Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Molecular interactions among tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), Beclin 1, and ubiquitin-specific protease 14 (USP14). (A) Truncated mutants of Beclin 1. Myc-tagged Beclin 1 truncated mutants were generated as described in Section “ .” BH3, Bcl-2 Homology 3; CCD, coiled coil domain; ECD, evolutionarily conserved domain. (B) Expression vectors of Myc-tagged Beclin 1 wild type (wt) and truncated mutants were co-transfected with Flag-tagged USP14 plasmid into HEK293T cells followed by immunoprecipitation (IP) and western blotting analyses. (C) Myc-tagged Beclin 1 wt, Myc-tagged Beclin 1 truncated mutants, and mock as control plasmid were co-transfected with Flag-tagged TRAF6 into HEK293T cells followed by IP and western blotting analyses. (D) Flag-tagged TRAF6 wt, Flag-tagged TRAF6 truncated mutants, and mock as control plasmid were co-transfected with Myc-tagged Beclin 1 into HEK293T cells followed by IP and western blotting analyses. (E) Flag-tagged TRAF6 wt, Flag-tagged TRAF6 truncated mutants, and mock as control plasmid were co-transfected with Myc-tagged USP14 into HEK293T cells followed by IP and western blotting analyses. (F) A schematic model showing molecular interactions among TRAF6, Beclin 1, and USP14. TRAF-C, C-terminal TRAF domain.

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Generated, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Ubiquitin-specific protease 14 (USP14) inhibits ubiquitination of Beclin 1. (A) Myc-tagged Beclin 1, Flag-tagged tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and mock as control plasmid were co-transfected with Flag-tagged USP14 plasmid at different concentrations. At 38 h post-transfection, transfected cells were extracted, immunoprecipitated with anti-Myc antibody, and subjected to immuno-blotting (IB) assay using anti-USP14, anti-TRAF6, anti-Flag, or anti-Myc antibody. *The intensity of Flag-USP14 was measured by ImageJ. Fold change relative to Flag-USP14 of lane 3 was calculated. **The intensity of Flag-TRAF6 was measured by ImageJ. Fold change relative to Flag-TRAF6 of lane 2 was calculated. (B) Myc-tagged Beclin 1, HA-tagged Ub, Flag-tagged TRAF6, and mock as control plasmid were co-transfected with Flag-tagged USP14 vector at different concentrations. At 38 h post-transfection, transfected cells were extracted, immunoprecipitated with anti-Myc antibody, and subjected to IB assay using anti-HA, anti-Myc, anti-USP14, or anti-TRAF6 antibody. (C) A schematic model showing the inhibition of Beclin 1 ubiquitination by USP14.

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Inhibition

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Ubiquitin-specific protease 14 (USP14) negatively regulates autophagy induction and toll-like receptor 4 (TLR4)-mediated signaling. (A) Upon TLR4 stimulation, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interacts with Beclin 1 through its coiled coil (CC) domain, induces ubiquitination of Beclin 1, and facilitate autophagy induction (upper). In contrast, USP14 competitively interacts with Beclin 1 to the CC domain of TRAF6. This leads to the suppression of Beclin 1 ubiquitination by TRAF6, resulting in the inhibition of autophagy induction (down). (B) Following TLR4 stimulation, ubiquitinated TRAF6 is associated with the TAB 2–TAK1–TAB 1 complex through an interaction between its polyubiquitinated chain and TAB 2. TAB 2 is then ubiquitinated and the complex facilitates the activation of TAK1. Simultaneously, the IKK complex is associated with the former complex through the polyubiquitinated chain and TAB 2, leading to the activations of nuclear factor-kappa B (NF-κB) and p38 (left). In contrast, the interaction between TAB 2 and USP14 may lead to the deubiquitination of TAB 2, thus inhibiting the association of IKKs, which inhibits the activation of NF-κB and p38 (right).

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Inhibition, Activation Assay

