Data from: Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms

SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active VSG expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a His-HA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes such as DNA replication and repair, transcription, rRNA biogenesis, and chromatin remodelling, among others.

SUMO化修饰（SUMOylation）是真核生物中保守存在的一类重要翻译后修饰。在布氏锥虫（Trypanosoma brucei）中，SUMO蛋白在前循环期（procyclic form）和血流期（bloodstream form）虫体中均为必需因子。此外，SUMO与抗原变异过程密切相关，近期研究在活性可变表面糖蛋白（Variant Surface Glycoprotein, VSG）表达位点的染色质结合蛋白中，鉴定到一个高度SUMO化的染色质焦点。本研究旨在建立一套可靠的策略，用于鉴定布氏锥虫中的SUMO结合物。我们从内源基因座中表达了多种带有标签的SUMO变体。其中，组氨酸-血凝素标签融合的布氏锥虫SUMO（His-HA-TbSUMO）可用于验证标签的功能，但经亲和纯化后，SUMO结合物的富集效果不佳，难以有效去除杂蛋白污染。为减少赖氨酸特异性内切酶Lys-C酶切产生的杂污染，我们构建了赖氨酸缺陷型SUMO变体，该变体虽解决了杂污染问题，但无法实现大量SUMO化位点的定位鉴定。后续利用该细胞系证实，多聚SUMO链对寄生虫的存活并非必需。最终，我们采用His-HA-TbSUMOT106K变体实现了SUMO结合物的高效纯化，经Lys-C酶切后，通过特异性抗体富集二甘氨酸-赖氨酸（diGly-Lys）肽段。基于该位点特异性蛋白质组学策略，我们明确鉴定出45个SUMO化蛋白与53个SUMO化受体位点。SUMO化蛋白主要参与细胞核内的诸多生物学过程，包括DNA复制与修复、转录调控、核糖体RNA（rRNA）生物发生以及染色质重塑等。