Dataset Information

Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection.

ABSTRACT: Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC_14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host-pathogen interactions.

SUBMITTER: Shin D

PROVIDER: S-EPMC7690952 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection.

Shin Donghyuk D Bhattacharya Anshu A Cheng Yi-Lin YL Alonso Marta Campos MC Mehdipour Ahmad Reza AR van der Heden van Noort Gerbrand J GJ Ovaa Huib H Hummer Gerhard G Dikic Ivan I

eLife 20201113

Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at ...[more]

PMID: 33185526

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Project description:Legionella spp. are the causative agents of a severe pneumonia known as Legionnaires' disease. Upon being engulfed by host cells, these environmental bacteria replicate intracellularly in a plasma membrane-derived niche termed Legionella-containing vacuole (LCV) in a way that requires the defective in organelle trafficking/intracellular multiplication (Dot/Icm) protein transporter. Our understanding of interactions between Legionella and its hosts was mostly based on studies of Legionella pneumophila. In this study, we found that the LCVs created by virulent Legionella longbeachae are similarly decorated by polyubiquitinated proteins to those formed by L. pneumophila and that the ubiquitin-proteasome system (UPS) is required for optimal intracellular growth of L. longbeachae. Furthermore, we utilized bioinformatics methods and the ubiquitin-vinylmethyl ester probe to obtain potential deubiquitinases (DUBs) encoded by L. longbeachae. These efforts led to the identification of 9 L. longbeachae DUBs that displayed distinct specificity toward ubiquitin chain types. Among these, LLO_1014 and LLO_2238 are associated with the LCVs and impact the accumulation of polyubiquitinated species on the bacterial phagosome. Moreover, LLO_1014 and LLO_2238 could fully restore the phenotypes associated with Δceg23 (lotB) and Δlem27 (lotC) mutants of L. pneumophila, indicating that these DUBs have similar functions. Together, these results reveal that L. longbeachae uses multiple DUBs to construct an intracellular niche for its replication. IMPORTANCE Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires' disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires' disease worldwide. However, our knowledge about the interactions between L. longbeachae and its hosts is very limited. Here, we found that, similar to L. pneumophila infection, the host ubiquitin proteasome system is also important for the intracellular replication of L. longbeachae. In addition, the bacterial phagosomes harboring L. longbeachae are enriched with polyubiquitinated proteins in a Dot/Icm system-dependent manner. We further identified 9 L. longbeachae proteins that function as DUBs with distinct ubiquitin chain specificity. Of note, several of the phagosome-associated L. longbeachae DUBs regulate the recruitment of polyubiquitinated proteins to the LCV.

Project description:ObjectivePlatelets express a functional ubiquitin-proteasome system. Mass spectrometry shows that platelets contain several deubiquitinases, but whether these are functional, modulate the proteome, or affect platelet reactivity are unknown.Approach and resultsPlatelet lysates contained ubiquitin-protein deubiquitinase activity hydrolyzing both Lys48 and Lys63 polyubiquitin conjugates that was suppressed by the chemically unrelated deubiquitinase inhibitors PYR41 and PR619. These inhibitors acutely and markedly increased monoubiquitination and polyubiquitination of the proteome of resting platelets. PYR41 (intravenous, 15 minutes) significantly impaired occlusive thrombosis in FeCl3-damaged carotid arteries, and deubiquitinase inhibition reduced platelet adhesion and retention during high shear flow of whole blood through microfluidic chambers coated with collagen. Total internal reflection microscopy showed that adhesion and spreading in the absence of flow were strongly curtailed by these inhibitors with failure of stable process extension and reduced the retraction of formed clots. Deubiquitinase inhibition also sharply reduced homotypic platelet aggregation in response to not only the incomplete agonists ADP and collagen acting through glycoprotein VI but also to the complete agonist thrombin. Suppressed aggregation was accompanied by curtailed procaspase activating compound-1 binding to activated IIb/IIIa and inhibition of P-selectin translocation to the platelet surface. Deubiquitinase inhibition abolished the agonist-induced spike in intracellular calcium, suppressed Akt phosphorylation, and reduced agonist-stimulated phosphatase and tensin homolog phosphatase phosphorylation. Platelets express the proteasome-associated deubiquitinases USP14 and UCHL5, and selective inhibition of these enzymes by b-AP15 reproduced the inhibitory effect of the general deubiquitinase inhibitors on ex vivo platelet function.ConclusionsRemodeling of the ubiquitinated platelet proteome by deubiquitinases promotes agonist-stimulated intracellular signal transduction and platelet responsiveness.

Dataset Information

Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection.

Publications

Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection.

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