Legionella maintains host ubiquitin homeostasis by effectors with unique catalytic mechanisms

Legionella maintains host ubiquitin homeostasis by effectors with unique catalytic mechanisms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Legionella maintains host ubiquitin homeostasis by effectors with unique catalytic mechanisms Zhao-Qing Luo, Jiaqi Fu, Siying Li, Wei Xian, Zhengrui Zhang, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4431542/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions. Biological sciences/Biochemistry/Chemical modification Health sciences/Pathogenesis/Infection Adenylylation posttranslational modification ubiquitination bacterial toxins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files LnaBinteractingproteins.xlsx Dataset 1 8IPJMavLADPRUbValidationreport.pdf 8IPWMavLADPRValidationreport.pdf 8J9Bvalreportfullupdated.pdf Editorialpolicychecklist1.pdf FuetalSupplementaryinformation.pdf Reportingsummary1.pdf UncroppedimagingNC.pdf Cite Share Download PDF Status: Published Journal Publication published 15 Jul, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4431542","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":303639735,"identity":"0da5713a-0c6e-41f2-938f-a0344f82ee13","order_by":0,"name":"Zhao-Qing Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYBACPhDxAUQcABFsRGgBqWGcQbIWZh7StEgkH/5su+NwPt+N5AMMH8oOE6MlLcE498xhy5k30hIYZ5wjSkuOQXJu22EDgxs5Bsy8bURqOWwJ1pL/gfkvkVoMmxkhtjAwMxKlhedZMmPvmXQDyTPPDA72nEsnrIWfPfnwh587rA34jic/fPCjzJqwFjBgbIDQB4hUj6RlFIyCUTAKRgFWAACxQDoPO9myVgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8890-6621","institution":"Purdue University West Lafayette","correspondingAuthor":true,"prefix":"","firstName":"Zhao-Qing","middleName":"","lastName":"Luo","suffix":""},{"id":303639736,"identity":"1117bb43-f4fa-4648-8c92-dbf5d449a201","order_by":1,"name":"Jiaqi Fu","email":"","orcid":"https://orcid.org/0000-0003-0081-6133","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Fu","suffix":""},{"id":303639737,"identity":"c0206b58-d785-455c-a60a-710884486408","order_by":2,"name":"Siying Li","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Siying","middleName":"","lastName":"Li","suffix":""},{"id":303639738,"identity":"6bd63ff9-4757-409a-8487-6f6da9842ca6","order_by":3,"name":"Wei Xian","email":"","orcid":"https://orcid.org/0000-0002-3683-7983","institution":"Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xian","suffix":""},{"id":303639739,"identity":"892e5f9c-4733-49d6-909c-95507314aa86","order_by":4,"name":"Zhengrui Zhang","email":"","orcid":"","institution":"Purdue University","correspondingAuthor":false,"prefix":"","firstName":"Zhengrui","middleName":"","lastName":"Zhang","suffix":""},{"id":303639740,"identity":"b74f0152-6002-494d-a786-bdfdeec67aa0","order_by":5,"name":"Tao-Tao Chen","email":"","orcid":"https://orcid.org/0000-0002-0660-2841","institution":"College of Life Sciences, Fujian Normal University","correspondingAuthor":false,"prefix":"","firstName":"Tao-Tao","middleName":"","lastName":"Chen","suffix":""},{"id":303639741,"identity":"61cbd690-14f2-4d6a-a483-095e701316ae","order_by":6,"name":"Yao Liu","email":"","orcid":"https://orcid.org/0000-0002-4330-2389","institution":"Purdue University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Liu","suffix":""},{"id":303639742,"identity":"5658099f-9f27-4f40-a9fd-1e9718f16662","order_by":7,"name":"Qiuhua Lu","email":"","orcid":"","institution":"Fujian Normal 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University","correspondingAuthor":false,"prefix":"","firstName":"Jinyu","middleName":"","lastName":"Li","suffix":""},{"id":303639752,"identity":"0f8f0d64-5443-4232-9f12-3248559c0b11","order_by":17,"name":"Lina Kang","email":"","orcid":"","institution":"Fujian Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Kang","suffix":""},{"id":303639753,"identity":"4e5c4d7e-7d4d-4d98-9e20-71bda1785c6b","order_by":18,"name":"Si-Ru Zheng","email":"","orcid":"","institution":"Fujian Normal University","correspondingAuthor":false,"prefix":"","firstName":"Si-Ru","middleName":"","lastName":"Zheng","suffix":""},{"id":303639754,"identity":"37435922-5807-414f-a91b-91cd3428027c","order_by":19,"name":"Shoujing Cao","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Shoujing","middleName":"","lastName":"Cao","suffix":""}],"badges":[],"createdAt":"2024-05-16 