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• W2469403673 abstract "Article1 July 2016Open Access Transparent process Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2-dependent antibacterial autophagy Teresa LM Thurston Teresa LM Thurston Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK Search for more papers by this author Keith B Boyle Keith B Boyle Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Mark Allen Mark Allen Division of Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Benjamin J Ravenhill Benjamin J Ravenhill Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Maryia Karpiyevich Maryia Karpiyevich Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Stuart Bloor Stuart Bloor Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Annie Kaul Annie Kaul orcid.org/0000-0002-9965-8026 Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jessica Noad Jessica Noad Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Agnes Foeglein Agnes Foeglein Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Sophie A Matthews Sophie A Matthews MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK Search for more papers by this author David Komander David Komander Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Mark Bycroft Mark Bycroft Division of Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Felix Randow Corresponding Author Felix Randow [email protected] orcid.org/0000-0003-0694-5315 Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK Search for more papers by this author Teresa LM Thurston Teresa LM Thurston Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK Search for more papers by this author Keith B Boyle Keith B Boyle Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Mark Allen Mark Allen Division of Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Benjamin J Ravenhill Benjamin J Ravenhill Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Maryia Karpiyevich Maryia Karpiyevich Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Stuart Bloor Stuart Bloor Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Annie Kaul Annie Kaul orcid.org/0000-0002-9965-8026 Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jessica Noad Jessica Noad Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Agnes Foeglein Agnes Foeglein Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Sophie A Matthews Sophie A Matthews MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK Search for more papers by this author David Komander David Komander Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Mark Bycroft Mark Bycroft Division of Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Felix Randow Corresponding Author Felix Randow [email protected] orcid.org/0000-0003-0694-5315 Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Teresa LM Thurston1,2, Keith B Boyle1, Mark Allen3, Benjamin J Ravenhill1, Maryia Karpiyevich1, Stuart Bloor1,5, Annie Kaul1, Jessica Noad1, Agnes Foeglein1, Sophie A Matthews2, David Komander1, Mark Bycroft3 and Felix Randow *,1,4 1Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK 2MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK 3Division of Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, UK 4Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK 5Present address: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK *Corresponding author. Tel: +44 1223 267161; Fax: +44 1223 268306; E-mail: [email protected] The EMBO Journal (2016)35:1779-1792https://doi.org/10.15252/embj.201694491 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti-bacterial autophagy relies on the core autophagy machinery, cargo receptors, and “eat-me” signals such as galectin-8 and ubiquitin that label bacteria as autophagy cargo. Anti-bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti-bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella-associated “eat-me” signals, including host-derived glycans and K48- and K63-linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host. Synopsis The recruitment of catalytically active TANK-binding