Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System.
Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System.
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DOI：
10.1371/journal.pone.0134950
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Alvarez VE
中科院分区：
文献类型：
--
作者：
Iribarren PA;Berazategui MA;Cazzulo JJ;Alvarez VE
SUMO（Small Ubiquitin-like Modifier）是真核生物中保守的蛋白质翻译后修饰，对多种细胞过程起着重要的调控作用。在布氏锥虫（Trypanosoma brucei）中，SUMO是真核生物进化中最早的分支之一，对正常细胞周期进程至关重要，并且可能参与寄生虫生存关键基因的表观遗传控制，例如编码变异表面糖蛋白的基因。蛋白质组学研究已经开始发现SUMO调控的分子途径;然而，功能后果的表征仅限于数量减少的靶点。在这里，我们提出了一个细菌菌株工程生产SUMO化的蛋白质，通过转移SUMO从T。布鲁氏菌及其活化和结合所必需的酶。由于缺乏E.该系统可用于通过免疫印迹直接在细胞裂解物中表达和鉴定SUMO化的蛋白，并且SUMO化的靶标可最终纯化用于生物化学或结构研究。我们应用这种策略来描述TbSUMO在体外形成链的能力，并检测来自酿酒酵母和T.布鲁塞。为了进一步验证靶标，我们使用T.布鲁氏菌SUMO特异性蛋白酶能够恢复修饰模式。该系统为锥虫SUMO化蛋白的靶点验证、突变体产生和功能研究提供了一个有价值的工具。
Post-translational modification with the Small Ubiquitin-like Modifier (SUMO) is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids.