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 <em>Trypanosoma brucei</em>, 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 <em>T. brucei</em>. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-<em>Tb</em>SUMO 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-<em>Tb</em>SUMOT106K 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重要翻译后修饰（post-translational modification）。在布氏锥虫（Trypanosoma brucei）中，SUMO在其循环前期型与血流型阶段均为不可或缺的因子。此外，SUMO与抗原变异（antigenic variation）过程紧密关联，近期研究在活性VSG表达位点（VSG expression site）的染色质相关蛋白（chromatin-associated proteins）中发现了高度SUMO化的富集区域。本研究旨在建立一套可靠的实验策略，以鉴定布氏锥虫中的SUMO结合底物。我们从内源基因座（endogenous locus）中表达了多种带有标签的SUMO变体。组氨酸-血凝素（His-HA）标记的TbSUMO可用于验证标签功能，但经亲和纯化（affinity purification）后，SUMO结合底物的富集度不足以有效清除杂蛋白污染。为减少赖氨酸内切酶（Lys-C）消化过程中产生的杂蛋白污染，我们构建了赖氨酸缺陷型SUMO变体；该变体虽能解决上述富集不足的问题，但无法实现多数SUMO化修饰位点的定位。利用该细胞系，我们进一步证实多聚SUMO链（polySUMO chains）对寄生虫的活力并非必需。最终，组氨酸-血凝素标记的TbSUMOT106K变体可实现SUMO结合底物的高效纯化，经赖氨酸内切酶消化后，可通过特异性抗体富集二甘氨酸修饰赖氨酸肽（diGly-Lys peptides）。该位点特异性蛋白质组学策略（proteomic strategy）使我们成功鉴定出45个SUMO化修饰蛋白及53个明确的修饰受体位点（acceptor sites）。SUMO化修饰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、核糖体RNA生物发生及染色质重塑等。