3.1 The importance of the dynamics of Herp for ERAD
3.1.3 Herp-K61R is stabilised and impairs the degradation of NHK
The experiments described above strongly suggest that the turnover of Herp is not mediated by Synoviolin based complexes. So far, only the UBL domain of Herp was demonstrated to determine its stability, as Herp lacking this domain was shown to be stable (Sai et al., 2003).
In more detail, the expression of a recombinant Herp-version (aa 1-259) together with the E2 enzyme Ube2g2 and the active C-terminus of the E3 enzyme Gp78 in E.coli revealed that lysine 61, located within the UBL domain, is the crucial polyubiquitination site of this Herp-version (Li et al., 2007). In fact, the UBL domain of Herp was demonstrated to be essential for the effective ERAD of CD3-δ (Schulze, 2006). Collectively, these data suggest a correlation between the stability of Herp, determined by the UBL domain, and the function of Herp in Synoviolin mediated ERAD processes. It was hypothesised that an enhanced stability of Herp is beneficial for ERAD. To check this hypothesis, cellular Herp levels were increased but the elevated expression of Herp had no effect on the stability of ERAD substrates, which is presumably due to an enhanced degradation of Herp in this situation (M.Seeger, personal communication). To circumvent this difficulty, lysine 61 was substituted by an arginine using site-directed mutagenesis in order to obtain a stabilised Herp (Herp-K61R). To test whether the resulting Herp mutant was more stable than wild type Herp, HeLa cells, expressing wild type Herp or Herp-K61R were subjected to cycloheximide chase analyses. Additionally, HeLa cells expressing Herp or Herp-K61R as fusion proteins with a C-terminal hexa-histidine-biotin (HTB)-tag were included in the assay. Degradation was assessed by Western blot analysis using an antibody specific for Herp.
Compared to the wild type Herp, Herp-K61R was shown to be stabilised (Figure 11, A). The same result was observed for the Herp-versions which were C-terminally fused with the HTB-tag. Therefore, the single amino acid substitution K61R within the UBL domain was sufficient to stabilise Herp.
Next, this stabilised Herp mutant was tested for having an impact on the degradation of an ERAD substrate. For this purpose, the Null Hong Kong mutant of α1-antitrypsin (NHK) was chosen as a substrate, because its degradation was shown before to be dependent on Synoviolin as sole E3 ligase (Christianson et al., 2008). Associated with Synoviolin, Herp was suggested to also influence the degradation of NHK. Considering that Herp positively affects the turnover of NHK, stabilisation of Herp was hypothesised to improve this process.
To test this hypothesis, degradation of NHK was investigated in HeLa cells co-expressing NHK and either the wild type Herp or Herp-K61R. In parallel, NHK-expressing HeLa cells were transfected with siRNA specific for Herp. Cycloheximide chases were performed for degradation analysis.
control Herp Herp-K61R Herp-HTB Herp-K61R-HTB IB:
MG 132
Figure 11: Analysis of the Herp-K61R mutant. (A) HeLa cells were transfected with plasmids encoding Herp or Hexahistidin-Biotin (HTB)-tagged Herp with or without the K61R mutation. At 48 h after transfection 50 μg/mL cycloheximide (chx) were added to all samples and MG132 to a final concentration of 10 μM where indicated.
After further incubation for the indicated times (chase), cells were lysed in RIPA buffer. Proteins were separated on SDS-PAGE and visualised by Western blot analysis using the indicated specific antibodies. (B) HeLa cells were cotransfected with plasmids encoding the Null Hong Kong mutant of α1-Antitrypsin (NHK) and the indicated Herp constructs. The experiment was proceeded as described in A. (C) HeLa cells were co-transfected with either a control siRNA or a siRNA specific for Herp and 24 h later with a plasmid encoding NHK. At 48 h after transfection 50 μg/mL cycloheximide were added and cells were further incubated as indicated. Further analysis was performed as described in A. (D) HeLa cells were transfected with plasmids encoding HTB-tagged Herp or Herp-K61R. At 48 h after transfection cells were lysed in DBC containing buffer. HTB-tagged proteins were precipitated using streptavidin agarose. Proteins were separated on SDS-PAGE and visualised by Western blot analysis using the specific antibodies as indicated. IB=immunoblot; av-precip.=streptavidin agarose precipitation, an unspecific cross reaction is marked by an asterisk.
Degradation of NHK occurred within six hours of chase and was dependent on the proteasome, since the incubation with MG132 impaired NHK degradation (Figure 11, B).
Utilisation of siRNA specific for Herp led to the depletion of the Herp protein by 50% and resulted in the impaired degradation of NHK indicating that the substrate’s degradation is Herp dependent (Figure 11, C). In addition, compared to the co-expression of wild type Herp with NHK, the co-expression of a dominant negative Herp-version lacking the UBL domain also resulted in the stabilisation of this ERAD substrate (Figure 11, B, left panels). These results showed for the first time that effective degradation of NHK depends on the presence of Herp. The UBL-domain of Herp was indispensable for its ERAD promoting function.
Regarding the correlation between the stability of Herp and the stability of the substrate NHK, a surprising observation was made: the stabilisation of Herp did not improve but rather impair the degradation of NHK as shown by the co-expression of HerpK61R-HTB in comparison with Herp-HTB (Figure 11, B, right hand panel). These results demonstrate that the effective degradation of the Herp and Synoviolin dependent ERAD substrate NHK depends on the presence of the Herp-UBL domain and is positively correlated to the degradation rate of Herp, whereas stabilisation of Herp seems to have a negative effect on the ERAD process.
According to its enhanced stability, Herp-K61R might be associated with Synoviolin for a longer time than wild type Herp. To ensure that Herp-K61R associates with Synoviolin at all, Herp-K61R-HTB and Herp-HTB were expressed in HeLa cells and precipitated using streptavidin agarose. Co-precipitated proteins were analysed by Western blotting. Herp- and Herp-K61R-HTB were found to be expressed at equal levels and comparable amounts of these proteins were precipitated (Figure 11, D, Herp). Endogenous Synoviolin and Derlin-1 were co-precipitated with both Herp versions. In addition, also Usp7 was co-precipitated with HTB and K61R-HTB (Figure 11, D, av-precip.). This result indicates that Herp-K61R as well as wild type Herp is associated with Synoviolin based complexes.
Taken together, this first part of the study shows that Herp reveals distinctive dynamics, when compared to other components of Synoviolin based complexes. Herp is induced earlier upon ER stress and degraded faster. Interestingly, Synoviolin associated Herp is constantly substituted by newly synthesised Herp molecules without being a substrate of this E3 ligase or the associated proteins Sel1L and Ube2j1. Degradation and reassociation with Synoviolin seems to be important for the function of Herp at the ERAD complexes, since the depletion as well as the stabilisation of Herp led to an impaired degradation of the ERAD substrate NHK.