13:55:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4431542/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4431542/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-50311-2","type":"published","date":"2024-07-15T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56746200,"identity":"072495fd-f244-422e-8428-72d3396e099b","added_by":"auto","created_at":"2024-05-20 01:56:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1314347,"visible":true,"origin":"","legend":"\u003cp\u003eMavL is a macro domain protein that converts ADPR-Ub into ADP-ribose and ubiquitin a. Hydrolysis of ADPR-Ub into ADPR and Ub by MavL DupA or DupB. Recombinant proteins were incubated with ADPR-Ub and the production of native Ub was detected by native polyacrylamide gel electrophoresis (upper panel). Native Ub, PR-Ub and ADPR- Ub were loaded separately as controls. Identical samples separated by SDS-PAGE were detected by CBB staining, phosphoprotein stain or immunoblotting with an ADPR-specific antibody (lower three panels). b. Mutational analysis of residues important for the de-ADP ribosylation activity of MavL. Recombinant MavL or its mutants were incubated with ADPR-Ub and the reduction of the reactant was detected by immunoblotting with an ADPR-specific antibody. c. Binding of Ub, ADPR-Ub and ADPR to MavL40-404 or its mutants. Binding affinity was evaluated using a low volume Nano ITC set at 20°C. d. Ribbon diagram representation of the MavL(40-404)D315A-ADPR-Ub complex. Ub, ADPR and MavL are colored in pink, yellow and cyan, respectively. The recognition of ADPR- Ub by MavL as well as the interactions between the two proteins are shown in the middle panel. The 2mFo−DFc(blue) and mFo−DFc (green) electron-density maps of the key residues of MavL, Ub and water surrounding ribose involved in forming the catalytic center are contoured at the 1.5σ and 3.0σ levels and shown in the right panel. It represents the transient state of the substrate catalyzed by MavL. The N-glyosidic bond between the side chain of R42 in Ub and the ADPR moiety was cleaved, as indicated by the red dashed lines and arrow. e. The overall structure of MavL(40-404)D315A-ADPR-Ub and its comparison to Apo MavL (gray) and MavL-ADPR (blue). The conformational changes that contribute to the opening of the catalytic pocket to facilitate the binding of ADPR-Ub and the subsequent reaction were shown. Residues that cause steric hindrance between Apo MavL and Ub were marked with red dashed circles. f. MavL reduces the level of ADPR-Ub in infected cells. The indicated L. pneumophila strains were used to infected cells expressing 3xHA-Ub and the accumulation of ADPR- Ub was detected after HA antibody immunoprecipitation. Expression of Flag-MavL and its mutant was detected with Flag antibody and isocitrate dehydrogenase (ICDH) was probed as a loading control.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4431542/v1/17aa3856ba2ab55f1919ccb9.png"},{"id":56746208,"identity":"203921fe-9809-4781-b12a-59ab9a0d1d85","added_by":"auto","created_at":"2024-05-20 01:56:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":460886,"visible":true,"origin":"","legend":"\u003cp\u003eConversion of PR-Ub into ADPR-Ub by LnaB requires Actin as a co-factor a. Native lysates of mammalian cells activate LnaB. Native (N) or boiled (B) lysates of 293HEK cells were added to reactions containing PR-Ub, MavK or LnaB and the production of ADPR-Ub was detected by immunoblotting. b. Identification of Actin as a LnaB-binding protein. Flag-LnaB expressed in HEK293T cells was isolated by immunoprecipitation and the bound proteins were identified by mass spectrometry. Similarly obtained samples with Flag-RavN were used as a control. alpha Actin (ACTC1) and beta Actin-like 2 (ACTBL2) were the among the most abundant proteins identified. c. Interactions between LnaB and Actin measured by ITC. Raw ITC curves (top panel) and binding isotherms with fitting curves (bottom panel) of LnaB titration by Actin. The thermogram is a monophasic curve with an inflection point at molar ratio of 0.84. The binding affinity is approximately 1.24 μM and the stoichiometry is 1:1 of Actin:LnaB. The thermodynamic parameters were also shown, ΔH: -6.88 kJ•mol-1 and ΔS: 88.75 J•mol- 1 •K-1 . d. LnaB and actin utilize ATP to convert PR-Ub into ADPR-Ub. Actin was added to a subset of reactions containing LnaB and PR-Ub. Samples separated by SDS-PAGE were probed for ADPR-Ub (upper panel), ubiquitin, LnaB or Actin by immunoblotting with antibodies specific for each protein or its epitope tag. e-f. LnaB transfers the AMP moiety of ATP to PR-Ub. The chemical structure of ADPR- Ub with the AMP moiety added to phosphate group on PR-Ub being highlighted (dashed box) (e). 