Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) HEK 293T cells expressing FLAG-STX17 were infected with different strains of Legionella for 2 h. Lysates were used for GST pulldown with the DupA trapping mutant GST-DupA (H67A), followed by western blots with antibodies against FLAG, GST, and ubiquitin. Whole-cell lysates were blotted with antibodies against FLAG and vinculin as a loading control. This experiment was repeated three times with similar results. ( B ) HEK 293T cells expressing V5-SNAP29 were infected with different Legionella strains for 2 h. Lysates were used for GST pulldown with the DupA trapping mutant GST-DupA (H67A) followed by western blotting with antibodies against V5, GST, and ubiquitin. Whole-cell lysates were probed with antibodies against V5 and vinculin as a loading control. This experiment was repeated three times with similar results. ( C ) Domain architecture of STX17 and SNAP29 showing the PR-ubiquitination sites. ( D ) A549 cells expressing GFP-tagged STX17, STX17TM or the PR-Ub deficient mutant of STX17 were infected with Legionella strains for 2 h before fixation and immunostaining with a Legionella -specific antibody for analysis by confocal microscopy. Control cells were treated with 300 nM Torin-1 for 4 h. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. Scale bar: 5 µm. Scale bar in inset: 2 µm. ( E ) The number of STX17 + bacteria per cell were counted for ~50 cells taken three different experiments. In the box plots, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. N = 52, 56 cells taken from three experimental replicates. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 7.43E-8. Scale bar: 5 µm. ( F ) The formation of WT and PR-Ub-deficient SNAP29-GFP puncta was monitored in Legionella -infected cells 4 h post-infection. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( G ) SNAP29 puncta were counted in 50-μm 2 regions of interest using FIJI. In the box plot, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n > 50 cells taken from three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, For the graph SNAP29 puncta/cell: *** P = 6.82E-20. For the graph % cells with SNAP29 recruitment to bacteria: *** P = 2.27E-5 (WT, SNAP29WT vs ΔS, SNAP29WT) *** P = 2.56E-6 (WT, SNAP29WT vs WT, SNAP29mut. Scale bar: 5 µm. .

Article Snippet: SNAP29-ser mutant , Lab generated using SNAP29 coding sequence of SNAP29 from addgene #69821 , .

Techniques: Expressing, Infection, Mutagenesis, Western Blot, Ubiquitin Proteomics, Control, Immunostaining, Confocal Microscopy, Bacteria, Software, Two Tailed Test

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) (i) HEK 293 T cells were cotransfected with FLAG-STX17 and GFP-tagged SdeA/SdeA(EE/AA) or a control vector for 16 h. FLAG-STX17 was immunoprecipitated using FLAG resin and analyzed by western blot using antibodies against FLAG to detect PR-Ub-modified and unmodified FLAG-STX17. The experiment was repeated 2 times with similar results. (ii) HEK 293T cells were cotransfected with FLAG-STX17 and GFP-tagged SdeA/SdeA(EE/AA) or a control vector and immunoprecipitated as shown in ( B ). The samples were then treated with or without pure DupA for 1 h before western blotting with antibodies against FLAG to detect PR-Ub-modified and unmodified FLAG-STX17. The experiment was repeated two times with similar results. ( B ) HEK 293T cells were cotransfected with GFP-SNAP29 and HA-tagged SdeA/SdeA(EE/AA) or a control vector for 16 h. GFP-SNAP29 was immunoprecipitated with anti-GFP beads, treated with or without pure DupA for 1 h and analyzed by western blot with antibodies against GFP to detect PR-Ub-modified and unmodified SNAP29. The experiment was repeated two times with similar results. ( C ) GST-STX17 and GST-STX17(S195AS202AS209A) were incubated with or without SdeA in the presence of 1 mM NAD + and ubiquitin for 1 h. The samples were analyzed by western blot using antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. ( D ) GST-STX17 and its PR-Ub-deficient mutant (S195AS202AS209A) were modified with or without SdeA, in the presence of 1 mM NAD+ and ubiquitin for 1 h. Samples were analyzed by western blot with antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. SdeA(EE/AA): mART mutant SdeA(E860AS862A) ( E ) GST-SNAP29 and its PR-Ub-deficient mutant (S61AS63AS70A) were modified with or without SdeA, in the presence of 1 mM NAD + and ubiquitin for 1 h. Samples were analyzed by western blot with antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. SdeA(EE/AA): mART mutant SdeA(E860AS862A).

Article Snippet: SNAP29-ser mutant , Lab generated using SNAP29 coding sequence of SNAP29 from addgene #69821 , .

Techniques: Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Modification, Incubation, Ubiquitin Proteomics, Mutagenesis

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) Mass spectrum and deduced sequence map of PR-Ub-modified SNAP29. ( B ) HEK 293 T cells were transfected with GFP-tagged WT STX17, STX17TM, or the STX17 serine mutant (S195AS202AS209A) followed by Legionella infection for 2 h. STX17 was then immunoprecipitated using anti-GFP beads followed by western blotting with antibodies against GFP and ubiquitin. Cell lysates were analyzed by western blot with an antibody against GFP to check the expression levels of GFP-STX17 constructs. The experiment was repeated three times with similar results.

