Conjugate formation of JunB and FAT10 under semi-endogenous conditions

Since conjugate formation between JunB and FAT10 was hardly detectable under completely endogenous conditions, the experimental setup was slightly modified and performed under semi-endogenous conditions. Hence we next aimed to determine whether endogenous FAT10, upregulated by TNF-α and IFN-γ, can build a stable conjugate with over-expressed JunB.

HEK293 cells were transiently transfected with the expression construct pCMV6-JunB-MYC-FLAG and treated with TNF-α and IFN-γ for one day, or left untreated, to carry out co-immunoprecipitation experiments. JunB was immunoprecipitated with FLAG-M2 affinity matrix and samples immunoblotted either with a FLAG-reactive antibody to reveal JunB expression (Figure 31 (a)), or a polyclonal FAT10 antibody to detect endogenous FAT10 and FAT10 conjugates (Figure 31 (b)).

Figure 31: Co-immunoprecipitation of endogenous FAT10 and transiently transfected JunB from HEK293 cells.

HEK293 cells were transiently transfected with 4 µg pCMV6-JunB-MYC-FLAG plasmid and subsequently treated with 200 U ml^{− 1} IFN-γ and 400 U ml^{− 1} TNF-α to up-regulate endogenous FAT10 expression, or left untreated.

After 24 h, cells were lysed and JunB was immunoprecipitated using anti-FLAG-agarose, followed by SDS-PAGE and Western blot analysis using a FLAG-reactive antibody conjugated to HRP (left panel) or a polyclonal FAT10 specific Ab (right panel) under reducing conditions (10 % β-mercaptoethanol). The arrow head indicates JunB-FAT10 conjugate formation.

(a) (b)

Results

117 JunB expression was apparently increased in INF-γ/TNF-α treated cells (Figure 31 (a), lane 3), whereas the total protein amount remained constant (WB: anti-ß-actin).

Conjugate formation between JunB and FAT10 could be detected under reducing conditions (10 % ß-mercaptoethanol) after immunoprecipitation against the FLAG-tag of JunB and Western Blot analysis against FAT10 in a high molecular weight band (~65 kDa), as indicated by the arrow head (Figure 31 (b)). Interestingly, only a single conjugate band could be detected in comparison to the previous experiments (see 6.2.3), where always a double-band appeared. However, these results indicate, that conjugate formation between JunB and FAT10 takes place under semi-endogenous condition.

6.2.13 In vitro auto-FAT10ylation assay

The experiments described above strongly indicate, that FAT10 becomes stably attached to JunB via an isopeptide linkage. The function of TRIM11 as a putative FAT10 E3 ligase remains hitherto unclear. In the performed experimental in vivo setups we could show, that co-expression of TRIM11 led to decreased protein amounts of either FAT10, ubiquitin or JunB. Moreover, overexpression of TRIM11 reduced severely the amount of JunB-FAT10 conjugate. To ensure in vitro, if TRIM11 is a FAT10 specific E3 ligase and to determine, whether TRIM11 mediates the transfer of FAT10 onto JunB, we performed in vitro activation experiments with recombinant proteins. We expected when TRIM11 acts as a FAT10 specific E3 ligase, FAT10 can be transferred on the putative substrate JunB only in presence of FAT10 specific E1, E2 and TRIM11 under conditions containing a rich source of renewable energy such as ATP, creatine phosphate, creatine phosphokinase and inorganic pyrophosphatase, which could serve as a cell free system for the FAT10ylation reaction in vitro. For this purpose immunoprecipitation was coupled with an in vitro FAT10ylation assay of FLAG-tagged JunB in the presence of the recombinant FAT10 E1 (UBA6), E2 (HIS-USE1) and the putative FAT10 specific E3 ligase GST-TRIM11.

JunB-MYC-FLAG was expressed in HEK293 cells and immunoprecipitated with anti-FLAG-agarose to purify JunB for the subsequent in vitro FAT10ylation assay. JunB bound to the beads was incubated together with purified recombinant FAT10, FLAG-UBA6 and 6HIS-USE1 in the presence of ATP at 37 °C for 1 h. Finally, JunB was visualized by Western blot analysis using anti-FLAG antibodies (Figure 32 (a)), USE1 was detected with an anti-6HIS-POX antibody (Figure 32 (b)), TRIM11 was detected with a monoclonal anti-GST-antibody (Figure 32 (c)) and FAT10 was visualized with a monoclonal anti-FAT10 antibody (Figure 32 (d)).

118

Figure 32: In vitro FAT10ylation assay of JunB

HEK293 cells expressing MYC-FLAG-tagged JunB were lysed in FAT10 IP-buffer buffer supplemented with 1 % NP40 and an immunoprecipitation was performed anti-FLAG-Agarose. Beads were washed 3 times with FAT10-IP buffer and immunoprecipitated. JunB was incubated with 10 μg FAT10, 1,25 μg FLAG-UBA6, 3 μg HIS-USE1, 50 μg GST-TRIM11 and ATP. After 60 min of incubation at 30°C, the beads were washed twice with FAT10-IP-buffer and boiled with reducing Laemmli FAT10-IP-buffer containing 10 % β-mercaptoethanol, followed by Western blot analysis with (a) a FLAG-reactive antibody conjugated to horseradish peroxidase (HRP) to detect either JunB-MYC-FLAG or FLAG-UBA6. (b) HIS-USE1 was detected with a peroxidase (POX) conjugated polyhistidine-reactive antibody. (c) GST-TRIM11 was detected with an anti-GST-antibody and (d) FAT10 was detected through a monoclonal FAT10-antibody. Input contains 10 % of JunB, recombinant FAT10, HIS-USE1, FLAG-UBA6 and GST-TRIM11 of the total protein amount used for the in vitro FAT10ylation assay. Asterisks indicate light and heavy antibody chains of the FLAG-reactive antibody used for the IP of FLAG-JunB.

Although all samples were treated under reducing conditions (10 % β-mercaptoethanol), conjugate formation between UBA6 and FAT10 could be detected in the anti-FLAG-blot (Figure 32 (a)), as well as in the anti-FAT10-blot (Figure 32 (d)), indicating a covalent linkage. The in vitro conjugation assay revealed, that JunB was not apparently FAT10ylated in vitro in the presence of recombinant FAT10, UBA6, USE1 and TRIM11, because no defined conjugate band was detectable neither on the anti-FLAG nor anti-FAT10 blot (Figure 32 (a), lane 11 and (d), lane 6).

(a) (b)

(c)

(d)

Results

119 Interestingly, a high molecular weight smear, indicating a poly-FAT10ylation of JunB was detectable when FAT10 and its E1, E2 enzyme and TRIM11 were present (Figure 32 (a) and (d)). To argue why the in vitro FAT10ylation assay with GST-TRIM11 failed seems to be very troublesome. One reason could be that the purification of recombinant GST-TRIM11 was not satisfactory and contained still impurities and degradation products (Figure 32 (c), lane 1, input). Another problem could be the poor solubility of recombinant TRIM11.