Deubiquitination of phosphoribosyl-ubiquitin conjugates by phosphodiesterase-domain–containing Legionella effectors

Liu

et al. 2020

Preprint

28Legionella pneumophila extensively modulates the host ubiquitin network to create the 29 Legionella-containing vacuole (LCV) for its replication. Many of its virulence factors function 30 as ubiquitin ligases or deubiquitinases (DUBs). Here we identified Lem27 as a DUB that 31 displays a preference for diubiquitin formed by K6, K11 or K48. Lem27 is associated with 32 the LCV where it regulates Rab10 ubiquitination in concert with SidC and SdcA, two 33 bacterial E3 ubiquitin ligases. Structural analysis of the complex formed by an active 34 fragment of Lem27 and the substrate-based suicide inhibitor ubiquitin-propargylamide (PA) 35 reveals that it harbors a fold resembling those in the OTU1 DUB subfamily with a Cys-His 36 catalytic dyad and that it recognizes ubiquitin via extensive hydrogen bonding at six contact 37 sites. Our results establish Lem27 as a deubiquitinase that functions to regulate protein 38 ubiquitination on L. pneumophila phagosomes by counteracting the activity of bacterial 39 ubiquitin E3 ligases.40 41 The Gram negative bacterium Legionella pneumophila is an opportunistic pathogen 42 ubiquitously found in natural and man-made water systems, often by association with 43 amoebae species 1 . Infection of humans by L. pneumophila occurs when susceptible 44 individuals inhale contaminated aerosols which introduce the bacteria to the lung where 45 alveolar macrophages engulf them by phagocytosis 2 . The bacterial phagosome called the 46 Legionella-containing vacuole (LCV) initiates a trafficking route that bypasses the endocytic 47 maturation pathway 3 . Instead, it appears to intercept vesicles originating from the 48 endoplasmic reticulum (ER) and is eventually converted into a compartment whose 49 membranes resemble those of the ER 4,5 . 50 51 The conversion of the plasma membranes from nascent phagosomes into the LCV with 52 ER properties largely is mediated by virulence factors delivered into host cells via the 53 Dot/Icm type IV secretion system of L. pneumophila 6 . These virulence factors，also called 54 effectors， interfere with such diverse cellular processes as membrane trafficking, autophagy, 55 protein translation and immunity by diverse mechanisms 7 . The modulation of these 56 processes is mediated by targeting key regulatory proteins via effector-induced 57 posttranslational modifications, including phosphorylation 8 , methylation 9 , 58 phosphorylcholination 10,11 , AMPylation 12,11,13 and ubiquitination 14 or by subverting the 59 metabolism of lipids key in cell signaling 15 , including the production of phosphatidylinositol-60 4-phosphate (PI4P) on the LCV 16 and the removal of phosphatidylinositol-3-phosphate from 61 distinct organelles by phospholipase 17 or PI phosphatases 18 . 62 63 Modulation of the ubiquitin network has emerged as an important theme in interactions 64 between L. pneumophila and its hosts. More than 10 Dot/Icm effectors have been found to 65 function as ubiquitin E3 ligases via various mechanisms 14 . Some of these proteins possess 66 structural domains s...

Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Interplay between bacterial deubiquitinase and ubiquitin E3 ligase regulates ubiquitin dynamics onLegionellaphagosomes

Liu

et al. 2020

Preprint

Front. Cell. Infect. Microbiol.

“…Recently, two paralogues of L. pneumophila effectors, DupA and DupB, that possess the PDE domain, were identified as enzymes that can specifically reverse unconventional ubiquitination mediated by SidEs (Wan et al, 2019a;Shin et al, 2020b) ( Figure 1B). The PDE domain is conserved in nine effectors of the Philadelphia strain of L. pneumophila; SidE, SdeA, SdeB, SdeC, DupA, DupB (SdeD), Lpg1496, Lpg2239, and Lpg2523 (Wan et al, 2019a). DupA and DupB are relatively small proteins, consisting of only the PDE domain.…”

Section: Dupa and Dupbmentioning

confidence: 99%

“…The enzymatic activities of DupA and DupB have been shown to suppress the Golgi fragmentation (Wan et al, 2019a) that has been reported to be caused by SdeA (Jeong et al, 2015). Furthermore, the proteomic identification of host proteins subjected to PR-ubiquitination was conducted utilizing a dupA and dupB double knock-out L. pneumophila strain (Wan et al, 2019a;Shin et al, 2020b).…”

Section: Dupa and Dupbmentioning

confidence: 99%

Divergence of Legionella Effectors Reversing Conventional and Unconventional Ubiquitination

Kitao

Nagai

Kubori

2020

The intracellular bacterial pathogen Legionella pneumophila employs bacteria-derived effector proteins in a variety of functions to exploit host cellular systems. The ubiquitination machinery constitutes a crucial eukaryotic system for the regulation of numerous cellular processes, and is a representative target for effector-mediated bacterial manipulation. L. pneumophila transports over 300 effector proteins into host cells through its Dot/Icm type IV secretion system. Among these, several effector proteins have been found to function as ubiquitin ligases, including unprecedented enzymes that catalyze ubiquitination through unconventional mechanisms. Recent studies have identified many L. pneumophila effector proteins that can interfere with ubiquitination. These effectors include proteins that are distantly related to the ovarian tumor protein superfamily described as deubiquitinases (DUBs), which regulate important signaling cascades in human cells. Intriguingly, L. pneumophila DUBs are not limited to enzymes that exhibit canonical DUB activity. Some L. pneumophila DUBs can catalyze the cleavage of the unconventional linkage between ubiquitin and substrates. Furthermore, novel mechanisms have been found that adversely affect the function of specific ubiquitin ligases; for instance, effector-mediated posttranslational modifications of ubiquitin ligases result in the inhibition of their activity. In the context of L. pneumophila infection, the existence of enzymes that reverse ubiquitination primarily relates to a fine tuning of biogenesis and remodeling of the Legionella-containing vacuole as a replicative niche. The complexity of the effector arrays reflects sophisticated strategies that bacteria have adopted to adapt their host environment and enable their survival in host cells. This review summarizes the current state of knowledge on the divergent mechanisms of the L. pneumophila effectors that can reverse ubiquitination, which is mediated by other effectors as well as the host ubiquitin machinery.

Development of ADPribosyl Ubiquitin Analogues to Study Enzymes Involved in Legionella Infection

Misra

González

et al. 2020

Chemistry A European J

Legionnaires’ disease is caused by infection with the intracellularly replicating Gram‐negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole‐linked UbADPr, and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr. The inhibitory effects of modified Ub on the canonical eukaryotic E1‐enzyme Uba1 are investigated and rationalized in the context of a high‐resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull‐down overexpressed DupA from cell lysate.