32 P-α-ATP was added to the indicated reactions and incubated at 37o C for 1 h. Samples separated by SDS-PAGE were detected by CBB staining (left) and autoradiograph, respectively. Note the presence of self-modified LnaB in the reaction without PR-Ub (f). g. ATP analogs with a cleavable α phosphate support LnaB activity. Samples of reactions receiving the indicated ATP analogs were resolved by SDS-PAGE, and ADPR-Ub and the reactants were detected by immunoblotting by antibodies specific for ADPR, Ub, LnaB or Actin. Note that ApCpp is uncleavable at the α position thus did not support the activity of LnaB. In each case, similar results were obtained in at least three independent experiments\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4431542/v1/9ecbf50b0c9b5d7010be0b9c.png"},{"id":56746206,"identity":"7d08f35e-6a28-4479-a358-5e53fe4fdca2","added_by":"auto","created_at":"2024-05-20 01:56:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220370,"visible":true,"origin":"","legend":"\u003cp\u003eLnaB and MavL sequentially convert PR-Ub into ADPR and active ubiquitin a-b. Detection of LnaB-mediated conversion of PR-Ub into ADPR-Ub by mass spectrometric analysis. Excised protein bands from SDS-PAGE gels corresponding to PR-Ub prior to the reaction or ADPR-Ub after incubated with ATP, LnaB and Actin were digested with trypsin and analyzed by mass spectrometry. A reference fragment T12ITLEVEPSDTIENVK27 was present in both samples with similar abundance (a left panel). The abundance of the fragment with PR-modified R42 was high in the PR-Ub samples but became almost undetectable after reaction with LnaB, ATP and Actin, which was accompanied by the increase of ADPR-modified fragment. A MS/MS spectrum indicating ADPR modification of R42 was shown in b. c. A reaction scheme depicting the conversion of PR-Ub into ubiquitin by LnaB and MavL. The AMPylation activity of LnaB first converts PR-Ub into ADPR-Ub, which is further reduced into ADP-ribose and ubiquitin by MavL. The AMP moiety defined by a dash line rectangle indicates the chemical group added to PR-Ub by LnaB. d. The use of ADPR-Ub produced from PR-Ub by LnaB in protein modification by the phosphodiesterase (PDE) activity of SdeA. PR-Ub was incubated in the indicated reactions and the ability to ubiquitinate Rab33b was detected by the formation of higher MW species detected by immunoblotting with the Flag-specific antibody. Native ADPR- Ub was included as a control (1st lane). e. Conventional ubiquitination by ubiquitin produced by MavL and LnaB from PR-Ub. A series of reactions containing PR-Ub and combinations of relevant proteins were allowed to proceed for 1 h at 37°C. The products were boiled for 5 min at 95°C and a cocktail containing E1, E2, SidC (E3) and ATP was added, self-ubiquitination of SidC was detected by immunoblotting with a ubiquitin-specific antibody.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4431542/v1/af58b3a0e10ab013a0ff6ccb.png"},{"id":56746204,"identity":"e6bd9b36-96cb-4a58-89ab-12d2a1e8b526","added_by":"auto","created_at":"2024-05-20 01:56:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":376028,"visible":true,"origin":"","legend":"\u003cp\u003eThe reaction catalyzed by LnaB required an S-HxxxE motif a-b. Conversion of PR-Ub into ADPR-Ub by LnaB requires an S-HxxxE motif. Samples of reactions containing ATP, PR-Ub, LnaB, Actin, LnaB or its mutants and resolved by SDS-PAGE were detected for the production of ADPR-Ub (top). Each reactant was detected by immunoblotting with the appropriate antibodies (a). Similar reactions with 32 P- α-ATP were established, proteins were detected by CBB staining (upper) and the production of 32 P-ADPR-Ub was detected by autoradiograph (lower) (b). c-d. LnaB functions to convert PR-Ub into ADPR-Ub in cells infected with L. pneumophila. HEK293 cells transfected to express 3xHA-Ub were infected with the indicated bacterial strains (I to V). Immunoprecipitation