Herp recruits Usp7 to Synoviolin

Since Usp7 and Synoviolin both directly interact with Herp, an association of Usp7 with Synoviolin is likely. Recent experiments revealed that only upon elevated expression of Herp, but not HerpΔUBL, Usp7 is associated with Synoviolin (M.Seeger, personal communication).

Therefore, it was suggested that the induction of Herp protein expression in case of ER stress leads to an association of Usp7 and Synoviolin. To test this hypothesis, HeLa cells expressing Synoviolin-HTB were treated with tunicamycin for up to six hours to induce ER stress. For Western blot analysis, HTB-tagged Synoviolin was precipitated and tested for co-precipitated proteins.

Within six hours of continuous ER stress the expression of Herp was increased, whereas the expression of Synoviolin and Usp7 remained even (Figure 15, A, lysate). Constant amounts of Synoviolin-HTB were precipitated at all tested time points, whereas the amount of co-precipitated Herp increased over time (Figure 15, A, av.precip.). At six hours of ER stress treatment a small subpopulation of endogenous Usp7 was also co-precipitated with Synoviolin-HTB indicating that the association of Usp7 and Synoviolin is mediated by Herp (Figure 15, A, av-precip.). An inverse observation was made for Derlin-1, another Synoviolin associated protein. Although Derlin-1 protein expression was marginally induced by ER stress, the amount of Derlin-1 which co-precipitated with Synoviolin declined during the period of stress treatment indicating that ER stress on the one hand leads to the increased association of proteins with Synoviolin and on the other hand to the dissociation of distinct proteins from these ERAD complexes.

To evaluate the stability of the association of Usp7 with Synoviolin, ER stress exposed HeLa cells expressing Synoviolin-HTB were treated with cycloheximide for up to six hours before streptavidin agarose precipitation and Western blot analysis was performed as before.

A

Figure 15: Association of Usp7 and Synoviolin. (A) HeLa cells, expressing hexahistidin-biotin (HTB)-tagged Synoviolin (clone 6, low expression level) were treated with 10 μg/mL tunicamycin (tu) for the indicated times.

Then the cells were lysed in DBC containing buffer and Synoviolin-HTB was precipitated with streptavidin agarose. Proteins of the lysates and the precipitations were separated on SDS-PAGE and visualised by Western blot analysis using the specific antibodies as indicated. (B) HeLa cells, stably expressing Synoviolin-HTB (clone 6) were treated with 10 μg/mL tunicamycin for four h. After that 50 μg/mL cycloheximide was added and cells were lysed in DBC containing buffer at the indicated time points. The experiment was continued as described in (A). Exp=exposure; IB=immunoblot; h=; chx=cycloheximide; av-precip.=streptavidin agarose precipitation.

Usp7 and p97 remained stable for six hours of the cycloheximide chase, whereas Herp was completely degraded within this time period (Figure 15, B, lysate). The amount of Usp7 that was co-precipitated with Synoviolin decreased over time, which went along with a decrease of the amount of Synoviolin co-precipitated Herp (Figure 15, B, av-precip.). In contrast, the amount of p97 that was co-precipitated with Synoviolin remained constant. Taken together, these data demonstrate that Usp7 is associated with Synoviolin. Furthermore, the interaction of Usp7 and Synoviolin is positively correlated to the interaction of Herp with Synoviolin indicating that the association of Usp7 and Synoviolin depends on Herp.

Usp7 binds to the UBL domain of Herp, whereas Synoviolin directly interacts with a region distal from the UBL domain (Schulze, 2006; Schulze et al., 2005). Thus, Usp7 and Synoviolin are likely to interact with Herp simultaneously. Particularly in an ER stress situation, Herp may function as a linker of these proteins. To test this assumption, endogenous Herp, Usp7 and Synoviolin were tested for their mutual interactions. For this purpose HeLa cells were metabolically labelled and Herp or Usp7 were immunoprecipitated.

Before the labelling process, cells were subjected to ER stress for four hours to induce Herp but not Synoviolin protein expression in order to investigate Herp dependent interactions.

The amount of co-precipitated Synoviolin or Herp was assessed by a second immunoprecipitation. Proteins were analysed by SDS-PAGE and autoradiography.

ER stress treatment led to the elevated expression of Herp, whereas the expression of Usp7 was not affected, which is in agreement with the previous results (Figure 16, left panel).

When Herp was increased through ER stress treatment, also an increased amount of Synoviolin was co-precipitated with Herp (Figure 16, lane 7). In the absence of ER stress Herp or Synoviolin were not co-precipitated with Usp7 (Figure 16, -Tg, lanes 6, 8). In contrast, induction of ER stress led to the co-precipitation of Herp and Synoviolin with Usp7 (Figure 16, +Tg, lanes 6, 8). These data revealed that Usp7 is associated with Synoviolin predominantly in case of ER stress. This finding strongly suggests that Herp mediates the interaction of both enzymes.

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Figure 16: Herp dependent association of Usp7 and Synoviolin. HeLa cells were left untreated or treated with 2 μM thapsigargin for four h. Then metabolic labelling with ³⁵S-methionine was followed by the lysis of the cells in DBC containing buffer. For the first immunoprecipitation (IP) either pre-immune serum (PI) or specific antibodies as indicated were utilised. The dissociation of the co-precipitated proteins was accomplished by incubation in RIPA buffer. The resulting supernatants were subjected to a second immunoprecipitation using the indicated specific antibodies. Precipitated proteins were separated on SDS-PAGE and visualised by autoradiography. First IPs are depicted in the left, second IPs in the right hand panels. Nonspecific cross reactions are marked by an asterisk. tg=thapsigargin; Synv=Synoviolin.