Phage Display to Identify Nedd8-Mimicking Peptides as Inhibitors of the Nedd8 Transfer Cascade

• Published in: Chembiochem : a European journal of chemical biology Vol. 14; no. 11; pp. 1323 - 1330
• Main Authors: Zhao, Bo, Zhang, Keya, Villhauer, Eric B., Bhuripanyo, Karan, Kiyokawa, Hiroaki, Schindelin, Hermann, Yin, Jun
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 22.07.2013; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Subjects: Amino Acid Sequence; Binding Sites; Catalytic Domain; Crystallography, X-Ray; enzyme inhibitors; Humans; Kinetics; Mutagenesis; Nedd8; Nedd8 activating enzymes; NEDD8 Protein; Peptide Library; Peptides; Peptides - chemistry; Peptides - metabolism; phage display; Proteins; Ubiquitins - antagonists & inhibitors; Ubiquitins - genetics; Ubiquitins - metabolism; peptides; Nedd8; enzyme inhibitors; phage display; Nedd8 activating enzymes
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201300234

The Nedd8 activating enzyme (NAE) launches the transfer of the ubiquitin‐like protein Nedd8 through an enzymatic cascade to covalently modify a diverse array of proteins, thus regulating their biological functions in the cell. The C‐terminal peptide of Nedd8 extends deeply into the active site of NAE and plays an important role in the specific recognition of Nedd8 by NAE. We used phage display to profile C‐terminal mutant sequences of Nedd8 that could be recognized by NAE for the activation reaction. We found that NAE can accommodate diverse changes in the Nedd8 C‐terminal sequence (71LALRGG76), including Arg and Ile replacing Leu71, Leu, Ser, and Gln replacing Ala72, and substitutions by bulky aromatic residues at positions 73 and 74. We also observed that short peptides corresponding to the C‐terminal sequences of the Nedd8 variants can be activated by NAE to form peptide∼NAE thioester conjugates. Once NAE is covalently loaded with these Nedd8‐mimicking peptides, it can no longer activate full‐length Nedd8 for transfer to the neddylation targets, such as the cullin subunits of cullin‐RING E3 ubiquitin ligases (CRLs). We have thus developed a new method to inhibit protein neddylation by Nedd8‐mimicking peptides. Trojan horse inhibitor of protein neddylation: The ubiquitin‐like protein Nedd8 goes through an enzymatic cascade to modify key cellular proteins, such as cullin‐RING ubiquitin ligase (CRL). Phage selection identified short peptides that function as a Trojan horse to covalently react with NAE and thus block Nedd8 transfer through the cascade.