel β-sheets wrapped around one α-helix (Bayer et al., 1998).

Reversible attachment of SUMO, namely SUMOylation, to its targets is controlled by a strict enzymatic energy-consuming reaction cascade that is analogous to the Ub pathway. First, the C-terminal end is processed by a protease (in human these are the SENP proteases and Ulp1 in yeast) to expose a C-terminal di-glycine motif, which is required for substrate protein binding. Subsequently, SUMO becomes activated by an E1 SUMO-activating enzyme (heterodimeric SAE1 and SAE2). Then, it is transferred to the E2 enzyme ubiquitin carrier 9 (Ubc9) and finally becomes attached to an ε-amino group of a specific lysine in the target proteins mostly via one of several E3 enzymes. Further, SUMO can be deconjugated from the target protein by the action of SUMO specific proteases (Hay, 2005; Johnson and Blobel, 1997; Su and Li, 2002).

During SUMOylation the target lysine residue is generally located within a recognizable consensus sequence, namely ψKxE, where ψ is an aliphatic and x an arbitrary amino acid (Sampson et al., 2001; Seeler and Dejean, 2003).

Introduction

33 The presence of a consensus site is not a strict requirement for SUMOylation of a target since several proteins have been found to be modified on non-consensus sites (Hoege et al., 2002; Pichler et al., 2005).

Interestingly, SUMO proteins do not have the lysine residue corresponding to K48 in the Ub molecule that is required for the most common formation of poly-ubiquitin chains, suggesting that SUMO does not build the same type of multi-chains as Ub (Bayer et al., 1998). In contrast to ubiquitin where all seven lysine residues of Ub have been implicated in linkage formation (Xu et al., 2009), SUMO chains are linked mainly through a single lysine residue at position 11 at the N-terminus, which is embedded in the ψKxE consensus sequence (Bencsath et al., 2002; Bylebyl et al., 2003; Knipscheer et al., 2007; Skilton et al., 2009;

Tatham et al., 2001). Protein SUMOylation is a highly regulated post-translational modification that is involved in versatile modes of regulation in widely different biological processes. Modification of target proteins with SUMO has been shown to be involved in the subcellular localization of proteins, maintenance of genome integrity, protein–protein interactions and mainly in the transactivation of transcription factors (Anckar and Sistonen, 2007; Garaude et al., 2008; Muller et al., 2004; Pichler and Melchior, 2002; Ulrich, 2008;

Yang et al., 2003; Yeh, 2009; Zhao, 2007).

For instance, the observation that RanGAP1, the first identified SUMO substrate and the promyelocytic leukemia protein (PML), also known as TRIM19, are targeted to distinct subcellular structures upon conjugation to SUMO-1 let suggest, that SUMOylation might play an important role in regulating the subcellular localization of proteins (Mahajan et al., 1997;

Matunis et al., 1996; Seeler and Dejean, 2001). Moreover, transcription factors such as AP-1 and p53 are two of many examples where modification with SUMO regulates their transcriptional activity. For instance, SUMOylation of the heterodimeric transcription factor complex composed of c-Jun and c-Fos, which belong to the AP-1 family, decreases their transactivation potency (Bossis et al., 2005; Muller et al., 2000), whereas SUMOylation of the AP-1 family member JunB in T lymphocytes can positively regulate cytokine gene transcription and likely plays a critical role in T cell activation (Garaude et al., 2008).

Furthermore, SUMOylation is influenced by other protein modifications like phosphorylation or acetylation (Urvalek et al., 2011; Yao et al., 2011). Interestingly, in addition to the structural relationship between the UBLs, Ub and SUMO, a crosstalk between SUMO and Ub-based signaling was described, sharing a multitude of functional interrelations.

This include the targeting of the same attachment sites in specific substrates, such as the inhibitor of NF-kBα (IκBα), the proliferating cell nuclear antigen (PCNA) and the NF-κB

This include the targeting of the same attachment sites in specific substrates, such as the inhibitor of NF-kBα (IκBα), the proliferating cell nuclear antigen (PCNA) and the NF-κB

Im Dokument Investigation of the FAT10 conjugation pathway (Seite 24-34)