kinase 1 to cytosol-invading Salmonella induces anti-bacterial autophagy via the upstream autophagy regulator WIPI2. Recruitment of enzymatically active TBK1 to cytosol-invading bacteria is essential for anti-bacterial autophagy. TBK1 and PI3 kinase independently control recruitment of WIPI2, itself essential for anti-bacterial autophagy. Promiscuity in recruiting TBK1 via several “eat-me” signals buffers TBK1 functionality from potential bacterial antagonism. Introduction Anti-bacterial autophagy provides potent cell-autonomous immunity against bacterial attempts to colonize the cytosol of mammalian cells (Kuballa et al, 2012; Deretic et al, 2013; Randow et al, 2013). The defense of the gut epithelium against bacteria in particular is crucially dependent on anti-bacterial autophagy, since mice lacking the essential autophagy gene Atg5 in enterocytes suffer from tissue invasion by commensal bacteria and from increased pathology upon infection with Salmonella enterica serovar Typhimurium (S. Typhimurium), a specialized enteropathogen (Benjamin et al, 2013). Macro-autophagy, hereafter autophagy, is an evolutionarily conserved quality control and degradation pathway that engulfs cytosolic content into double-membrane vesicles called autophagosomes. Autophagosome biogenesis requires the concerted activity of about 15 core AuTophaGy genes (ATGs), among them the VPS34 lipid kinase complex (Mizushima et al, 2011). VPS34 produces membrane patches rich in phosphatidylinositol 3-phosphate (PI(3)P) that recruit PI(3)P-binding proteins such as WIPI and DFCP1 to the site of phagophore formation (Axe et al, 2008). In contrast to the non-selective engulfment of cytosol into starvation-induced autophagosomes, anti-bacterial autophagy is mediated by cargo receptors including NDP52, optineurin, and p62 (Thurston et al, 2009; Zheng et al, 2009; Wild et al, 2011). Cargo receptors bind members of the LC3/GABARAP family of ubiquitin-like proteins on the autophagosomal membrane and specific “eat-me” signals associated with cytosol-invading bacteria, thereby selectively tethering bacteria to phagophore membranes (Weidberg et al, 2011; Rogov et al, 2014). Salmonella enterica serovar Typhimurium reaches the cytosol from a vesicular compartment, the Salmonella-containing vacuole (SCV). Damage to the limiting membrane of the SCV during bacterial escape exposes host glycans otherwise hidden inside the vacuole as ligands for a family of cytosolic lectins, the galectins (Dupont et al, 2009; Paz et al, 2010). By binding the cargo receptor NDP52, galectin-8 provides an “eat-me” signal for anti-bacterial autophagy (Thurston et al, 2012). The dense layer of poly-ubiquitylated proteins that accumulates on cytosol-exposed S. Typhimurium serves as an alternative “eat-me” signal, which is sensed by multiple cargo receptors, namely NDP52, optineurin, and p62 (Perrin et al, 2004; Thurston et al, 2009; Zheng et al, 2009; Wild et al, 2011). Failure of “eat-me” signals to associate with cytosolic bacteria or interference with cargo receptor function prevents efficient anti-bacterial autophagy and allows hyper-proliferation of cytosolic S. Typhimurium (Boyle & Randow, 2013). Restricting the proliferation of S. Typhimurium also requires the kinase TBK1, a member of the IKK (inhibitor of nuclear factor κB kinase) family (Radtke et al, 2007; Thurston et al, 2009). The anti-bacterial function of TBK1 is distinct from its well-characterized role of inducing type I interferons by phosphorylating IRF3 in virally infected cells (Randow et al, 2013; Wu & Chen, 2014). TBK1 accumulates in the vicinity of cytosol-exposed bacteria together with its adaptor proteins Nap1, Sintbad, and their binding partner NDP52 (Fujita et al, 2003; Ryzhakov & Randow, 2007; Thurston et al, 2009; Verlhac et al, 2015). TBK1 also associates with optineurin and it has been reported to phosphorylate both optineurin and p62, thereby enhancing their affinity for LC3B and ubiquitin, respectively (Morton et al, 2008; Wild et al, 2011; Pilli et al, 2012; Heo et al, 2015; Richter