Ambra1 co-localizes with FAT10 in punctuated structures

In co-immunoprecipitation assays we could clearly show, that Ambra1 co-immunoprecipitates with either ubiquitin or FAT10 in a non-covalent manner.

The subcellular localization of Ambra1 has been determined to occur in cytoplasmic vesicle and autophagosomes. FAT10 has been suggested to be involved in autophagic processes given that p62/SQSTM, a autophagosomal receptor and signaling adapter was found to be covalently as well as non-covalently linked to FAT10 (A. Aichem et al., manuscript submitted). Moreover, Kalveram et al. showed a localization of FAT10 in aggresomes.

FAT10 interacts and co-localizes with histone deacetylase 6 (HDAC6) in aggresomes under conditions of proteasome inhibition in a microtubule dependent manner, suggesting that HDAC6 functions as a linker between the dynein motor complex and FAT10 and FAT10ylated protein cargos (Kalveram et al., 2008).

To further investigate the localization and fate of either Ambra1 or FAT10 when co-expressed together and to analyze the role of non-covalent interaction in this process, we conducted confocal laser-scanning microscopy. We transfected HEK293 cells with either a pcDNA3.1-HA-FAT10 or pCMV6-Ambra1-MYC-FLAG construct alone, or co-transfected them together. Cells were treated with or without MG132 for 6 hours to inhibit the proteasome or amino acid starved for 4 h, to induce autophagy and a series of co-localization experiments were carried out, using a directly labelled HA-coupled Alexa Fluor 488 antibody to visualize HA-FAT10 expression and a rabbit polyclonal antibody to Ambra1 (ab59141) followed by a Alexa Fluor 546-coupled secondary goat-anti-mouse antibody, to stain for Ambra1.

Results

141

Figure 45: Co-localization of FAT10 and Ambra1

293 cells were transiently transfected with pCMV6-Ambra1-MYC-FLAG or pcDNA3.1-HA-FAT10 alone or were co-transfected together and fixed with 4 % PFA. (a) FAT10 transfected cells were stained with a 6,85 µg/ml dilution of directly labelled Alexa488-HA antibody (green). (b) Ambra1 transfected cells were stained with a 5 µg/ml dilution of a rabbit polyclonal antibody to Ambra1 (ab59141) followed by an Alexa Fluor 546-coupled secondary goat-anti-mouse antibody. (c) Double transfected cells were treated subsequently with Alexa488-HA antibody and rabbit polyclonal antibody to Ambra1 (ab59141), followed by an Alexa Fluor 546-coupled secondary goat-anti-mouse antibody. Confocal microscopy images are shown. Scale bar: 25 µm. Images are representatives of several cells examined in two independent experiments.

In singly transfected cells, FAT10 was evenly distributed throughout the cytosol and nucleus (see Figure 45 (a)). Ambra1 was detectable primarily in the cytosol and showed varying degrees of localization to the nucleus. Moreover, formation of punctuated structures, presumably autophagosomes, mainly in the cytosol, was detectable in Ambra1 transfected cells (see Figure 45 (b)). Strikingly, co-expression of FAT10 and Ambra1 resulted in a clear co-localization of Ambra1 and FAT10 in punctuated structures mainly in the cytosol, suggesting that Ambra1 and FAT10 indeed interact and this leads to their trans-location into punctuated structures.

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142

Figure 46: Co-localization of FAT10 and Ambra1 after serum starvation

293 cells were transiently transfected with pCMV6- Ambra1-MYC-FLAG or pcDNA3.1-HA-FAT10 alone or were co-transfected together, starved for 4 h in Earle´s Balanced salt solution and fixed with 4 % PFA. (a) HA-FAT10 transfected cells were stained with a 6,85 µg/ml dilution of directly labelled Alexa488-HA antibody (green). (b) Ambra1-MYC-FLAG transfected cells were stained with a 5 µg/ml dilution of a rabbit polyclonal antibody to Ambra1 (ab59141) followed by an Alexa Fluor 546-coupled secondary goat-anti-mouse antibody. (c) Doubly transfected cells were treated subsequently with Alexa488-HA antibody and rabbit polyclonal antibody to Ambra1 (ab59141), followed by an Alexa Fluor 546-coupled secondary goat-anti-mouse antibody. Confocal microscopy images are shown. Scale bar: 25 µm. Images are representatives of several cells examined in two independent experiments.

