TRIM11 turnover in presence of FAT10 is slightly accelerated

In order to investigate whether TRIM11 degradation is affected in presence of FAT10, HEK293 cells were transiently transfected with a HA-tagged FAT10 and a HIS-tagged TRIM11 construct. Cycloheximide (50 µg ml^-1) was added to the indicated timepoints for 2.5 and 5 h or left untreated treated. Moreover, cells were treated with or without MG132 for 6 h before cell lysis.

Figure 28: Determination of TRIM11 turnover rate in the presence of FAT10

HEK293 cells were transiently transfected with pcDNA3-HIS/-A-TRIM11and pcDNA3.1-HA-FAT10 plasmids.

Before cell lysis, cells were treated with the proteasome inhibitor MG132 (10 µM) for 6 hours and with cycloheximide (50 µg ml^-1) for different time periods, as indicated. Samples were subjected to Western analysis using a HIS-reactive antibody to evaluate TRIM11 expression. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. A representative blot out of three independent experiments is shown. Mean values ± SEM of three independent experiments are depicted as relative expression of transfected cells without cycloheximide treatment.

After cell lysis, whole-cell lysates were subjected to Western blot analysis with a anti-6HIS-reactive antibody, to illustrate TRIM11 expression. The cycloheximide data illustrate, that the HIS-TRIM11 half life in presence of FAT10 is about 4 h and therefore slightly accelerated in comparison to TRIM11-FLAG alone (see 6.2.8), where 5 h after cycloheximide treatment TRIM11 protein level decreased about 50 %. Besides, MG132 treatment enhanced accumulation of HIS-TRIM11.

Results

113 6.2.10 Co-expression of TRIM11 does not change protein turnover rates of

JunB and FAT10

We next aimed to determine the protein turnover rate of JunB and FAT10 in presence of TRIM11. Moreover, we wanted to analyze whether TRIM11 degradation is affected in presence of JunB and FAT10. HEK293 cells were transiently transfected with a HA-tagged FAT10 and FLAG-tagged JunB construct, or together with a HIS-tagged TRIM11 construct.

Cycloheximide (50 µg ml^-1) was added for the indicated time periods, 2.5 and 5 h, or left untreated. Moreover, cells were treated with or without MG132 for 6 h.

Aliquots of lysates from transfected cells were analyzed for expression of the respective proteins (Figure 29 (a) for FAT10, (b) for TRIM11 and (c) for JunB and JunB-FAT10 conjugates). After immunoprecipitation with anti-HA-agarose beads, we looked for immunoprecipitated FAT10 (Figure 29 (b)) and co-immunoprecipitated JunB (Figure 29 (c)).

(a)

(b)

114

Figure 29: Determination of the turnover rate of the JunB-FAT10 conjugate

HEK293 cells were transiently co-transfected with a pCMV6-JunB-MYC-FLAG and a pcDNA3.1-HA-FAT10 plasmid. Before cell lysis, cells were treated with/without the proteasome inhibitor MG132 (10 µM) for 6 hours and with cycloheximide (50 µg ml^-1) at different time points, as indicated. After immunoprecipitation against the HA-tag of FAT10 with anti-HA agarose, samples were subjected to Western analysis using a directly horseradish peroxidase (HRP) -linked anti-FLAG mAb to evaluate JunB expression and conjugate formation. The plasmid content in all transfections was balanced with empty expression vector pcDNA3. All samples were analyzed under reducing conditions (10% β-mercaptoethanol). β-actin served as a loading control. ECL signals of all experiments were quantified with Quantity One Software (BioRad). A representative blot out of four independent experiments is shown. Mean values ± SEM of three independent experiments are depicted as relative expression to the ECL signal of transfected cells without cycloheximide treatment, which was set to unity.

Quantitave analysis resulted in a quite similar degradation rate for either FAT10 or JunB, in presence or absence of overexpressed HIS-TRIM11 (data not shown). The JunB-FAT10 conjugate turnover rate between JunB and FAT10 in presence of TRIM11 could not be calculated, because co-expression with TRIM11 almost completely abolished conjugate formation (Figure 29 (c), lane 6-9).

As we could show previously, co-expression of TRIM11 resulted in severely decreased protein levels either for JunB or FAT10 and TRIM11 itself, without changing the velocity of degradation, which might suggest a role in regulating and controlling protein stability and abundance and might be involved in protein quality control as has been shown for Pax6 (Tuoc and Stoykova, 2008).

(c)

Results

115 6.2.11 Conjugate formation of endogenous JunB and FAT10

To test whether a JunB-FAT10 conjugate can also be found in absence of overexpression, we treated HEK293 cells with TNF-α and IFN-γ to induce FAT10 expression and then performed immunoprecipitation with a FAT10-specific monoclonal antibody (mAb) (designated 4F1), followed by Western blot analysis with either a FAT10-specific polyclonal antibody (pAB) or a polyclonal JunB antibody under reducing conditions (10 % β-mercaptoethanol). This cell line, despite low JunB expression, was chosen because FAT10 can easily induced with the proinflammatory cytokines TNF-α and IFN-γ.

Figure 30: Co-immunoprecipitation of endogenous FAT10 and JunB from HEK293 cells.

HEK293 cells were 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 FAT10 was immunoprecipitated using a monoclonal FAT10-reactive antibody (4F1), followed by SDS-PAGE and Western blot analysis using a FAT10-reactive polyclonal Ab (a) or a JunB-reactive polyclonal Ab (ab314221) (b) under reducing conditions (10 % β-mercaptoethanol). The arrow head indicates JunB-FAT10 conjugate formation. Asterisks indicate heavy and light antibody chains of the FAT10-reactive antibody used for the immunoprecipitation.

Whole-cell lysates were also directly immunoblotted with a polyclonal FAT10 and JunB-antibody to reveal the expression of induced FAT10 and endogenous JunB. FAT10 (~18 kDa) was clearly induced, although better detectable after immunoprecipitation (Figure 30 (a)). Due to various background bands it was difficult to detect the appropriate band showing endogenous JunB (Figure 30 (b), input). However, the blot in Figure 30 (b) reveals, that endogenous JunB (~48 kDa) becomes co-immunoprecipitated with FAT10, which indicates a non-covalent interaction between the two proteins.

(a)

(b)

116 Endogenous JunB-FAT10 conjugate was detectable in a very low grade, in the presence but not in the absence of FAT10 induction by proinflammatory cytokines (Figure 30 (b), lane 4, arrow head).

6.2.12 Conjugate formation of JunB and FAT10 under semi-endogenous