A Simplein SituCell-Based SUMOylation Assay with Potential Application to Drug Screening

Abstract

We found that the small ubiquitin-related modifier (SUMO) conjugation reaction can be visualized simply by incubating green fluorescent protein (GFP)-SUMO-1 with permeabilized cells in the presence of ATP for 15 min. Neither special equipment for protein purification nor highly developed skills for recombinant technologies is required, making the assay potentially applicable to large-scale drug-screening strategies for the identification of drugs that can inhibit or enhance SUMOylation.

Construction of a mouse Aos1-Uba2 chimeric SUMO-E1 enzyme, mAU, and its expression in baculovirus-insect cells

“…2C). As shown in Figure 2D, the baculovirus-insect expressed mAU showed enzymatic activity of SUMO-E1, at least, in the in situ SUMOylation assay: 29,30 the assay revealed enhanced nuclear rim staining, which represented an accumulation of GFP-SUMO-1 conjugated to the nuclear pore complex, when the digitonine-permeabilized and paraformaldehyde-fixed HeLa cells were treated with the SUMOylation buffer containing His 6 -mAU. In contrast, GFP signals in the reaction containing the SUMOylation buffer without His 6 -mAU exhibited negligible GFP signals.…”

“…(D) application of His 6 -maU recombinant protein to the in situ SUMOylation assay. 29,30 HeLa cells were permeabilized with 0.01% digitonin followed by 4% paraformaldehyde fixation. twenty μl of the in situ SUMOylation buffer containing Ubc9, GFp-SUMO-1, and Mg 2+ -atp, supplemented with (+, left column) or without (−, right column) the His 6…”

Molecular Cloning and Bacterial Expression of the Catalytic Domain of the SENP1 Gene ofOryzias latipes

“…3A and B, GFP-SUMO-1 signals were detected in multiple nuclear regions and were co-stained with either anti-PML (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-fibrillarin antibody (Santa Cruz Biotechnology) in Triton X100-permeabilized human cervical epidermoid carcinoma CaSki cells, indicating that in situ SUMOylation assay allowed visualization of the subcellular regions in which SUMOylation took place, such as promyelocytic leukemia (PML)-associated nuclear bodies and the nucleolus. [9][10][11] While the GFP signals remained unchanged in the cells treated with recombinant OlSENP1-CDc/s, the GFP signals in both the PML bodies and nucleolus were less strong when the in situ SUMOylation reaction was followed by incubation with recombinant OlSENP1-CD. Similar results were obtained with GFP-SUMO-3 (data not shown).…”

High-Yield Expression of Mouse Aos1-Uba2-Fusion SUMO-Activating Enzyme, mAU, in a Baculovirus-Insect Cell System

“…[3][4][5][6] A key component of these assays is, of course, the recombinant protein that shows SUMO-E1 activity.…”

“…Secondly, to evaluate further whether purified His 6 -mAU acts as SUMO-E1, another type of in vitro assay, designated the in situ SUMOylation assay, 5,6) was performed. Briefly, HeLa cells were grown on a coverslip and then permeabilized for 5 min on ice with PBS plus 0.1% of triton X-100.…”