Inactive and Active States of the Interferon-inducible Resistance GTPase, Irga6, in Vivo

Papic, Natasa; Hunn, Julia P.; Pawlowski, Nikolaus; Zerrahn, Jens; Howard, Jonathan C.

doi:10.1074/jbc.m804846200

Cited by 51 publications

(86 citation statements)

References 31 publications

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“…We used bioinformatic tools to predict transmembrane regions of IRG proteins, which might provide insights into their ability to recruit to membrane compartments. We found that the IRG family members, including IRGB10, contain two putative myristoylation sites at the amino terminus region (Figure S5B), consistent with previous studies reporting the presence of a myristoylation motif in IRGB10 and IRGA6 (also known as IIGP1) (Haldar et al, 2013; Martens et al, 2004; Papic et al, 2008). We further identified two putative transmembrane helices on the carboxyl terminus of IRGB10 and several other IRG proteins, of which putative transmembrane helix 1 is an amphipathic helix and predicted to show antimicrobial potential (Figures S5C and S5D).…”

Section: Resultssupporting

confidence: 91%

IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3 Inflammasomes

Man

Karki

Sasai

et al. 2016

Cell

255

292

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SUMMARY The inflammasome is an intracellular signaling complex, which on recognition of pathogens and physiological aberration, drives activation of caspase-1, pyroptosis, and the release of the pro-inflammatory cytokines IL-1β and IL-18. Bacterial ligands must secure entry into the cytoplasm to activate inflammasomes, however, the mechanism by which concealed ligands are liberated in the cytoplasm have remained unclear. Here, we showed that the interferon-inducible protein IRGB10 is essential for activation of the DNA-sensing AIM2 inflammasome by Francisella novicida, and contributed to the activation of the LPS-sensing caspase-11 and NLRP3 inflammasome by Gram-negative bacteria. IRGB10 directly targeted cytoplasmic bacteria through a mechanism requiring guanylate-binding proteins. Localization of IRGB10 to the bacterial cell membrane compromised bacterial structural integrity and mediated cytosolic release of ligands for recognition by inflammasome sensors. Overall, our results reveal IRGB10 as part of a conserved signaling hub at the interface between cell-autonomous immunity and innate immune sensing pathways.

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Section: Resultssupporting

confidence: 91%

IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3 Inflammasomes

Man

Karki

Sasai

et al. 2016

Cell

255

292

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“…Immunoreagents used in this study were 3E2 mouse monoclonal antibody against ROP5 isoforms (Leriche and Dubremetz, 1991), anti‐ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity‐purified rabbit sera 87558 against (pT108)Irga6 (Steinfeldt et al ., 2010), 10D7 and 10E7 mouse monoclonal antibodies (Papic et al ., 2008) or 165 rabbit antiserum (Martens et al ., 2004) against Irga6, B34 mouse monoclonal antibody (Carlow et al ., 1998) against Irgb6, 940/6 rabbit antiserum (unpublished) against Irgb10, 2078 rabbit antiserum (Martens et al ., 2004) against Irgd, 3.1.2 and 2.4.21 rat monoclonal antibodies against T. gondii GRA7 (unpublished), and rabbit anti‐calnexin antiserum (Calbiochem).…”

Section: Methodsmentioning

confidence: 99%

TheToxoplasma gondiirhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

Hermanns

Müller

Könen‐Waisman

et al. 2015

Cellular Microbiology

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SummaryIn mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.

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“…Parasites were allowed to invade for 30 min at 37°C after which the coverslips were washed 3 times with PBS, fixed with 4% formaldehyde PBS, and permeabilized with 0.05% Triton X-100 in PBS. Coverslips were blocked in 10% goat serum (Life Technologies) PBS and then stained with the mouse monoclonal antibody 10D7, which recognized GTP-bound Irga6 59 , at a 1:500 dilution and the rabbit polyclonal anti-TgAldolase 60 at 1:1,000. Alexa-fluor conjugated secondary antibodies were applied at 1:1,000 and counterstained with 0.1 μg/ml Hoechst 33258.…”

Section: Methodsmentioning

confidence: 99%

Identification of Small Molecule Inhibitors That Block the Toxoplasma gondii Rhoptry Kinase ROP18

Simpson

Jones²,

Hull-Ryde

et al. 2016

ACS Infect. Dis.

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The protozoan parasite Toxoplasma gondii secretes a family of serine-threonine protein kinases into its host cell in order to disrupt signaling and alter immune responses. One prominent secretory effector is the rhoptry protein 18 (ROP18), a serine-threonine kinase that phosphorylates immunity related GTPases (IRGs) and hence blocks interferon gamma-mediated responses in rodent cells. Previous genetic studies show that ROP18 is a major virulence component of T. gondii strains from North and South America. Here, we implemented a high throughput screen to identify small molecule inhibitors of ROP18 in vitro and subsequently validated their specificity within infected cells. Although ROP18 was not susceptible to many kinase-directed inhibitors that affect mammalian kinases, the screen identified several sub micromolar inhibitors that belong to three chemical scaffolds: oxindoles, 6-azaquinazolines, and pyrazolopyridines. Treatment of interferon gamma-activated cells with one of these inhibitors enhanced immunity related GTPase recruitment to wild type parasites, recapitulating the defect of Δrop18 mutant parasites, consistent with targeting ROP18 within infected cells. These compounds provide useful starting points for chemical biology experiments or as leads for therapeutic interventions designed to reduce parasite virulence.

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Inactive and Active States of the Interferon-inducible Resistance GTPase, Irga6, in Vivo

Cited by 51 publications

References 31 publications

IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3 Inflammasomes

IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3 Inflammasomes

TheToxoplasma gondiirhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

Identification of Small Molecule Inhibitors That Block the Toxoplasma gondii Rhoptry Kinase ROP18

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