et al, 2016). While these findings imply that TBK1 strengthens the tethering function of cargo receptors, TBK1 has also been suggested to promote autophagosome maturation (Pilli et al, 2012). Here we show that in order to restrict Salmonella proliferation TBK1 activity is required in the proximity of cytosolic bacteria for the recruitment of WIPI2, a PI(3)P-binding upstream autophagy component itself essential for anti-bacterial autophagy. To investigate the recruitment requirements for TBK1 in restricting bacterial proliferation, we deployed a TBK1 variant unable to bind any of its known adaptors. Recruitment of TBK1 to S. Typhimurium via any of several eat-me signals, including galectin-8 and K48- or K63-linked ubiquitin, is sufficient to provide TBK1 functionality for anti-bacterial autophagy, suggesting that robust and promiscuous recruitment of TBK1 to cytosol-invading bacteria may be beneficial in thwarting potential bacterial evasion attempts. Results The autophagic capture of Salmonella requires enzymatically active TBK1 in the bacterial vicinity TBK1 is essential for anti-bacterial autophagy but its precise function in the pathway, as well as its mode of activation, remain poorly understood. TBK1 comprises an N-terminal kinase domain, a ubiquitin-like domain, and two C-terminal coiled-coils. To explore the role of TBK1 in antagonizing S. Typhimurium replication inside host cells we utilized TBK1 knockout mouse embryonic fibroblasts (MEFs). We confirmed previous findings of unrestricted proliferation of S. Typhimurium in Tbk1−/− MEFs, a phenotype complemented with wild-type but not catalytically inactive TBK1K38M (Figs 1A and EV1A) (Pomerantz & Baltimore, 1999; Radtke et al, 2007). We have previously shown that TBK1 physically associates with those intracellular Salmonella that are positive for the TBK1 adaptor proteins Nap1 and Sintbad and the autophagy cargo receptor NDP52 (Thurston et al, 2009). To test whether the function of TBK1 in anti-bacterial autophagy requires interactions with its adaptor proteins we truncated TBK1 at its C-terminus (TBK1N685 hereafter referred to as TBK1ΔC), thereby generating a molecule deficient in binding to all its known adaptors, that is Nap1, Sintbad, Tank, and optineurin (Fig EV1B) (Goncalves et al, 2011), while maintaining kinase activity as indicated by the activation of an ISRE reporter (Fig EV1C). Complementation of Tbk1−/− MEFS with TBK1ΔC failed to restrict proliferation of S. Typhimurium (Figs 1A and EV1A). The double mutant lacking catalytic activity and adaptor binding had a phenotype no more severe than either single mutant in the Salmonella assay (Figs 1A and EV1A). We therefore conclude that the catalytic activity of TBK1 and its ability to bind adaptor proteins are equally important to protect cells against S. Typhimurium, most likely because adaptor binding controls TBK1 spatially and/or temporally. Figure 1. TBK1 kinase activity and C-terminal domain are required for restriction of Salmonella enterica serovar Typhimurium replication Kinetics of S. Typhimurium replication in Tbk1−/− MEFs stably expressing the indicated TBK1 alleles. Intracellular bacteria were enumerated by their ability to form colonies on agar plates following cell lysis at the indicated time points. Statistical analysis comparing Tbk1−/− MEFs and cells expressing TBK1 alleles. Western blot for Flag-tagged TBK1 variants in post-nuclear cell lysates. Replication of S. Typhimurium in Atg5−/− MEFs complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 (10 nM) or DMSO. Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post-inoculation (p.i.) following cell lysis. Analysis of Tbk1−/− MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium. Percentage of S. Typhimurium coated with ubiquitin (detected by FK2 antibody) at 1 or 4 h p.i. Western blot for Flag-tagged TBK1 variants in post-nuclear cell lysates. Data information: Mean and SD of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats (A, B). Mean and SEM of three independent experiments with duplicate coverslips. > 200 bacteria counted per coverslip (C). *P < 0.05, ***P < 0.001, one-way ANOVA with Dunnett's multiple comparisons test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Characterization of TBK1 alleles Replication of Salmonella enterica serovar Typhimurium in Tbk1−/− MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Replication normalized to cells expressing GFP. Mean and SEM. N = 3, ***P < 0.001 one-way ANOVA with Dunnett's multiple comparisons test. Lumier binding assay. Binding of luciferase-tagged Nap1, Tank, or optineurin to beads coated with Flag:GFP, Flag:TBK1, or Flag:TBK1ΔC. Western blot for luciferase-tagged or Flag-tagged proteins in total cell lysates. ISRE reporter induction upon transient expression of the indicated Flag:TBK1 alleles into 293ET cells. Western blot for Flag-tagged proteins. Mean and SEM of four independent experiments. *P < 0.05, **P < 0.01, Student's t-test. ISRE reporter induction upon stimulation of 293ET cells with polyI:C (8 h) in the presence of DMSO or TBK1 inhibitors MRT68843 or MRT68601. Download figure Download PowerPoint Precisely how TBK1 restricts bacterial proliferation is controversial; the enhanced bacterial load in TBK1-deficient cells has been suggested to be caused by either the cells' inability to maintain SCV integrity (Radtke et al, 2007) or the cells' inability to execute anti-bacterial autophagy (Thurston et al, 2009). The TBK1 inhibitor MRT68843, which inhibits poly(I:C)-induced ISRE reporter activity similar to the related TBK1 inhibitor MRT68601 (Newman et al, 2012) (Fig EV1D), was used to analyze the relationship between TBK1 activity and autophagy. As expected, Atg5−/− cells failed to suppress proliferation of S. Typhimurium (Fig 1B). Addition of MRT68843 increased bacterial replication only 1.5-fold in Atg5−/− MEFs but more than eightfold in cells complemented with ATG5, consistent with TBK1 controlling anti-bacterial autophagy due to its kinase activity. To substantiate this finding, we next investigated where in the anti-bacterial autophagy pathway TBK1 acts. By complementing Tbk1−/− MEFs, we confirmed that lack of TBK1 increased the percentage of ubiquitin-coated cytosolic S. Typhimurium at 4 h post-infection (Fig 1C) (Radtke et al, 2007; Thurston et al, 2009). TBK1K38M and TBK1ΔC, which are catalytically inactive and deficient in binding adaptor proteins, respectively, did not complement the ubiquitin phenotype, in line with the lack of these alleles to control proliferation of S. Typhimurium in the cytosol of host cells (Fig 1A and C). The recruitment of WIPI2, itself essential for anti-bacterial autophagy, is controlled by TBK1 The anti-bacterial autophagy attack can be visualized by assessing the association of S. Typhimurium with LC3B, a mammalian Atg8 ortholog. However, complementation of Tbk1−/− MEFs with wild-type TBK1 did not significantly alter the percentage of GFP:LC3B-positive S. Typhimurium at 1 h post-infection, nor did complementation with TBK1ΔC or TBK1K38M (Fig 2A). Such apparently normal recruitment of LC3B to S. Typhimurium in cells failing to restrict bacterial proliferation (Fig 1A and C) may be due to conjugation of LC3 to the remnants of SCV membranes rather than anti-bacterial phagophores, a phenotype well-documented for MEFs with defects in upstream autophagy components such as FIP200 or ATG9 (Kageyama et al, 2011). The phenotype in Tbk1−/− MEFs therefore points to an upstream defect in the autophagy pathway. Phagophore formation requires the PI3 kinase VPS34 to generate PI(3)P as a recruitment signal for WIPI proteins, the mammalian orthologs of yeast Atg18 (Proikas-Cezanne et al, 2004). Since wortmannin, a potent inhibitor of VPS34, prevents colocalization of WIPI1 but not LC3 with S. Typhimurium (Kageyama et al, 2011), we tested whether TBK1 was similarly required for the recruitment of WIPI proteins to bacteria. We found that GFP-tagged WIPI1 and WIPI2B but not WIPI3 and WIPI4 accumulated on S. Typhimurium, as did endogenous WIPI2 (Figs 2B and C, and EV2A and B). The