Article Snippet: SNAP29-ser mutant , Lab generated using SNAP29 coding sequence of SNAP29 from addgene #69821 , .

Techniques: Sequencing, Modification, Transfection, Mutagenesis, Infection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing, Construct

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) A549 cells expressing STX17-GFP were infected with WT Legionella for 2 h in the presence or absence of 100 nM brefeldin A before fixation and staining the intracellular bacteria by DAPI. STX17-positive bacteria was counted in 50 cells per set, taken from three independent experiments. Error bars indicate SEM. Difference between sets was non-significant from p value calculated by two-tailed, type 3 Student’s t test. Scale bar:10 µm. ( B ) A549 cells expressing STX17-GFP were infected with Legionella (WT/ΔS) for 2 h in the presence or absence of 100 nM wortmannin before fixation and immunostaining with antibodies against Legionella . p value was calculated by two-tailed, type 3 Student’s t test. *** P = 4.45E-6(WT and ΔS sets without wortmannin), *** P = 2.05E-5 (WT +/-wortmannin), Graph represents n = 50 cells taken from three experiments, error bars indicate SEM. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( C ) Immuno-electron microscopy of HeLa cells transfected with STX17-GFP and infected with WT Legionella-DsRed for 4 h. Ultrathin cryosection immunogold labeled for STX17-GFP by protein A–10-nm gold. Colors are added by Photoshop: Yellow marks a STX-17.GFP-positive ER cisterna closely aligned with the Legionella (Leg) containing vacuole. Green marks the space between the vacuolar membrane and enclosed Legionella. Bar, 200 nm. ( D ) A549 cells were infected with WT or ΔRΔS Legionella for 1 h, fixed and immunostained with the STX17 and Legionella antibodies to check for the recruitment of STX17 to intracellular bacteria. White arrows mark intracellular bacteria with STX17 recruitment. The data are means ± SEM of 118 cells from three independent experiments. p value was calculated by two-tailed, type 3 Student’s t test. *** P = 4.21E-6. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( E ) A549 cells were infected with WT or ΔRΔS Legionella for 1 h, fixed and immunostained with the SNAP29 and Legionella antibodies to check for the recruitment of SNAP29 to intracellular bacteria. White arrows mark intracellular bacteria with SNAP29 recruitment. The data are means ± SEM of 120 cells from three independent experiments. p value was calculated by two-tailed, type 3 Student’s t test. *** P = 2.21E-4. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( F ) HeLa cells were cotransfected with RFP-tagged SdeA or its catalytic mutant (E860AE862A) and GFP-tagged WT SNAP29 or its PR-Ub-deficient mutant. Cells were treated with 300 nM Torin-1 for 4 h to induce autophagy before fixation and confocal imaging. Scale bar:5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( G ) The graph shows the number of cells with SNAP29-GFP puncta (from panel d) counted in FIJI. In the box plot, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n > 30 cells taken from three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 0.00032. In bar graph, the data are means ± SEM of n > 30 cells from three independent experiments. Scale bar:5 µm. ( H ) A549 cells were treated with SNAP29 or control siRNA for 48 h followed by infection Legionella. Intracellular bacterial replication was assessed after 0, 24 and 48 h. Data are means ± SEM of three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 2.1E-4 (ΔR), ** P = 0,.031(ΔRΔS). (ni not infected, WT wild-type, Legionella , ΔS-ΔSidE Legionella ).

Article Snippet: SNAP29-ser mutant , Lab generated using SNAP29 coding sequence of SNAP29 from addgene #69821 , .

Techniques: Expressing, Infection, Staining, Bacteria, Two Tailed Test, Immunostaining, Immuno-Electron Microscopy, Transfection, Labeling, Membrane, Mutagenesis, Imaging, Software, Control

