Departamento de Ciências Biológicas, Núcleo de Pesquisas em Ciências Biológicas, Universidade Federal de Ouro Preto, Morro do Cruzeiro, Ouro Preto, MG, Brazil.
Parasitol Res. 2011 Dec;109(6):1537-46. doi: 10.1007/s00436-011-2394-4. Epub 2011 May 15.
SUMO-dependent post-translational modification is implicated in a variety of cellular functions including gene expression regulation, nuclear sub-localization, and signal transduction. Conjugation of SUMO to other proteins occurs in a similar process to ubiquitination, which involves three classes of enzymes: an E1 activating, an E2 conjugating, and an E3 target-specific ligase. Ubc9 is the unique SUMO E2 enzyme known to conjugate SUMO to target substrates. Here, we present the molecular characterization of this enzyme and demonstrate its expression profile during the S. mansoni life cycle. We have used bioinformatic approaches to identify the SUMO-conjugating enzyme, the SmUbc9-like protein, in the Schistosoma mansoni databases. Quantitative RT-PCR was employed to measure the transcript levels of SUMO E2 in cercariae, adult worms, and in vitro cultivated schistosomula. Furthermore, recombinant SmUbc9 was expressed using the Gateway system, and antibodies raised in rats were used to measure SmUbc9 protein levels in S. mansoni stages by Western blotting. Our data revealed upregulation of the SmUbc9 transcript in early schistosomula followed by a marked differential gene expression in the other analyzed stages. The protein levels were maintained fairly constant suggesting a post-transcriptional regulation of the SmUbc9 mRNA. Our results show for the first time that S. mansoni employs a functional SUMO E2 enzyme, for the conjugation of the SUMO proteins to its target substrates.
SUMO 依赖性翻译后修饰参与多种细胞功能，包括基因表达调控、核亚定位和信号转导。SUMO 与其他蛋白质的缀合过程类似于泛素化，涉及三类酶：E1 激活酶、E2 连接酶和 E3 靶标特异性连接酶。Ubc9 是已知将 SUMO 缀合到靶标底物的唯一 SUMO E2 酶。在这里，我们介绍了这种酶的分子特征，并证明了其在 S. mansoni 生命周期中的表达谱。我们使用生物信息学方法在曼氏血吸虫数据库中鉴定了 SUMO 连接酶 SmUbc9 样蛋白。定量 RT-PCR 用于测量在尾蚴、成虫和体外培养的曼氏血吸虫幼体中 SUMO E2 的转录水平。此外，使用 Gateway 系统表达了重组 SmUbc9，并使用大鼠产生的抗体通过 Western blot 测量 SmUbc9 蛋白水平在曼氏血吸虫各阶段的表达。我们的数据显示，SmUbc9 转录物在早期幼体中上调，随后在其他分析阶段出现明显的差异基因表达。蛋白水平保持相当稳定，表明 SmUbc9 mRNA 受到转录后调控。我们的结果首次表明，S. mansoni 采用功能性 SUMO E2 酶，将 SUMO 蛋白缀合到其靶标底物上。
