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. 2018 Aug 30;2018:6062520.
doi: 10.1155/2018/6062520. eCollection 2018.

Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes

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Free PMC article

Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes

Bo Jin et al. Biomed Res Int. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
ATP-PPi exchange rates of peptides with Ube1. C1P<0.05 versus P1, C2P<0.05 versus P1, and C7P<0.05 versus P1, n=3.
Figure 2
Figure 2
(a) ATP-PPi exchange rates of wild-type ubiquitin and CP1, CP2, and CP7 proteins with Ube1. CP1P<0.05 versus wt ub, CP2P<0.05 versus wt ub, and CP7P<0.05 versus wt ub, n=3. (b) Western-blot of E1 transfer of wild-type ubiquitin and CP1, CP2, and CP7 proteins with Ube1.
Figure 3
Figure 3
Transfer of wild-type ubiquitin and ubiquitin mutants to Ube1 and UbcH7. (a) Reducing SDS-PAGE. (b) Nonreducing SDS-PAGE.
Figure 4
Figure 4
(a) Transfer of PCP-peptides to Ube1 and UbcH7. (b) Transfer of PCP-P2, PCP-P3, and PCP-P4 to Ube1 and UbcH7.
Figure 5
Figure 5
Transfer of PCP-peptides to Ube1 and E2 enzymes UbcH5a and UbcH5b.

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