Is Transthyretin a Regulator of Ubc9 SUMOylation?

Ageing and mutations of transthyretin (TTR), the thyroid hormones and retinol transporting protein lead to amyloidosis by destabilizing the structure of TTR. Because protein structure is regulated through posttranslational modifications, we investigated the Small Ubiquitin-like Modifier (SUMO)ylation of TTR. We chose the widely used Ubc9 fusion-directed SUMOylation system, which is based on a fusion of the SUMOylation substrate of interest with Ubc9, a sole SUMO conjugating enzyme. Surprisingly, despite our presumptions, we found that Ubc9 fused to TTR was SUMOylated at a unique set of lysine residues. Three unknown SUMOylation sites of Ubc9-K154, K18 and K65-were revealed by mass spectrometry (MS). The previously reported SUMOylation at K49 of Ubc9 was also observed. SUMOylation of the lysine residues of TTR fused to Ubc9 was hardly detectable. However, non-fused TTR was SUMOylated via trans-SUMOylation by Ubc9 fused to TTR. Interestingly, mutating the catalytic residue of Ubc9 fused to TTR did not result in complete loss of the SUMOylation signal, suggesting that Ubc9 linked to TTR is directly cross-SUMOylated by the SUMO-activating enzyme E1. Ubc9, TTR or fusion proteins composed of TTR and Ubc9 specifically affected the global SUMOylation of cellular proteins. TTR or Ubc9 alone increased global SUMOylation, whereas concomitant presence of TTR and Ubc9 did not further increase the amount of high-molecular weight (HMW) SUMO conjugates. Our data suggest that TTR may influence the SUMOylation of Ubc9, thereby altering signalling pathways in the cell.

Multidomain sumoylation of the ecdysone receptor (EcR) from Drosophila melanogaster.

The 20-hydroxyecdysone receptor (EcR) is a transcription factor belonging to the nuclear receptor superfamily. Together with the ultraspiracle nuclear receptor (Usp) it coordinates critical biological processes in insects such as development and reproduction. EcR and its ligands are used in commercially available ecdysone-inducible expression systems and are considered to be artificial gene switches with potential therapeutic applications. However, the regulation of EcR action is still unclear, especially in mammals and as far as posttranslational modifications are concerned. Up until now, there has been no study on EcR sumoylation. Using bioinformatic predictors, a Ubc9 fusion-directed sumoylation system and mutagenesis experiments, we present EcR as a new target of SUMO1 and SUMO3 modification. Our research revealed that EcR undergoes isoform-specific multisumoylation. The pattern of modification remains unchanged in the presence of the ligand and the dimerization partner. The SUMO acceptor sites are located in the DNA-binding domain and the ligand-binding domain that both exhibit structural plasticity. We also demonstrated the existence of a sumoylation site in the F region and EcRA-A/B region, both revealing characteristics of intrinsically disordered regions. The consequences of modification and the resulting impact on conformation and function may be especially crucial for the disordered sequences in these two areas. The isoform-specificity of sumoylation may explain the differences in the transcriptional activity of EcR isoforms.

Alternative sumoylation sites in the Drosophila nuclear receptor Usp.

The ultraspiracle protein (Usp), together with an ecdysone receptor (EcR) forms a heterodimeric ecdysteroid receptor complex, which controls metamorphosis in Drosophila melanogaster. Although the ecdysteroid receptor is considered to be a source of elements for ecdysteroid inducible gene switches in mammals, nothing is known about posttranslational modifications of the receptor constituents in mammalian cells. Up until now there has been no study about Usp sumoylation. Using Ubc9 fusion-directed sumoylation system, we identified Usp as a new target of SUMO1 and SUMO3 modification. Mutagenesis studies on the fragments of Usp indicated that sumoylation can occur alternatively on several defined Lys residues, i.e. three (Lys16, Lys20, Lys37) in A/B region, one (Lys424) in E region and one (Lys506) in F region. However, sumoylation of one Lys residue within A/B region prevents modification of other residues in this region. This was also observed for Lys residues in carboxyl-terminal fragment of Usp, i.e. comprising E and F regions. Mass spectrometry analysis of the full-length Usp indicated that the main SUMO attachment site is at Lys20. EcR, the heterodimerization partner of Usp, and muristerone A, the EcR ligand, do not influence sumoylation patterns of Usp. Another heterodimerization partner of Usp - HR38 fused with Ubc9 interacts with Usp in HEK293 cells and allows sumoylation of Usp independent of the direct fusion to Ubc9. Taken together, we propose that sumoylation of DmUsp can be an important factor in modulating its activity by changing molecular interactions.