Regulation of phosphoribosyl-linked serine ubiquitination by deubiquitinases DupA and DupB, Shin et al

The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacterium causing Legionnaires’ disease. Here we describe identification of two bacterial effectors (LaiE and LaiF) that actively reverse PR-ubiquitination during infection and are thus named DeUbiquitinases for PR-ubiquitination (DUPs; DupA/LaiE and DupB/LaiF). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which dictates its catalytic activity allowing DupA to predominantly cleave PR-ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we next exploited a catalytically inactive DupA mutant to trap and subsequently identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates uncovered by DupA-mediated trapping strategy provides critical insights into host-pathogen interactions during Legionella infection.

细菌SidE酶家族可催化非经典磷酸核糖连接（phosphoribosyl-linked, PR）丝氨酸泛素化，并增强嗜肺军团菌（Legionella pneumophila）——一种引发军团病（Legionnaires’ disease）的致病菌——的侵染能力。本研究鉴定出两种可在侵染过程中主动逆转PR泛素化的细菌效应因子（LaiE与LaiF），因此将其命名为PR泛素化去泛素酶（DeUbiquitinases for PR-ubiquitination, DUPs；DupA/LaiE和DupB/LaiF）。结构分析显示，DupA与SidE泛素连接酶共享高度同源的催化磷酸二酯酶（phosphodiesterase, PDE）结构域。但与SidE泛素连接酶不同的是，DupA对PR泛素化底物具有更高的亲和力，这决定了其催化活性，使DupA可主要从底物上切割PR泛素。干扰DupA与泛素的结合可使其催化活性转向SidE型连接酶。鉴于DupA对PR泛素化底物的高亲和力，本研究进一步利用催化失活的DupA突变体进行捕获，随后在军团菌侵染的细胞中鉴定出180余种PR泛素化宿主蛋白。参与内质网（endoplasmic reticulum, ER）片段化以及膜招募至含军团菌液泡（Legionella-containing vacuoles, LCV）的蛋白，是SidE的主要作用靶点。通过DupA介导的捕获策略所揭示的PR泛素化底物全局图谱，为军团菌侵染过程中的宿主-病原体相互作用研究提供了关键见解。