Determination of SUMO1 and ATP affinity for the SUMO E1by quantitative FRET technology

Abstract

SUMOylation plays important roles in many key physiological and pathological processes. The SUMOylation cascade involves a heterodimer of activating enzyme, E1 (Aos1/Uba2); a conjugating enzyme, E2 (Ubc9); and many ligase enzymes, E3. Focusing on the activation step of the SUMOylation process, we examined the interaction of E1 with its substrates. Previous studies reported the Km of E1 enzymes in ubiquitin and other ubiquitin-like pathways, but the Km of the SUMO paralogs (SUMO2 and SUMO3) is unknown. Here, by using quantitative FRET to measure the SUMO E1 enzyme kinetics of SUMO1, 2, and 3 and ATP under steady state conditions, we found that the enzyme kinetics from the quantitative FRET method are comparable to those from conventional radioactive assays. Additionally, the kinetic constants, Km , of SUMO2 (3.418 ± 0.9131 μM) and SUMO3 (2.764 ± 0.75 μM) [FW1] are approximately four to five times higher than that of SUMO1 Km (0.7458 ± 0.1105 μM). These results demonstrate the advantages of FRET technology for determining Km , including the ability to monitor reaction progress in real-time with high-throughput and high-sensitivity in an environmentally friendly manner. The processes discussed here extend the utility of quantitative FRET in characterizing protein-protein interactions and enzyme kinetics.

The ubiquitin-like modifier FAT10 interferes with SUMO activation

“…7). The measured K d value of 1.8 µM for the interaction of FAT10 and AOS1/UBA2 is similar to the previously published K m value for SUMO-1 and AOS1/UBA2 of 0.74508 ± 0.1105 µM 41 , which is in good agreement with the FRET-based inhibition assay shown in Fig. 3a.…”

The SUMO activating enzyme subunit, SAE2, contributes SUMO protein bias for mitotic fidelity

“…Fig. 2a), consistent with K m measurements of SUMO1 Vs SUMO2 with AOS1/UBA2 (Wiryawan, Dan et al, 2015). In contrast, the K164Q-SAE2 E1 had a greater affinity (lower K d ) for the SUMO2 isoform and reduced affinity for SUMO1 (Fig.…”

Knockdown of Uba2 inhibits colorectal cancer cell invasion and migration through downregulation of the Wnt/β‐catenin signaling pathway

“…Small ubiquitin-like modifiers (SUMO) have been shown to play important roles in many physiological processes, including protein stability, DNA repair, chromatic structure regulation, protein translocation, and protein-protein interactions. [4][5][6][7][8] Furthermore, recent data suggest that cancers are associated with alterations in SUMOylation. 4,[9][10][11] Ubiquitin-like modifier activating enzyme 2 (Uba2), also known as SUMO-activating enzyme subunit 2 (SAE2), has been identified as a critical component of the SUMOactivating enzyme (Aos1/Uba2 heterodimeric pair) necessary for the SUMO pathway.…”

“…SUMOylation is a common process observed in many cellular proteins and the majority of transcription factors. Small ubiquitin‐like modifiers (SUMO) have been shown to play important roles in many physiological processes, including protein stability, DNA repair, chromatic structure regulation, protein translocation, and protein‐protein interactions . Furthermore, recent data suggest that cancers are associated with alterations in SUMOylation …”