Qiu Jiazhang, Luo Zhao-Qing
Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.
Department of Biological Sciences, Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN, USA.
Methods Mol Biol. 2019;1921:267-276. doi: 10.1007/978-1-4939-9048-1_17.
Protein ubiquitination is one of the most prevalent posttranslational modifications; it regulates a wide range of critical cellular processes in eukaryotes. This modification occurs by covalent attachment of the ubiquitin molecule to other proteins via an isopeptide bond in reactions typically catalyzed by sequential actions of three enzymes, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Ubiquitination is a reversible process catalyzed by a group of proteins known as deubiquitinase (DUB), which specifically cleaves the isopeptide bond between ubiquitin and modified proteins. Recently, a novel form of ubiquitination catalyzed by the SidE family of effectors from the bacterial pathogen Legionella pneumophila was reported. These proteins ubiquitinate structurally diverse host proteins such as reticulons and ER-associated Rab small GTPases by a two-step mechanism that uses NAD as the energy source for ubiquitin activation prior to being transferred to serine residues in target proteins. This process bypasses the need for E1 and E2 enzymes. Intriguingly, ubiquitination induced by SidEs is regulated by SidJ, another L. pneumophila effector protein which reverses the modification by functioning as an unconventional DUB. Here, we summarize the experimental details of Rab small GTPases (use Rab33b as an example) ubiquitination catalyzed by SidEs (use SdeA as an example) as well as deubiquitination catalyzed by SidJ.
蛋白质泛素化是最普遍的翻译后修饰之一；它调节真核生物中广泛的关键细胞过程。这种修饰通过泛素分子经由异肽键与其他蛋白质共价连接而发生，该反应通常由三种酶（包括泛素激活酶（E1）、泛素结合酶（E2）和泛素连接酶（E3））的顺序作用催化。泛素化是一个由一组称为去泛素酶（DUB）的蛋白质催化的可逆过程，其特异性切割泛素与修饰蛋白质之间的异肽键。最近，报道了一种由细菌病原体嗜肺军团菌的SidE家族效应蛋白催化的新型泛素化形式。这些蛋白质通过两步机制使结构多样的宿主蛋白（如网质蛋白和内质网相关的Rab小GTP酶）泛素化，该机制在将泛素转移到靶蛋白中的丝氨酸残基之前，使用NAD作为泛素激活的能量来源。这个过程不需要E1和E2酶。有趣的是，SidEs诱导的泛素化受另一种嗜肺军团菌效应蛋白SidJ调节，SidJ作为一种非常规的DUB发挥作用，使这种修饰发生逆转。在这里，我们总结了由SidEs（以SdeA为例）催化的Rab小GTP酶（以Rab33b为例）泛素化以及由SidJ催化的去泛素化的实验细节。
