(A) Schematics of the regulation of NEDD8 substrates by modification with either WT- (left panel) or L73P-Nedd8 (right panel), and deneddylation by NEDD8-specific proteases. CSN is the deneddylase responsible for deconjugating NEDD8 from cullin substrates, but proteases regulating deneddylation of non-cullin substrates are largely uncharacterized. (B) Surface representation of NEDD8 (pdb: 1NDD) and details of its C-terminal tail, showing its proteolytic cleavage site and location of the L73P mutation. (C) Recombinant CRL1/Rbx1 was in vitro neddylated by purified His-NEDD8-WT or His-NEDD8-L73P, in the presence of E1 and E2 enzymes and ATP. Reactions were quenched, and recombinant CSN was added at increasing concentrations to monitor the ability of each NEDD8 moiety to be deconjugated from CUL1. OPT (1,10-orthophenatroline, 1 mM) was added to samples containing the highest concentration of CSN (last lane) to completely inhibit CSN activity. (D) FLAG-NEDD8-WT or FLAG-NEDD8-L73P was induced in HeLa-FlpIn-N8 cells using 1 ug/mL doxycycline for 48 hr prior to collection. Whole-cell lysates of untreated or Dox-treated cells were incubated with anti-FLAG beads to purify FLAG-NEDD8-conjugates. Immunoblots of input and IP samples were analyzed for FLAG-NEDD8-modified CUL1 and CUL2. (E) HeLa-FlpIn-N8 cells were treated with or without Dox as in D to induce FLAG-NEDD8-WT or FLAG-NEDD8-L73P, and subsequently incubated with or without the of the CRL inhibitor MLN4924 (5 µM for 4 hr) before harvesting. Whole-cell extracts were analyzed for FLAG-NEDD8-conjugated CUL1 and CUL2. (F) (left panel) Workflow for expression and purification of FLAG-NEDD8-WT and FLAG-NEDD8-L73P for MS analysis. (right panel) Percentages of total spectral counts detected in FLAG-IPs from cells expressing either FLAG-NEDD8-WT (orange bars) or FLAG-NEDD8-L73P (purple bars). The numbers in the columns indicate actual spectral counts. The IPs were performed on lysates from the same number of cells.