We next aimed to determine, if autophagy induction through amino acid starvation changes the localization of FAT10 or Ambra1. In singly transfected and amino acid starved cells, FAT10 was evenly distributed throughout the cytosol and nucleus (see Figure 46 (a)), showing similar distribution in comparison to non-starved cells (see Figure 45 (a)).

Previous reports indicated, that autophagy induction results in an increased localization of Ambra1 in the perinuclear region and co-localization studies revealed that a high percentage of Ambra1 relocates to the endoplasmic reticulum, where it partially co-localizes with the omegasomes, the sites of autophagosome production (Di Bartolomeo et al., 2010; Fimia et al., 2011).

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Results

143 Our data show, that Ambra1 was detectable primarily in the cytosol and showed increased formation of punctuated structures after amino-acid starvation (see Figure 46 (b)).

Co-expression of FAT10 and Ambra1 resulted in a clear co-localization of Ambra1 and FAT10 in punctuated structures mainly in the cytosol (see Figure 46 (c)), similar to the case when FAT10 and Ambra1 were co-expressed in unstarved cells (see Figure 45 (c)).

Figure 47: Co-localization of FAT10 and Ambra1 after MG132 treatment

293 cells were transiently transfected with constructs encoding for Ambra1-MYC-FLAG or HA-FAT10 or were co-transfected together and treated with MG132 (10 μM) for 6 h and subsequently fixed with 4 % PFA. (a) HA-FAT10 transfected cells were stained with a 6,85 µg/ml dilution of directly labelled Alexa488-HA antibody (green). (b) Ambra1-MYC-FLAG transfected cells were stained with a 5 µg/ml dilution of a rabbit polyclonal antibody to Ambra1 (ab59141) followed by an Alexa Fluor 546-coupled secondary goat-anti-mouse antibody. (c) Double transfected cells were treated subsequently with Alexa488-HA antibody and rabbit polyclonal antibody to Ambra1 (ab59141), followed by an Alexa Fluor 546-coupled secondary goat-anti-mouse antibody. Confocal microscopy images are shown. Scale bar: 25 µm. Images are representatives of several cells examined in two independent experiments.

Our previous data indicated that the proteasome is involved in Ambra1 as well as FAT10 degradation (see chapter 6.2.21). To further investigate, if proteasome inhibition has an impact on either FAT10 or Ambra1 localization or distribution in the cell, HEK293 transfected cells were treated with MG132 (10 μM) for 6 h.

(a) (b)

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144 Singly transfected cells expressing FAT10 (see Figure 47 (a)) or Ambra1 (see Figure 47 (b)) reveal an indistinguishable localization pattern compared to untreated cells (see Figure 45 (a) + (b)). Co-transfection of Ambra1 and FAT10 led to a clear co-localization in punctuated structures widely distributed in the cytosol as well as in the nucleus (see Figure 47 (c)) in contrast to untreated or starved cells, where these structures were mainly detectable in the cytosol.

To investigate the subcellular distribution of either FAT10 or Ambra1 when the proteasome is inhibited and additionally autophagy is induced, we treated Ambra1, FAT10 or co-transfected cells for 6 with MG132 and subsequently starved the cells in amino acid and serum-free medium for 4 h.

Figure 48: Co-localization of FAT10 and Ambra1 after amino acid starvation and MG132 treatment

293 cells were transiently transfected with pCMV6-Ambra1-MYC-FLAG or pcDNA3.1-HA-FAT10 constructs alone or were o-transfected together, treated with MG132 (10 μM) for 6 h, starved for 4 h in Earle´s Balanced salt solution and fixed with 4 % PFA. (a) HA-FAT10 transfected cells were stained with a 6,85 µg/ml dilution of directly labelled Alexa488-HA antibody (green). (b) Ambra1-MYC-FLAG transfected cells were stained with a 5 µg/ml dilution of a rabbit polyclonal antibody to Ambra1 (ab59141) followed by an Alexa Fluor 546-coupled secondary goat-anti-mouse antibody. (c) Doubly transfected cells were treated subsequently with Alexa488-HA antibody and rabbit polyclonal antibody to Ambra1 (ab59141), followed by an Alexa Fluor 546-coupled secondary goat-anti-mouse antibody. Confocal microscopy images are shown. Scale bar: 25 µm. Images are representatives of several cells examined in two independent experiments.

