Short heptapeptides were identified to function as ubiquitin (UB) mimics that are activated by E1 and form thioester conjugates with E1, E2 and HECT type E3 enzymes. The activities (kcat/K1/2) of E1 with the UB-mimicking peptides are 100–300 fold higher than the equally long peptide with the native C-terminal sequence of UB. By forming covalent conjugates with E1, E2 and E3 enzymes, the UB-mimicking peptides can block the transfer of native UB through the cascade.
The ubiquitin-like protein SUMO is transferred through a core E1-E2 cascade composed of the SUMO activating enzyme (SAE) and Ubc9 to modify cellular proteins and transmit important biological signals. SAE primarily recognizes the C-terminal tail of SUMO and catalyzes ATP condensation with the SUMO C-terminal carboxylate to activate its transfer through the cascade. Here, we used phage display to show that a broad profile of SUMO C-terminal sequences can be activated by SAE. Based on this, we developed 7-mer peptides that can (1) form thioester conjugates with SAE, (2) be transferred from SAE to Ubc9, and (3) be further transferred to the sumoylation target protein RanGAP1. Since these peptides recapitulate the action of SUMO in protein modification, we refer to them as “SUMO-mimicking peptides”. We found that once the peptides are conjugated to SAE and Ubc9, they block full-length SUMO from entering the cascade. These peptides can thus function as mechanism-based inhibitors of the protein sumoylation reaction.
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 specific recognition of Nedd8 by NAE. We used phage display to profile C-terminal sequences of Nedd8 that can be recognized by NAE for the activation reaction. We found that besides the native Nedd8 sequence ending with 71LALRGG76, NAE can accommodate diverse changes at the Nedd8 C-terminus 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, they can no longer activate full length Nedd8 for its 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 via Nedd8-mimicking peptides.
The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB∼NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a “gate-keeping” role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE.
The back cover picture shows that the SUMO protein has been downsized by protein engineering to a heptamer short peptide that can be activated and transferred through the E1 and E2 enzymes to RanGAP1, a SUMO modification target in the cell. For details, see the paper by J. Yin et al. on .
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