Journal: PLoS Pathogens

Article Title: Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy

doi: 10.1371/journal.ppat.1006671

Figure Lengend Snippet: A , A549 cells were treated with or without FAK inhibitor or were transfected with control siRNA or Src siRNA followed by challenge with RH T . gondii . Expression of total Akt and phospho-S473 Akt were examined by immunoblot. A vertical line was inserted between densitometry data of lysates from cells treated with control or FAK inhibitor, or transfected with control or Src siRNA to indicate that relative densities of phospho-Akt from infected cells were compared to bands from their respective uninfected (control) cells. Relative density of phospho-Akt for uninfected samples was given a value of 1. B , Human RPE cells were challenged with RH (type I) or ME49 (type II) strains of T . gondii . Expression of total STAT3 and phospho-Y705 STAT3 were examined by immunoblot. Relative densities of phospho-STAT3 in lysates from infected cells were compared to those from their respective uninfected (control) cells. Relative density of phospho-STAT3 for uninfected samples was given a value of 1. C , BV-2 cells were challenged with RH T . gondii followed by assessment of total STAT3 and phospho-Y705 STAT3 by immunoblot. D , A549 cells were challenged with either WT or Δrop16 T . gondii . Cell lysates were probed for total STAT3 and phospho-Y705 STAT3. Band densities in lysates from infected cells were compared to those from uninfected (control) cells. Densitometries for control bands were given a value of 1. E , A549 cells transfected with control siRNA or Src siRNA were challenged with RH T . gondii . Immunoblot and densitometries were assessed as above. F , A549 cells transfected with plasmid encoding either WT-EGFR or Y845F-EGFR were infected with T . gondii 48 h after transfection. Immunoblot and densitometries were assessed as above. G , NMuMG that express WT EGFR or EGFR AA mutant were challenged with WT or Δrop16 T . gondii . Immunoblot and densitometries were assessed as above. Densitometry data represent means ± SEM of 3 independent experiments.

Article Snippet: Cells were transfected with Src siRNA [ ], ULK1 siRNA (Life Technologies), Beclin 1 siRNA [ ], STAT3 siRNA [ ], MyD88 siRNA [ ] or control siRNA (Dharmacon, Lafayette, CO) as well as plasmids encoding WT EGFR, Y845F EGFR [ ], WT STAT3, Y705 STAT3 (gift from Jim Darnell; Addgene plasmids # 8707 and 8709) [ ], FLAG-tagged WT-PKR, dominant negative (DN)-PKR (K296R) or empty plasmid (gifts from Bill Sugden, University of Wisconsin) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. mHEVc cells were transduced with lentivirus vectors encoding FAK or control shRNA (Open Biosystems, Lafayette, CO) followed by selection with puromycin (10 μg/ml; Sigma-Aldrich).

Techniques: Transfection, Control, Expressing, Western Blot, Infection, Plasmid Preparation, Mutagenesis

Journal: PLoS Pathogens

Article Title: Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy

doi: 10.1371/journal.ppat.1006671

Figure Lengend Snippet: A , A549 cells were transfected with control siRNA or STAT3 siRNA and challenged with RH T . gondii . Monolayers were examined microscopically at 2 and 24 h to determine the percentages of infected cells, and at 24 h to ascertain the numbers of T . gondii tachyzoites, T . gondii -containing vacuoles per 100 cells and parasites per vacuole. B , A549 cells transfected with control siRNA or STAT3 siRNA were challenged with T . gondii -RFP (RH). Expression of LC3 was examined by immunofluorescence 5 h post-challenge to determine the percentage of cells with LC3 accumulation around the parasite. Arrowheads indicate accumulation of LC3 around the parasite. Original magnification X630. C , A549 cells transfected with control siRNA or STAT3 siRNA were challenged with T . gondii -YFP (RH). Expression of LAMP-1 was examined by fluorescence microscopy 8 h post-challenge. The percentage of cells with LAMP-1 accumulation around the parasite was determined. Arrowheads indicate accumulation of LAMP-1 around the parasite. D , A549 cells transfected with Beclin 1 siRNA were transfected with STAT3 siRNA followed by challenge with RH T . gondii . Monolayers were examined 24 h post-challenge. E , mHEVc cells transfected with WT STAT3 or Y705F STAT3 were challenged with T . gondii -RFP (RH). Expression of LC3 was examined by immunofluorescence 5 h post-challenge. LC3 accumulation around the parasite was assessed as above. F , mHEVc cells transfected with WT STAT3 or Y705F STAT3 were challenged with RH T . gondii followed by addition of leupeptin/pepstatin (lysosomal inhibitors, LI). Monolayers were examined at 2 and 24 h post-challenge. Results are shown as the mean ± SEM of 3 independent experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: Cells were transfected with Src siRNA [ ], ULK1 siRNA (Life Technologies), Beclin 1 siRNA [ ], STAT3 siRNA [ ], MyD88 siRNA [ ] or control siRNA (Dharmacon, Lafayette, CO) as well as plasmids encoding WT EGFR, Y845F EGFR [ ], WT STAT3, Y705 STAT3 (gift from Jim Darnell; Addgene plasmids # 8707 and 8709) [ ], FLAG-tagged WT-PKR, dominant negative (DN)-PKR (K296R) or empty plasmid (gifts from Bill Sugden, University of Wisconsin) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. mHEVc cells were transduced with lentivirus vectors encoding FAK or control shRNA (Open Biosystems, Lafayette, CO) followed by selection with puromycin (10 μg/ml; Sigma-Aldrich).

Techniques: Transfection, Control, Infection, Expressing, Immunofluorescence, Fluorescence, Microscopy

Journal: PLoS Pathogens

Article Title: Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy

doi: 10.1371/journal.ppat.1006671

Figure Lengend Snippet: A-C , mHEVc cells transduced with lentiviral vectors that express either FAK shRNA or control shRNA ( A ), NMuMG cells with stable expression of WT EGFR or EGFR AA mutant ( B ) or NMuMG cells with stable expression of WT EGFR cells transfected with WT STAT3 or Y705F STAT3 ( C ) were infected with RH T . gondii . Cell lysates were obtained to probe for total eIF2α and phospho-S51 eIF2α. Relative densities of phospho-eIF2α in lysates from infected cells were compared to those from their respective uninfected (control) cells. Relative density of phospho-eIF2α for uninfected samples was given a value of 1. D , mHEVc cells transduced with lentiviral vectors that express either FAK shRNA or control shRNA or NMuMG cells with stable expression of WT EGFR or EGFR AA mutant were challenged with RH T . gondii . Cell lysates were obtained at 5 min post-challenge to probe for total PKR and phospho-T451 PKR. Relative density of phospho-PKR was calculated as above. E , mHEVc cells transduced with lentiviral vectors that express FAK shRNA were transfected with WT or DN PKR followed by challenge with RH T . gondii . Cell lysates were probed for total eIF2α and phospho-S51 eIF2α. F , mHEVc cells were transfected with WT PKR or DN PKR followed by incubation with or without PP2 and infection with RFP- T . gondii (RH). Expression of LC3 was examined by immunofluorescence 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite. Original magnification X630. G , mHEVc cells transfected with WT PKR or DN PKR were treated with or without PP2, challenged with RH T . gondii . Monolayers were examined 2 and 24 h post-challenge. Results are shown as the mean ± SEM of 3 independent experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: Cells were transfected with Src siRNA [ ], ULK1 siRNA (Life Technologies), Beclin 1 siRNA [ ], STAT3 siRNA [ ], MyD88 siRNA [ ] or control siRNA (Dharmacon, Lafayette, CO) as well as plasmids encoding WT EGFR, Y845F EGFR [ ], WT STAT3, Y705 STAT3 (gift from Jim Darnell; Addgene plasmids # 8707 and 8709) [ ], FLAG-tagged WT-PKR, dominant negative (DN)-PKR (K296R) or empty plasmid (gifts from Bill Sugden, University of Wisconsin) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. mHEVc cells were transduced with lentivirus vectors encoding FAK or control shRNA (Open Biosystems, Lafayette, CO) followed by selection with puromycin (10 μg/ml; Sigma-Aldrich).

Techniques: Transduction, shRNA, Control, Expressing, Mutagenesis, Transfection, Infection, Incubation, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy

doi: 10.1371/journal.ppat.1006671

Figure Lengend Snippet: During invasion of mammalian cells, the formation of the moving junction, characterized by expression of RON4, appears to be accompanied by activation of FAK in the mammalian cell. In turn, FAK activates Src causing Src-dependent transactivation of EGFR (Y845 phosphorylation). This unique form of EGFR activation recruits STAT3 signaling that prevents activation of PKR and eIF2α. Blockade of the signaling cascade would result on activation of PKR and eIF2α leading to the formation of an autophagosome around the parasitophorous vacuole and killing of the parasite mediated by canonical autophagy.

Article Snippet: Cells were transfected with Src siRNA [ ], ULK1 siRNA (Life Technologies), Beclin 1 siRNA [ ], STAT3 siRNA [ ], MyD88 siRNA [ ] or control siRNA (Dharmacon, Lafayette, CO) as well as plasmids encoding WT EGFR, Y845F EGFR [ ], WT STAT3, Y705 STAT3 (gift from Jim Darnell; Addgene plasmids # 8707 and 8709) [ ], FLAG-tagged WT-PKR, dominant negative (DN)-PKR (K296R) or empty plasmid (gifts from Bill Sugden, University of Wisconsin) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. mHEVc cells were transduced with lentivirus vectors encoding FAK or control shRNA (Open Biosystems, Lafayette, CO) followed by selection with puromycin (10 μg/ml; Sigma-Aldrich).

Techniques: Expressing, Activation Assay, Phospho-proteomics