ng Arg74 and Gly75 block UB transfer from E2 to E3s of either HECT type for the formation of UB,HECT thioester conjugate or RING/U-box types for self-ubiquitination. These results suggest that the Cterminal sequences of UB and UB variants are closely monitored by the cascade enzymes during their passage through the UB or the Nedd8 cascade. 8 Nedd8-Like Ubiquitin Variants 9 Nedd8-Like Ubiquitin Variants Inhibition of Nedd8 Transfer through the NAE-Ubc12 Cascade by the Nedd8-mimicking MedChemExpress ML-128 peptides We also found that 7-mer peptides corresponding to the Cterminal sequence of NAE selected UB mutants are reactive with NAE in the ATP-PPi assay. These peptides can form thioester conjugates with NAE. Peptide pN26 derived from N26 with the native C-terminal sequence of Nedd8 can be further transferred to Ubc12 and cullin to form covalent conjugates. Peptide pN11 with the sequence 71 VAAAAGG76 is not reactive with NAE, although 19053768 the corresponding UB variant N11 can be activated by NAE with a similar efficiency as the wt Nedd8 or N26 and be transferred through the NAE-Ubc12 cascade for cullin modification. This suggests that the N11 variants of UB should rely on interfaces other than its C-terminal peptide for binding to NAE. The pN11 peptide itself cannot be recognized by NAE for the activation reaction. Peptides pN1 and pN7 derived from UB variants N1 and N7 were found to be activated by NAE at a similar efficiency as the pN26 peptide. They also form peptide,NAE conjugates at a similar level as pN26. However, these peptides are less efficient for transferring to Ubc12 and none of them can form peptide-cullin conjugates at a detectable level. Both peptides have bulky aromatic substitutions of the wt residues at positions 71, 73 and 74. These results again suggest that the C-terminal sequence of UB would greatly affect the activity of peptide transfer to Ubc12 and cullin. Finally we showed that NAE loaded with the Nedd8-mimicking peptides pN1, pN7 and pN26 is prevented from transferring Nedd8 through the Nedd8-Ubc12 cascade for cullin modification. Since these peptides rely on NAE catalyzed activation and peptide,NAE thioester formation to inhibit cullin modification, they should be classified as mechanism-based inhibitors of the Nedd8 transfer 17372040 cascade. Recently the compound MLN4924 was developed by Millennium as a potent inhibitor of NAE. MLN40924 binds to the nucleotide binding pocket of NAE as an AMP analog with a sulfamate group replacing the phosphate moiety of AMP. The amino group of the sulfamate attacks the thioester linkage between Nedd8 and the catalytic Cys residue of NAE leading to the formation of a 
Nedd8-MLN4924 adduct that binds to the adenylation domain of NAE with high affinity. The Nedd8 mimicking peptides block the catalytic Cys residues of NAE and prevent the loading of NAE with full-length Nedd8. Peptide pN26 with the native C-terminal sequence of Nedd8 can also be transferred to Ubc12 to block Nedd8 conjugation to E2. It can be further transferred to cullin to block Nedd8 modification of cullin. Peptides pN1 and pN7, however, are stalled at the stage of forming the peptide,NAE conjugate and are significantly less active in further transfer to Ubc12 or cullin. Thus, these peptides can function as road blocks of Nedd8 transfer at different stages of the NAE-Ubc12 cascade. They provide a mechanism for inhibiting cullin modification that is distinct from MLN4924. It will be of interest to further probe the effects of the Nedd8-mimickin