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Results

145 In singly transfected, MG132 treated and amino acid starved cells, FAT10 was evenly distributed throughout the cytosol and nucleus (see Figure 48 (a)). Increased formation of punctuated structures was visible in Ambra1 transfected cells (see Figure 48 (b)), distributed in the cytosol. Compared to MG132 treated or serum starved cells, appearing punctae were strongly augmented in size.

Co-expression of FAT10 and Ambra1 resulted in a clear co-localization of Ambra1 and FAT10 in punctuated structures mainly in the nucleus (see Figure 48 (c)), and these structures cannot consist of autophagosomes. Our results substantiate the precedent data that Ambra1 and FAT10 interact in cells and these data strongly suggest that interaction of Ambra1 and FAT10 regulates localization (co-localization of FAT10 and Ambra1 in punctuated structures) and probably function of both proteins.

Unfortunately, no quantitative analysis for all microscopy experiments was carried out due to time limitations. However, all microscopy results verified the interaction of Ambra1 and FAT10, as has previously been determined in co-immunoprecipitation assays.

146

7 Discussion

FAT10, a very young member of the ubiquitin protein family, is encoded in the major histocompatibility complex class I locus and is synergistically inducible with the proinflammatory cytokines IFN-γ and TNF-α in cells of nearly every tissue origin. It consists of two ubiquitin-like domains, which are connected by a short linker, and bears a free di-glycine-motif at its C-terminus through which it can become covalently conjugated to target proteins (Chiu et al., 2007; Raasi et al., 2001). FAT10 is so interesting to investigate because it is beside ubiquitin so far the only described ubiquitin-like modifier (ULM), where conjugation to target proteins serves as a signal for proteasomal degradation (Hipp et al., 2005). Moreover, FAT10 was shown to be as potent as ubiquitin in targeting artificial fusion proteins for proteasomal degradation (Hipp et al., 2005; Hipp et al., 2004), which is independent of poly-ubiquitination (Schmidtke et al., 2009) but can be accelerated by the UBL-UBA domain protein, NEDD8 ultimate buster 1-long (NUB1L) (Hipp et al., 2005; Hipp et al., 2004; Schmidtke et al., 2009). FAT10 and FAT10 conjugates are rapidly degraded by the proteasome and most likely not recycled as is usually the case for ubiquitin (Hipp et al., 2005).

In order gain a more detailed insight into the FAT10-conjugation pathway it is crucial to identify the FAT10 specific E1, E2, and E3 enzymes as well as the physiological substrates of FAT10 conjugation. The identification of enzymes involved in FAT10 conjugation and the identification of FAT10-interacting proteins or substrates is very important for the investigation of biological functions of FAT10ylation and FAT10-dependent protein degradation.

With respect to the conjugation cascade, Ubiquitin-like modifier activating enzyme 6 (UBA6) has been characterized not only as a second E1 for ubiquitin but also as an E1 for FAT10 (Chiu et al., 2007; Pelzer et al., 2007). Given that UBE1 and UBA6 are co-expressed in many tissues (although UBE1 is up to 10 fold more abundant than UBA6), these two enzymes may act in concert or in sequence to affect various signaling pathways. One possibility is that UBE1 and UBA6 might use a different spectrum of E2-enzymes and eventually different E3-enzymes with their corresponding substrates. UBA6 is highly expressed in testis, therefore an organ specific function is expected.

At the beginning of the doctoral thesis, no E2, E3 enzymes nor FAT10 specific substrates had been identified yet. In 2010, the UBA6 specific E2 enzyme (USE1) has been confirmed to be not only the first identified E2 enzyme for FAT10, but also the first identified substrate in the FAT10 conjugation pathway (Aichem et al., 2010). Very recently, two further substrates of FAT10, namely huntingtin (Nagashima et al., 2011) and p53 (Li et al., 2011) have been described.

Discussion

147 In order to identify new interaction partners of UBA6 and putative E2 enzymes for FAT10 we performed a yeast two-hybrid screen using a human thymus cDNA library as FAT10 was found to be most highly expressed in the thymus (Lukasiak et al., 2008). Among 96 bait dependent clones, which could be confirmed by retransformation, 69 different clones encoded for full length fat10 and 3 Plasmids encoded for the C-terminal half of bruce containing the entire ubiquitin conjugating (UBC) domain, which was further investigated in terms of beeing a putative FAT10 E2 conjugating enzyme. The putative association of UBA6 with BRUCE might argue for an UBE1- independent loading of BRUCE with either ubiquitin or FAT10 by this newly identified ortholog. Hence, we tested the possibility of interaction between BRUCE and either ubiquitin or FAT10.