products obtained by HA antibody from lysates of infected cells were analyzed by mass spectrometry to detect differently modified ubiquitin (c). Recombinant LnaB was added to a subset of similar prepared lysates of infected cells and the accumulation of PR-Ub was assessed by detecting LnaB-mediated ADPR-Ub production (d). e. The ratio of modified ubiquitin (PR-Ub) in cells infected with the ∆lnaB mutant. Cells expressing HA-ubiquitin was infected with strain Lp02∆lnaB for 2 h. HA-ubiquitin isolated by immunoprecipitation was analyzed by mass spectrometry to determine the ratio of modified ubiquitin. f. Overexpression of SdeA in the ∆lnaB mutant affects intracellular bacterial growth D. discoideum was infected with the indicated L. pneumophila strains and the growth of the bacteria was evaluated. Note that strain ∆lnaB(pSdeA) displayed significant defects in intracellular growth (upper panel). The expression of SdeA in the testing strains was probed by immunoblotting (lower panel). Data shown were one representative of three independent experiments done in triplicate with similar results.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4431542/v1/509b17746eafbfd9b32e744c.png"},{"id":56746211,"identity":"877f0a19-d8d0-4fad-9781-723d9735e4e7","added_by":"auto","created_at":"2024-05-20 01:56:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1206462,"visible":true,"origin":"","legend":"\u003cp\u003eSelf-AMPylation activity of members of the S-HxxxE toxin family a. LnaB self-AMPylates at Y196 and Y247. Protein bands corresponding to LnaB from the indicated reactions were analyzed to identify the modified residues by mass spectrometry. b-c. Mutations of the AMPylated Tyr residues abolished the activity of LnaB. LnaB or its mutants was incubated with 32 P-α-ATP and Actin and production of self-modified protein was detected by autoradiograph (b). Similar reactions receiving PR-Ub were established to probe the impact of the mutations on the conversion of PR-Ub into ADPR-Ub, which was detected by immunoblotting (top) and the proteins in the reactions were detected by CBB staining (lower). d. Self-AMPylation by members of the S-HxxxE family. Recombinant proteins of the indicated toxins were incubated with 32 P-α-ATP and Actin. Samples resolved by SDS- PAGE were detected for AMPylation by autoradiograph (lower) and for the proteins by CBB staining (upper). Red arrows indicated AMPylated proteins. Note that in each case, self-AMPylation required an intact S-HxxxE motif. e. Yeast toxicity by the toxins required an intact S-HXXXE motif. Serially diluted cells of yeast strains expressing the indicated toxin genes or their S-HxxxE mutants were spotted on medium containing glucose or galactose. Images were acquired after 3-day incubation at 30o C (left). The expression of the proteins was probed by immunoblotting with the Flag- specific antibody. The phosphoglycerate kinase (PGK) was probed as a loading control.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4431542/v1/678d7203ff2b378c4a5ff458.png"},{"id":56746213,"identity":"44e0cc64-61f9-4f88-b69d-07e7931545be","added_by":"auto","created_at":"2024-05-20 01:56:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1658494,"visible":true,"origin":"","legend":"\u003cp\u003eLnaB-Actin binary complex structure reveals a unique catalytic mechanism \u0026nbsp;on AMPylation \u0026nbsp;a. Cylindrical cartoon diagram representation of the LnaB-Actin complex. The top panels represent schematic diagrams of the regions for domain organization of LnaB and Actin. LnaB consists of the N-terminal domain (NTD, purple), the catalytic domain (CD, orange) and the C-terminal domain (CTD, grey); Actin is composed of NTD (green) and CTD (Limon). S261, H305 and E309 of the S-HxxxE motif are shown in red. The bottom panel shows the LnaB-Actin binary structure. Domains of LnaB and Actin were colored in accordance with the diagrams (top). The interfaces involved in LnaB-Actin interactions were highlighted in two dashed line circles. b. S261, H305 and E309 formed a platform in the structure of LnaB. Residues were represented as sticks and LnaB was depicted in surface, colored according to the electrostatic surface potential [contoured from -6kBT (red) to +6kBT (blue)]. c-d. The interfaces involved in LnaB-Actin interactions. LnaB and Actin were shown as orange and