recruitment of WIPI1 and WIPI2B was sensitive to wortmannin treatment and abrogated by mutations in their PI(3)P-binding sites (GFP:WIPI1FTTG and GFP:WIPI2BFTTG). Importantly, accumulation of WIPI1 and WIPI2B also required expression of wild-type TBK1 and was not supported by either catalytically inactive TBK1K38M or TBK1ΔC deficient in binding adaptor proteins (Fig 2B). In contrast, recruitment of DFCP1 did not require TBK1, although it was also sensitive to wortmannin treatment and mutational inactivation of its PI(3)P-binding site (DFCP1FYVE*) (Fig 2D–F). WIPI proteins also bind PI(3,5)P2 (Baskaran et al, 2012). However, the accumulation of PI(3,5)P2 on S. Typhimurium was independent of TBK1, as revealed by the normal recruitment of GFP:ML1N*2, a PI(3,5)P2-specific probe (Li et al, 2013b) (Fig 2G and H). We therefore conclude that TBK1 and VPS34 independently control the recruitment of WIPI1 and WIPI2 to S. Typhimurium and that TBK1 functionality requires catalytic activity as well as its C-terminal adaptor-binding coiled-coil domain. Figure 2. TBK1 kinase activity and C-terminal adaptor-binding domain are required to recruit WIPI1 and WIPI2 to Salmonella enterica serovar Typhimurium A–H. Analysis of Tbk1−/− MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium for 1 h. Percentage of S. Typhimurium coated with GFP:LC3B (A), the indicated GFP:WIPI alleles (B), GFP:DFCP1 (FYVE* denotes a PI(3)P-binding mutant of DFCP1) (D, E), or GFP:ML1N*2, a probe for PI(3,5)P2 (G). Where indicated, wortmannin (Wort) was added at 100 nM. Confocal micrographs of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry-expressing S. Typhimurium (C, F and H). Mean and SEM of at least three independent experiments with duplicate coverslips. > 200 bacteria counted per coverslip. *P < 0.05, **P < 0.01 one-way ANOVA with Dunnett's multiple comparisons test. Scale bar, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Recruitment of WIPI1 and WIPI2B to Salmonella enterica serovar Typhimurium requires TBK1 but not optineurin A. Western blots of GFP-tagged WIPI alleles and Flag-tagged TBK1 alleles corresponding to Fig 2B. B. Percentage of WIPI2-positive S. Typhimurium at 1 h p.i., in Tbk1−/− MEFs complemented with the indicated Flag-tagged TBK1 proteins. Wortmannin (Wort) was added as indicated at 100 nM. Mean and SEM. N = 3, *P < 0.05, one-way ANOVA with Dunnett's multiple comparisons test. Western blot of TBK1 alleles in post-nuclear lysates. C, D. Analysis of MEFs, stably expressing GFP:WIPI1 or GFP:WIPI2B, treated with the indicated siRNAs and infected with S. Typhimurium for 1 h. Mean and SEM. N = 3. Western blot for optineurin and tubulin upon the indicated siRNA treatment. Download figure Download PowerPoint To further investigate the mechanism of how TBK1 recruits WIPI1 and WIPI2 to cytosol-invading bacteria, we depleted cells of optineurin, the only known TBK1 substrate in anti-bacterial autophagy (Wild et al, 2011). Cells lacking optineurin recruited WIPI1 and WIPI2B normally to S. Typhimurium, suggesting that phosphorylation of a substrate other than optineurin is essential for WIPI1/2 recruitment in anti-bacterial autophagy (Fig EV2C). We also tested the interdependence of WIPI1 and WIPI2B recruitment and found that neither protein was required for the recruitment of the other (Fig EV2D). We next investigated whether WIPIs are essential to protect cells against bacterial proliferation. Cells lacking WIPI2 failed to restrict proliferation of S. Typhimurium, confirming a recent finding (Dooley et al, 2014), while the presence of WIPI1 was not required (Fig 3). We therefore conclude that the recruitment of WIPI2 to cytosol-invading bacteria is likely an essential function of TBK1 in cell-autonomous defense. Figure 3. WIPI2 restricts Salmonella proliferation A, B. Kinetics of Salmonella enterica serovar Typhimurium replication in MEFs depleted of WIPI1 (A) or WIPI2 (B). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. Western blot for GFP:WIPI1 and quantitative RT–PCR for WIPI2 upon the indicated siRNA treatments. Mean and SEM. N = 6 (A), n = 3 (B). *P < 0.05, Student's t-test. Download figure Download PowerPoint NDP52-mediated recruitment of TBK1 to S. Typhimurium suffices to restrict bacterial proliferation Since the C-terminal domain of TBK1 is required to restrict bacterial proliferation and mediates adaptor binding (Fig EV1A) we thought to repair TBK1ΔC by fusing it directly to individual adaptor proteins. This strategy enables the evaluation of individual adaptors in the TBK1-mediated restriction of S. Typhimurium and structure–function analyses without interference from potentially redundant adaptor function. As cytosol-exposed S. Typhimurium recruit Nap1 but not Tank (Thurston et al, 2009), we compared these two adaptors by fusing them to TBK1ΔC. Consistent with their differential recruitment to cytosolic Salmonella, TBK1ΔC:Tank did not restrict Salmonella proliferation in Tbk1−/− MEFs; in contrast, TBK1ΔC:Nap1 reduced bacterial replication as efficiently as full-length TBK1 (Figs 4A and EV3A). Complementation of Tbk1−/− MEFs with TBK1ΔC:Nap1 but not with TBK1ΔC:Tank also restored localization of GFP-WIPI1 to Salmonella (Fig 4B). Figure 4. Recruitment of TBK1 to Salmonella enterica serovar Typhimurium via NAP1 or NDP52, but not TANK, restricts bacterial proliferation and recruits WIPI1 A–D. Analysis of Tbk1−/− MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A, D). Infected cells were lysed at the indicated time points post-inoculation (p.i.), and bacteria were enumerated by their ability to form colonies on agar plates. Mean and SD of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1−/− MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post-nuclear cell lysates. (B) Percentage of GFP:WIPI1-positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and SEM of four independent experiments. > 200 bacteria counted per coverslip. *P < 0.05, ***P < 0.001, one-way ANOVA with Dunnett's multiple comparisons test. (C) TBK1–Nap1–NDP52 complex. N- and C-termini, domains and binding partners are indicated. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Recruitment of TBK1 to Salmonella enterica serovar Typhimurium via NAP1 or NDP52, but not TANK, restricts bacterial proliferation A, B. Replication of S. Typhimurium in Tbk1−/− MEFs complemented with the indicated Flag-tagged TBK1ΔC alleles. Replication normalized to cells expressing GFP. Mean and SEM. N = 3, *P < 0.05, **P < 0.01, one-way ANOVA with Dunnett's multiple comparisons test. Download figure Download PowerPoint How does Nap1 contribute to TBK1 function? Considering the overall structural similarity between Nap1 and Tank (Fig 4C) (Ryzhakov & Randow, 2007), efficient complementation with TBK1ΔC:Nap1 but not TBK1ΔC:Tank suggested a role for the N-terminal coiled-coil region of Nap1. Indeed, TBK1ΔC:Nap1ΔN85, in contrast to TBK1ΔC:Nap1, did not prevent hyper-proliferation of S. Typhimurium in Tbk1−/− MEFs (Figs 4A and EV3A). Complementation with TBK1ΔC:Nap1N85 also restricted bacterial proliferation; the N-terminal 85 residues of Nap1 are therefore required and sufficient to provide functionality to TBK1ΔC. Nap1N85 forms a coiled-coil that contributes to the dimerization of Nap1 and binds the autophagy cargo receptor NDP52 (Thurston et al, 2009). We therefore speculated that recruitment of TBK1 to Salmonella via NDP52 antagonizes bacterial replication. To test this hypothesis, we fused TBK1ΔC directly to NDP52 and found that it restricted bacterial proliferation in Tbk1−/− MEFs as efficiently as full-length TBK1 (Figs 4D and EV3B). However, although endogenous Nap1 cannot bind TBK1ΔC directly (Fig EV1B), Nap1 may be recruited to TBK1ΔC:NDP52 via its binding site in the NDP52 SKICH domain. To test whether such indirect recruitment of Nap1 was required for the complementation of Tbk1−/− MEFs with TBK1ΔC:NDP52, we examined TBK1ΔC:NDP52SKICH, which was inactive, and TBK1ΔC:NDP52ΔSKICH, which retained activity (Figs 4D and EV3B). We therefore conclude that in anti-bacterial autophagy, the interaction of TBK1 with