BRUCE interacts non-covalently with UBA6 and FAT10

To verify the yeast-two-hybrid findings and confirm the interaction with UBA6, we examined the ability of human UBA6 to interact with the human native full-length BRUCE in HEK293T cells (see 6.1.1). In co-immunoprecipitation experiments under non-reducing and reducing conditions (10 % ß-mercaptoethanol) we could detect a clear non-covalent interaction of BRUCE with UBA6, since UBA6 becomes co-immunoprecipitated to a higher amount with BRUCE under reducing conditions. UBA6 is probably a specialized E1 that can only transfer ubiquitin or FAT10 to a small number of E2s like USE1 and probably BRUCE.

FAT10 could be detected after co-immunoprecipitation with BRUCE in 4-12 % Bis-Tris-gels in a higher amount under reducing conditions than under non-reducing conditions, indicating that also FAT10 becomes thioester-linked to BRUCE and presumably can be transferred from UBA6 onto BRUCE in vivo.

Moreover, on 3-8 % Tris-Acetate gels, which allow the detection of high molecular weight bands, a very faint conjugate band at the height of ~530 kDa could be observed under non-reducing conditions, which strengthens the hypothesis that FAT10 becomes thioester-linked to BRUCE. Only a weak interaction between BRUCE and FAT10 could be previously detected in a yeast two-hybrid interaction assay with FAT10 and the C-terminal half of BRUCE. Further, a covalent interaction of ubiquitin and BRUCE was observed. BRUCE has been described to be a highly unusual enzyme of the ubiquitin conjugation system as it combines in a single poly-peptide ubiquitin conjugating enzyme (E2) with ubiquitin E3 ligase forming a chimeric E2/E3 ubiquitin ligase. It could be shown in vitro that BRUCE mediated the ubiquitination of the substrates Smac/Diablo, HtrA2 and active caspase-9 only in presence of functional UBE1 (Bartke et al., 2004; Hao et al., 2004).

148 To further test, if BRUCE can become thioester-linked to endogenous FAT10, a semi-endogenous co-immunoprecipitation assay with FLAG-BRUCE, HA-UBA6 and semi-endogenous FAT10 was performed, where FAT10 was induced with the pro-inflammatory cytokines TNF-α and IFN-γ (6.1.2). Immunoprecipitation assays revealed that HA-UBA6 immunoprecipitates with BRUCE, as previously shown and the amount of co-immunoprecipitated HA-UBA6 decreased under non-reducing conditions, which strongly suggest, that both proteins become thioester-linked to each other. Moreover, endogenous up-regulated FAT10 co-immunoprecipitated with BRUCE, but no difference between reducing or non-reducing conditions on protein level could be observed. Further, no conjugate formation between BRUCE and FAT10 at the height of ~530 kDa neither under non-reducing nor reducing conditions could be detected, and therefore provide no evidence, that FAT10 becomes thioester-linked to the active site cysteine of the UBC domain of BRUCE. However, FAT10 linked proteins have been described to be difficult to be detect, as so far identified, but unpublished FAT10 substrates were only to 5-10 % covalently modified with FAT10. Moreover, BRUCE has been described to be one of the largest proteins and the detection limit can be reached because of limited gel resolution.

We further tested in vitro whether activated FAT10 can be transferred from UBA6 onto BRUCE. For this purpose immunoprecipitation was coupled with an in vitro FAT10ylation assay of FLAG-tagged BRUCE in the presence of the recombinant FAT10 and recombinant UBA6 (E1). Unfortunately, no transfer of FAT10 on BRUCE could be detected in the presence of UBA6 (E1), FAT10 and the putative FAT10 E2 enzyme BRUCE (data not shown). In a modified in vitro assay, HA-FAT10 was coupled on HA-agarose beads and incubated with recombinant UBA6 and FLAG-BRUCE, which was eluted from anti-FLAG M2 affinity gel with FLAG-peptide. Also in this approach, no FAT10 transfer on BRUCE could be detected. However, the amounts of thioester linked UBA6~FAT10 adducts were quite low.

For further experiments it is essential to develop a reliable in vitro system to clarify the role of BRUCE as a putative FAT10 E2 conjugating enzyme.

So far it can only be concluded that BRUCE is interacting with UBA6 and FAT10 in a yeast two-hybrid assay and in co-immunoprecipitation assays. To gain absolute certainty about the identity of the BRUCE-FAT10 conjugate it could also be analyzed by mass spectrometry.

