Wrestling with SUMO

Continuing the theme of our ‘research area of the week’ from last week, we’re looking at ubiquitin’s cousin- SUMO, and some of the major stumbling blocks in cracking the SUMO signalling code.

SUMOylation is a post-translational modification that regulates numerous cellular pathways and facilitates protein-protein interactions. To understand how SUMOylation can specifically control protein activity, it is crucial to explore the individual and global processes regulated by this PTM. When studying SUMOylation some of the first questions we should answer are: Which technical approaches can be considered? Which biological model and experimental designs may be optimal?, and Which physiological conditions/stimuli can provide conclusive results? Assessment of both the advantages and inconveniences of the methods used to explore SUMOylation are crucial in obtaining the right answers.
It’s not that kind of SUMO.
It’s not that kind of SUMO.
There are two key problems in the detection of SUMO targets within cell systems. Firstly, many SUMOylated proteins such as transcription factors are present only at low abundance. Because only a small fraction of these targets are normally SUMOylated at steady state, detection by direct immunoblotting is often impossible. Secondly, SUMOylated species are rapidly lost upon cell lysis in nondenaturing buffers, owing to highly active SUMO isopeptidases. Consequently, large efforts have been made during recent years to develop protocols and tools for the identification and analysis of rare SUMOylated proteins.
One common strategy to work-around some of these issues is to overexpress tagged versions of SUMO along with a putative target protein in cells and evaluate target modification either by immunoblotting or immunoprecipitation followed by immunoblotting. To further boost SUMOylation, parts of the SUMOylation machinery (for example, the E2-conjugating enzyme Ubc9 or PIAS E3 ligases) can be coexpressed. Although these strategies are useful to test whether proteins can, in principle, be SUMOylated and can help in the development of SUMOylation-deficient protein variants, they obviously provide little insight into the endogenous regulation of target SUMOylation and are limited to transfectable material and genetically modifiable organisms.
The generation of affinity reagents, whether antibodies or aptamers, has remained a critical bottleneck in SUMOylation research. In contrast to ubiquitin neither antibodies nor aptamers against SUMO have been deeply explored. This is perhaps due to the poor capacity of the first reported antibodies to immunoprecipitate SUMO-modified proteins. Researchers have instead turned to HA, FLAG and Myc tagged versions of SUMO to pull-down SUMO conjugates or analysed SUMO through its inhibition in an attempt to determine function. Despite the dearth of investment in SUMO antibodies and aptamers, affinity reagents for use in this area are now available in the form of Affimer reagents. 
Affimer technology has already been shown to be successful in SUMOylation research, with binders to human SUMO-1 and SUMO-2 proving to be the first truly novel isoform-specific SUMO/SUMO Interaction Motif-dependent inhibitors. These binders have been characterised by both ITC and co-crystallisation with the SUMO target and been shown to express intracellularly to co-localise with SUMO. What’s more the people who’ve tried our SUMO Affimer reagents are delighted with the results and aren’t looking back to the antibodies and aptamers of old.
Dr David Hughes of St. Andrews University recently told us: “My laboratory studies SUMO-dependent protein-protein interactions (PPIs), which is complicated by the existence of several, highly similar isoforms of SUMO. We turned to Affimer technology to develop isoform-specific inhibitors of SUMO-PPIs, which has transformed our ability to understand these essential cellular processes. Very rapidly, we were able to generate a range of reagents that we’ve used in vitro, in cell-based assays and in X-ray crystallography studies to gain an understanding of SUMO-PPIs at the atomic level”.