Identification Of SUMO E2-specific Substrates By Orthogonal SUMO Transfer And The Reactive Mechanism Between Fusion Proteins With SUMO/UB Active Peptide And E1-E2

The modification of small ubiquitin-like modifier(SUMO),known as sumoylation,regulates protein-protein interactions and diverse cellular processes.SUMO is transferred to substrate proteins through either a relay of E1-E2 enzymes or a sequential E1-E2-E3 cascade composed of a single E1(SAE),a sole E2(Ubc9)and several E3 s,therefore SUMO substrates are correspondingly divided into E2-specific and E3-specific.Although various methods have been developed to identify SUMO substrates,it is difficult to differentiate these substrates to E2 or an individual E3 specific family in the cell.To achieve this goal,we have developed a novel strategy to engineer an Orthogonal SUMO Transfer(OST)by which an engineered SUMO(xSUMO)is transferred exclusively through engineered E1,E2,E3 enzymes(x E1,x E2,x E3)to modify the substrates of E2 or E3.The OST shares no reactivity with native SUMO E1,E2 and E3 s and uniquely attaches xSUMO to the substrates in the cell.Our work on engineered OST cascade will distinguish SUMO E2 specific substrates in the first time,and come up to discover SUMO E3 substrates further.This study will help to understand the mechanism of sumoylation in the cell,and the identification of E2 or E3 specific substrates will also provide information for drug discovery.UB and SUMO share similar three-dimension structures: a β-grasp folding structure and a C-terminal X-X-[R / A / E / K]-X-X-[G / X]-G motif.We have previously reported that the 7-amino acid C-terminal peptides(p S90,p4)from SUMO and UB mutants can be delivered to their corresponding E1 and E2.In this study,the C-terminal peptides of SUMO and UB with wild and mutant type was fused to UB/SUMO framework to construct and express four fusion proteins(UB-p SUMO,SUMO-p UB,UBp S90,SUMO-p4),and measure their reactivity with SUMO E1 and E2 by Western blot.The results showed that UB-p SUMO and SUMO-p UB with wild-type C-terminal peptides cannot be transferred to SUMO E1 and E2,and UB-p S90 with mutant C-terminal peptides can only be transmitted to SUMO E1,and SUMO-p4 can be conjugated to SUMO E1 and E2.To sum up,these results indicated that the main framework and C-terminal sequence of the fusion proteins together determined its reactivity in SUMO E1-E2 transfer.