Fig. 6
The N-terminus of FAT10 is crucial for its VCP-independent degradation. a, b HeLa cells were transiently transfected with HA-ubiquitin-(AV)-GFP or HA-FAT10-(AV)-GFP expression constructs. After 16 h cells were metabolically labeled with 35S-methionine for 1 h and chased in the presence of 5 μM of VCP inhibitors CB-5083 or NMS-873 for the indicated time periods. Then, cells were lysed and HA-tagged proteins were immunoprecipitated, separated by SDS-PAGE, and visualized by autoradiography. c HEK293 cells were transiently transfected with expression constructs for WT untagged FAT10 or a FAT10 variant lacking the six N-terminal amino acids APNASC (FAT10-ΔAPNASC). 24 h after transfection, cells were treated with 50 µg/mL CHX in the presence or absence of 10 µM proteasome inhibitor MG132 or 10 µM VCP inhibitor CB-5083 for 5 h. Cleared lysates were subjected to immunoprecipitation for FAT10 and degradation of FAT10 was visualized by western blot. Endogenous ubiquitin in total lysates was monitored by western analysis as well (IB: Ub). β-actin served as loading control. One representative experiment out of three experiments with similar outcomes is shown. d ECL signals of immunoprecipitated FAT10 and FAT10-ΔAPNASC were quantified and normalized to signals of the loading control β-actin. The values of untreated cells were set to 100% and the other values were calculated accordingly. The mean of three independent experiments with similar outcomes is shown as means ± s.e.m. e Molecular dynamics simulations of the N-terminal ubiquitin-like domain (UBD) of human FAT10 (WT) showing that the N-terminal unstructured region of FAT10 is highly flexible. The graph shows the distance between the N-terminus and center of the N-domain during the course of two simulations with different protonation states of histidine (deprotonated: black, protonated: blue). The flexible N-terminus forms contacts with the domain but is repeatedly released in both simulations. Inset: N-domain in cartoon representation colored in red according to high RMSF values. The arrow indicates the distance shown in time series
