title: Identification of enzymes involved in SUMOylation in Trypanosoma brucei.
abstract: Small ubiquitin-like modifier (SUMO), a reversible post-translational protein modifier, plays important roles in diverse cellular mechanisms. Three enzymes, E1 (activating enzyme), E2 (conjugating enzyme) and E3 (ligase), are involved in SUMO modification. SUMOylation system and process in higher eukaryotes have been well studied. However, in protozoa, such as Trypanosoma brucei (T. brucei), these remain poorly understood. Herein, we identified the E1 (TbAos1/TbUba2) and E2 (TbUbc9) enzymes of SUMOylation pathway in T. brucei by sequence analysis and GST pull-down assay. Furthermore, we successfully reconstructed the SUMOylation system in vitro with recombinant enzymes. Using this system, the active site of TbUba2 and TbUbc9 was revealed to be located at Cys343 and Cys132, respectively, and a centrin homologue (TbCentrin3) was identified to be a target of SUMOylation in T. brucei. Altogether, our results demonstrate that TbAos1/TbUba2 and TbUbc9 are the bona fide E1 and E2 enzymes of the SUMOylation system in T. brucei.
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China.
authors.forename: Kaiqin
authors.initials: K
authors.lastname: Ye
authors.affiliation: 1] School of Life Sciences, Anhui University, Hefei, Anhui 230039, P.R. China [2] Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, 111 Jiulong Road, Hefei, Anhui 230601, P.R. China.
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Ni
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China.
authors.forename: Shanhui
authors.initials: S
authors.lastname: Liao
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Tu
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D058207:Sumoylation; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; D003545:Cysteine