title: Is Transthyretin a Regulator of Ubc9 SUMOylation?
abstract: 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.
authors: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, Wrocław, Poland.
authors: Elżbieta
authors: http://orcid.org/0000-0003-1420-1439
authors: E
authors: Wieczorek
authors: Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors: Sylwia
authors: S
authors: Kędracka-Krok
authors: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, Wrocław, Poland.
authors: Katarzyna
authors: K
authors: Sołtys
authors: Department of Structural Biology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
authors: Urszula
authors: U
authors: Jankowska
authors: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, Wrocław, Poland.
authors: Rafał
authors: R
authors: Hołubowicz
authors: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, Wrocław, Poland.
authors: Justyna
authors: J
authors: Seliga
authors: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, Wrocław, Poland.
authors: Andrzej
authors: A
authors: Ożyhar
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011228:Prealbumin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D058207:Sumoylation; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D011228:Prealbumin; D011993:Recombinant Fusion Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine