Inhibiting the Protein Ubiquitination Cascade by Ubiquitin-Mimicking Short Peptides

Zhao, Bo; Choi, Chan Hee J.; Bhuripanyo, Karan; Villhauer, Eric B.; Zhang, Keya; Schindelin, Hermann; Yin, Jun

doi:10.1021/ol3027736

Cited by 13 publications

(14 citation statements)

References 36 publications

(37 reference statements)

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“…We previously found that the 7-mer peptides derived from the C-termini of UB variants from phage selection with UAE can form thioester conjugates with UAE and the E2 enzymes of the UB transfer cascade [30]. We referred to these peptides as “UB-mimicking peptides” and we further showed that once the peptides are loaded on UAE and the E2 enzymes, the cascade enzymes are blocked from transferring UB to E3 for protein ubiquitination.…”

Section: Resultsmentioning

confidence: 96%

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

et al. 2013

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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.

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Section: Resultsmentioning

confidence: 96%

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

et al. 2013

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“…We synthesized C1-C8 peptides (7mers based on the motif) and ascertained whether they were reactive with human E1 Ube1. For comparison, we used the C-terminal sequence of wild-type ubiquitin (P1) as negative control and the short peptides P3 (VQRYWGG) and P4 (VYRFYGG) we previously reported as positive controls [ 12 ].…”

Section: Resultsmentioning

confidence: 99%

“…Ubiquitin and UBLs share the same three-dimensional core structure: the β -grasp fold and a similar C-terminal motif X-X-[R/A/E/K]-X-X-[G/X]-G, where X represents any amino acid. We have previously reported short free peptides derived from the C-terminal sequences of ubiquitin and ubiquitin mutants could form the thioester intermediates with E1, E2, and E3 enzymes as full length ubiquitin [ 11 , 12 ]. Encouraged by these results, we continued to investigate whether proteins carrying such peptides as C-terminal fusion tags could still be recognized by ubiquitin pathway enzymes.…”

Section: Introductionmentioning

confidence: 99%

Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes

Jin

Wang

Liu

et al. 2018

BioMed Research International

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Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: β-grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the β-grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames.

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“…[36] We showed that short peptides derived from the C-terminal sequence of the UB variants are reactive with UAE to form thioester conjugates with UAE and E2s. [48] We referred to these peptides as “UB-mimicking peptides”. We found that they can effectively block the transfer of UB through the cascade enzymes due to their covalent attachment to UAE and E2s.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2013

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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.

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Inhibiting the Protein Ubiquitination Cascade by Ubiquitin-Mimicking Short Peptides

Cited by 13 publications

References 36 publications

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes

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

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