This is however only possible if it can be identified on a silver-stained SDS gel. To prove the possibility that BRUCE is an E2 enzyme, which mediates beside ubiquitin conjugation also charging with the ULM FAT10, a reasonable experiment would be siRNA mediated downregulation of BRUCE. Hereby it could be proven, if silencing of BRUCE leads to a reduction of either ubiquitin or FAT10 conjugates and therefore to determine, whether BRUCE has a central role in ubiquitin or FAT10 conjugate formation.

Discussion

149 Strong evidence for the function of FAT10 as a tumor suppressor comes from the finding that expression of FAT10 is able to induce caspase-dependent apoptosis in a variety of models.

Studies in a murine fibroblast line (Raasi et al., 2001) and renal tubular epithelial cells (Ross et al., 2006) demonstrated that overexpression of FAT10 resulted in massive caspase dependent apoptosis dependent on the conjugation of FAT10 to so far unidentified target proteins. BRUCE contains a single BIR-domain and an ubiquitin-conjugating enzyme (UBC) E2 domain and has been described to function as an inhibitor of apoptosis protein in mammalian cells (Bartke et al., 2004; Hao et al., 2004). It can bind and inhibit activated initiator caspases-8 and -9 and executioner caspases -3, -6 and -7 and moreover, both Smac and HtrA2 are able to compete for BRUCE-bound caspases. Interestingly, BRUCE is also a substrate of caspases and the serine protease HtrA2, pointing to a role in regulating apoptosis at early stages when proteolytic activity mediated by these enzymes is still low.

These interesting characteristics of both proteins leads to the hypothesis that binding of FAT10 to BRUCE might prevent caspase mediated apoptosis of FAT10 through binding to active caspases or FAT10 itself.

As a next step, functional studies could be performed. An important feature of BRUCE is its association with membranes including the Golgi apparatus (Hauser et al., 1998). To test if FAT10 has an influence on the subcellular localization of BRUCE, transiently transfected cells could be analyzed with confocal immunofluorescence microscopy in the presence and absence of FAT10.

BRUCE is a survival-like BIR domain containing protein (BIRP) with a role in cell cycle regulation and cytokinesis and BRUCE depletion causes cytokinesis defects and cytokinesis associated apoptosis. Especially, BRUCE has an indispensible role at final stages of cytokinesis and particularly controls proper midbody ring formation which is required for cell cycle continuation (Pohl and Jentsch, 2008). Since the expression of FAT10 as well as BRUCE is cell cycle regulated, co-localization studies should be performed with synchronized cells to exactly determine protein localization at different cellular states.

The RING finger containing ubiquitin ligase Nrdp1/FLRF has been described to catalyze BRUCE ubiquitination and proteasomal degradation, which contributes to apoptosis induction. Despite the UBC E2 motif, BRUCE did not function with Nrdp1 as an E2 in its own ubiquitination (Qiu et al., 2004), but it remains possible that BRUCE may function as an E2 for ubiquitination of other proteins under other conditions. In vitro experiments with the RING finger containing ubiquitin E3 and putative FAT10 E3 ligase TRIM11 would be interesting to investigate, to test whether TRIM11 mediates either FAT10 or ubiquitin ligation to BRUCE dependent on the UBC domain of BRUCE.

150 The ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway are the two main routes for eukaryotic intracellular protein clearance. Depending on the physiological and pathological conditions, autophagy has been shown to act as a pro-survival or pro-death mechanism in vertebrates (Levine and Klionsky, 2004).

Interestingly, drosphila BRUCE (dBRUCE) was shown to regulate autophagy and cell death during early and mid-oogenesis in Drosophila (Hou et al., 2008). In this case, dBRUCE and caspase activity were shown to influence autophagy, which provided first evidence for a mechanism by which autophagy regulates dBRUCE and cell death. Moreover, BRUCE itself becomes degraded by autophagy in the degenerating nurse cells during late oogenesis

Interestingly, drosphila BRUCE (dBRUCE) was shown to regulate autophagy and cell death during early and mid-oogenesis in Drosophila (Hou et al., 2008). In this case, dBRUCE and caspase activity were shown to influence autophagy, which provided first evidence for a mechanism by which autophagy regulates dBRUCE and cell death. Moreover, BRUCE itself becomes degraded by autophagy in the degenerating nurse cells during late oogenesis