Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.
Key Laboratory of Innate Immune Biology of Fujian Province, Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China; Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao 266237, China.
J Biol Chem. 2020 Feb 7;295(6):1646-1657. doi: 10.1074/jbc.RA119.011758. Epub 2020 Jan 6.
is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the -containing vacuole (LCV) that permits intracellular replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63-linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63-chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. deletion increased the association of Lys-63-linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63-linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of and Ceg23's role in bacterial virulence.
是肺部疾病军团病的病原体，它调节宿主功能，创造一个被称为含有空泡（LCV）的小生境，允许细胞内复制。这种调节的一个重要方面是宿主泛素网络与一组效应蛋白的协同作用。在这里，我们使用重组表达和纯化的蛋白质、分析超速离心、结构分析和计算建模，以及去泛素化酶（DUB）和细菌感染实验，发现细菌细胞器运输/细胞内增殖效应物 Ceg23 是卵巢肿瘤（OTU）DUB 家族的一员。我们发现 Ceg23 对 Lys-63 连接的多泛素链具有高度特异性，并且定位于 LCV 上，在那里它从 Lys-63 链型泛素化的蛋白质上去除泛素部分。对缺乏两个假定跨膜结构域的 Ceg23 变体的晶体结构进行分析，分辨率为 2.80 Å，结果表明，尽管在一级序列水平上与已建立的 OTU 家族成员非常有限的同源性，但 Ceg23 具有类似于典型 OTU 型 DUB 相关的催化基序。Ceg23 的缺失增加了 Lys-63 连接的多泛素与细菌吞噬体的结合，表明 Ceg23 调节 LCV 上 Lys-63 连接的泛素信号。总之，我们的研究结果表明，Ceg23 有助于调节 Lys-63 型多泛素与吞噬体的结合。未来鉴定 Ceg23 靶向的宿主底物，可以阐明这些多泛素链在和 Ceg23 在细菌毒力中的作用。