green cartoons, respectively. Residues important for binding were shown as sticks (Actin in green and LnaB in orange). Hydrogen bonds were marked by blue dashed lines. e. Optimal binding to Actin is required for maximal activity of LnaB. Indicated LnaB mutants were individually incubated with Actin, ATP, and PR-Ub for 30 min at 37o C and their activity in converting PR-Ub into ADPR-Ub was evaluated by immunoblotting with an ADPR-specific antibody. Proteins in the reactions were detected by CBB staining. f. Evaluation of the binding of Actin to LnaB and its mutants by Ni2+ beads pulldown. His6- LnaB and its mutants were individually incubated with Actin at 4o C for 6 h prior to pulldown with Ni2+ beads. Actin was detected using anti-Actin antibodies and proteins were detected by CBB staining. g-i. An ATP-binding pocket in LnaB identified by molecular docking. LnaB was displayed in a grey surface model. Residues potentially involved in binding ATP was indicated as orange sticks. ATP was shown as a cyan stick-ball model and hydrogen bonds were represented by blue dashed lines (G). LnaB mutants were evaluated for the ability to convert PR-Ub into ADPR-Ub with reactions described above. Proteins were detected by CBB staining (H). The affinity between ATP and LnaB and its mutants was determined using isothermal titration calorimetry (ITC). The binding constant (Kd) was calculated by the NanoAnayze software package. Data shown are one representative of three independent experiments with similar results (E, F, H and I).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4431542/v1/8c65dcb970dd8068e18a6292.png"},{"id":56746205,"identity":"8391ff3e-afc2-4340-9627-6d6808ca15b9","added_by":"auto","created_at":"2024-05-20 01:56:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":231732,"visible":true,"origin":"","legend":"\u003cp\u003eThe cycling of ubiquitin by Dot/Icm effectors in cells infected by L. pneumophila. Ubiquitin is converted into ADPR-Ub by the mART activity of SidEs, which is used to modified proteins by phosphoribosyl ubiquitination. The reversal of the modification produced PR-Ub, which is converted into native ubiquitin by sequential reactions catalyzed by LnaB and MavL. Note that both ADPR-Ub and PR-Ub may interfere with canonical ubiquitin signaling and that ADPR-Ub produced from PR-Ub by LnaB may be used by the PDE activity of SidEs for protein modification.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4431542/v1/d6044789e19c2228e8c4e392.png"},{"id":60380225,"identity":"521b6ed3-a64b-401c-b4b3-c529fb71d675","added_by":"auto","created_at":"2024-07-16 07:08:18","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3138258,"visible":true,"origin":"","legend":"","description":"","filename":"FuetalLnaBNCR1clean20240516.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4431542/v1_covered_4718e774-de49-4f09-8f08-9b03c0a3fb6e.pdf"},{"id":56746476,"identity":"ca11bcfb-8137-42b3-b148-74c34f122b3a","added_by":"auto","created_at":"2024-05-20 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toxins","lastPublishedDoi":"10.21203/rs.3.rs-4431542/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4431542/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions.","manuscriptTitle":"Legionella maintains host ubiquitin homeostasis by effectors with unique catalytic mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-20 01:56:48","doi":"10.21203/rs.3.rs-4431542/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6dc820da-28eb-4527-b60e-3d18dfd0e081","owner":[],"postedDate":"May 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":32054739,"name":"Biological sciences/Biochemistry/Chemical modification"},{"id":32054740,"name":"Health sciences/Pathogenesis/Infection"}],"tags":[],"updatedAt":"2024-07-16T07:08:03+00:00","versionOfRecord":{"articleIdentity":"rs-4431542","link":"https://doi.org/10.1038/s41467-024-50311-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-07-15 04:00:00","publishedOnDateReadable":"July 15th, 2024"},"versionCreatedAt":"2024-05-20 01:56:48","video":"","vorDoi":"10.1038/s41467-024-50311-2","vorDoiUrl":"https://doi.org/10.1038/s41467-024-50311-2","workflowStages":[]},"version":"v1","identity":"rs-4431542","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4431542","identity":"rs-4431542","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}