adaptor proteins can be replaced entirely by fusing TBK1 directly to NDP52. Recruitment of TBK1 to S. Typhimurium via either galectin-8 or ubiquitin suffices to restrict bacterial proliferation We speculated that the function of NDP52 in TBK1ΔC:NDP52 might be provided by its ability to sense cytosol-invading bacteria via autophagy-inducing “eat-me” signals, that is the bacterial ubiquitin coat and/or galectin-8 on damaged bacteria-containing vacuoles. Binding of NDP52 to galectin-8 is understood in structural detail; it is mediated by a hook-like structure formed by residues 371–381 and is abrogated in NDP52L374A (Kim et al, 2013; Li et al, 2013a). NDP52 binds ubiquitin via its C-terminal zinc finger and structural information on the interaction has been recently published (Xie et al, 2015). We also determined the solution structure of the C-terminal ubiquitin-binding zinc finger of NDP52 by NMR spectroscopy, which confirmed the existence of an UBZ-like fold consisting of an α-helix and a two-stranded β-sheet (Fig 5A) (Xie et al, 2015). The zinc ion is coordinated by residues His440 and His444 as well as residues Cys422 and Cys425, which are located in the helix and in the loop connecting the two β-strands, respectively. This fold is found in several ubiquitin-binding proteins with the NDP52 structure most similar to the C-terminal ubiquitin-binding Zn fingers of Nemo and optineurin (Fig 5B). Addition of mono-ubiquitin to the NDP52 zinc finger produced changes in the chemical shift primarily of residues in the helix (Fig 5C and Appendix Fig S1). Analysis of the chemical shift changes as a function of ubiquitin concentration revealed a dissociation constan" @default.
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• W2469403673 title "Recruitment of <scp>TBK</scp> 1 to cytosol‐invading <i>Salmonella</i> induces <scp>WIPI</scp> 2‐dependent antibacterial autophagy" @default.
• W2469403673 cites W1523693806 @default.
• W2469403673 cites W1946723857 @default.
• W2469403673 cites W1969776705 @default.
• W2469403673 cites W1969840664 @default.
• W2469403673 cites W1972726560 @default.
• W2469403673 cites W1976765363 @default.
• W2469403673 cites W1982328089 @default.
• W2469403673 cites W1986278788 @default.
• W2469403673 cites W1988042982 @default.
• W2469403673 cites W1989165480 @default.
• W2469403673 cites W1995700909 @default.
• W2469403673 cites W2001045944 @default.
• W2469403673 cites W2006714910 @default.
• W2469403673 cites W2009225398 @default.
• W2469403673 cites W2012625663 @default.
• W2469403673 cites W2016962320 @default.
• W2469403673 cites W2018302034 @default.
• W2469403673 cites W2023994136 @default.
• W2469403673 cites W2029607802 @default.
• W2469403673 cites W2030280505 @default.
• W2469403673 cites W2030755802 @default.
• W2469403673 cites W2032601500 @default.
• W2469403673 cites W2037506480 @default.
• W2469403673 cites W2052080816 @default.
• W2469403673 cites W2052247259 @default.
• W2469403673 cites W2052470191 @default.
• W2469403673 cites W2054795369 @default.
• W2469403673 cites W2060951315 @default.
• W2469403673 cites W2061541746 @default.
• W2469403673 cites W2093670875 @default.
• W2469403673 cites W2094992876 @default.
• W2469403673 cites W2095249521 @default.
• W2469403673 cites W2102420546 @default.
• W2469403673 cites W2106195012 @default.
• W2469403673 cites W2114847797 @default.
• W2469403673 cites W2122157526 @default.
• W2469403673 cites W2125548716 @default.
• W2469403673 cites W2127215376 @default.
• W2469403673 cites W2130350123 @default.
• W2469403673 cites W2136026438 @default.
• W2469403673 cites W2136054419 @default.
• W2469403673 cites W2140915719 @default.
• W2469403673 cites W2144652247 @default.
• W2469403673 cites W2148281700 @default.
• W2469403673 cites W2149200223 @default.
• W2469403673 cites W2150168624 @default.
• W2469403673 cites W2156171182 @default.
• W2469403673 cites W2207263914 @default.
• W2469403673 cites W2323582440 @default.
• W2469403673 cites W2469403673 @default.
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