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) A549 cells were treated with control or STX17 siRNA for 48 h followed by infection with WT Legionella . Cells were fixed and stained for the indicated autophagy markers 1 h after infection. The data are means ± SEM of 50 cells representing three experiments. which were analyzed per sample to quantify recruitment of FIP200 and ATG14L to bacteria. P value was calculated using two-tailed type 3 Student’s t test, *** P = 5.83E-7 (FIP200), *** P = 1.92E-12 (ATG14L), Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( B ) A549 cells were treated with STX17 or control siRNA for 48 h followed by infection with WT or ΔS Legionella for 12 h (MOI = 1). Cells were fixed for immunostaining with a Legionella -specific antibody followed by confocal microscopy. The LCV size was estimated in FIJI. In the box plot, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 32, 31 cells taken from three independent experiments. p value was calculated using two-tailed, type 3 Student’s t test, *** P = 8.13E-16. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( C ) A549 cells were treated with control or STX17 siRNA for 48 h followed by infection with WT or ΔS Legionella . Intracellular bacterial replication was assessed after 0, 24, and 48 h. Data are means ± SEM of three independent experiments. P value was calculated using two-tailed type 3 Student’s t test, ** P = 0.00526 (WT, control vs STX17siRNA, 24 h), ** P = 0.00815 (WT, control vs STX17siRNA, 48 h). ( D ) A549 cells were treated with STX17 or control siRNA for 48 h followed by transfection with WT or PR-Ub-deficient STX17 for 24 h. Intracellular bacterial replication was assessed after 0, 24, and 48 h. Data are means ± SEM of three independent experiments ** P = 0.0077 (WT, STX17siRNA versus STX17 mutant, STX17siRNA, 48 h). Western blotting with STX17 antibody shows knockdown efficiency of STX17 siRNA and reconstitution with WT or mutant STX17. ( E ) Proximity labeling assay workflow. HeLa cells expressing doxycycline-inducible APEX-STX17 and CD32(for increasing the efficiency of Legionella uptake) were infected with Legionella for 2 h before treatment with biotin-tyramide and H 2 O 2 followed by streptavidin pulldown. The samples were reduced, alkylated and digested with trypsin before MS analysis. Samples representing three biological replicates each of non-infected and Legionella -infected cells were analyzed in a single reaction by 6-plex TMT labeling. ( F ) Volcano plot showing changes in the biotin-labeled proteome following the infection of HeLa cells expressing APEX2-FLAG-STX17 with ΔR and ΔRΔS Legionella for 2 h, GO analysis of the biotin-labeled proteome showing pathways upregulated by infection with ΔR vs ΔRΔS Legionella . Data represents mean fold change of three experimental replicates per infection set ( n = 3). P value was calculated using two-tailed type 3 Student’s t test and significant candidates were chosen having P value ≤ 0.01 and log2 (fold change) value minimum of ±0.5. Red and green indicate compartments containing proteins enriched following infection with ΔR and ΔRΔS Legionella , respectively. ( G ) Cells expressing doxycycline-inducible APEX-STX17 were infected with Legionella for 2 h before treatment with biotin-tyramide and H 2 O 2 followed by streptavidin pulldown. The samples were analyzed by western blot with antibodies against proteins of the autophagic and endosomal pathways. The data represent means ± SD of three independent experiments. P value was calculated using two-tailed type 3 Students t test. WT vs ΔS P values: * P = 0.01006 (FIP200), ** P = 0.0206 (ULK1), P = ***0.0007 (ATG13) ** P = 0.005 (ATG14), * P = 0.0111(Beclin1), * P = 0.0219 (WIPI2), ** P = 0.0038 (ATG5), * P = 0.018 (ATG12), ** P = 0.001 (ATG16), ** P = 0.0036 (VAMP8), * P = 0.0424 (SNAP29). (n.i. not infected, WT wild-type, Legionella , ΔS ΔSidE Legionella ). .

Article Snippet: SNAP29-ser mutant , Lab generated using SNAP29 coding sequence of SNAP29 from addgene #69821 , .

Techniques: Control, Infection, Staining, Bacteria, Two Tailed Test, Immunostaining, Confocal Microscopy, Software, Transfection, Mutagenesis, Western Blot, Knockdown, Labeling, Expressing

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) PR-Ub (PDB ID: 5M93) was manually attached to residue S63 of SNAP29 in the structure of the STX17-SNAP29-VAMP8 complex (7BV6) using Pymol. ( B ) Schematic showing the formation of the autophagosomal SNARE complex, and the effect of PR-ub on its formation. ( C ) Untagged SNAP29 and STX17-GST was purified from E. coli and incubated with His-tagged Ub in an in vitro PR-ub assay. PR-Ub modified STX17 and SNAP29 were enriched by nickel-NTA-based affinity purification and incubated with 100 µg protein lysate from HEK 293T cells. This was followed by GST-pulldown of STX17 and antibody-based immunoprecipitation of SNAP29 to check for alterations in protein-protein interactions upon PR-Ub. The data represents means ± SD of three independent experiments P value was calculated using two-tailed type 3 Students t test 0.001 < ** P ≤ 0.01). ** P = 0.004 (VAMP8), ** P = 0.001 (ATG14L), ** P = 0.022 (PR-Ub STX17). ( D ) WT and PR-Ub-modified SNAP29 were incubated with equimolar amounts of purified GST-STX17, VAMP8 and ATG14L. Interactors of PR-Ub-modified SNAP29 were observed by immunoblotting. The data represent means ± SD of three independent experiments. P value was calculated using two-tailed type 3 Students t test. ** P = 0.0068 (STX17), ** P = 0.0059 (VAMP8). ( E ) WT or PR-Ub modified GST-STX17 were incubated with equimolar amounts of purified GST-STX17, VAMP8 and ATG14L. Interactors of PR-Ub modified STX17 were observed by immunoblotting. The data represent means ± SD of three independent experiments. P value was calculated using two-tailed type 3 Students t test. ** P = 0.042 (ATG14L), ** P = 0.029 (SNAP29). ( F ) WT or PR-Ub modified forms of both STX17 and SNAP29 were incubated with VAMP8 and ATG14L followed by immunoprecipitation of GST-STX17 and immunoblotting to check for protein-protein interactions. Pure proteins were run as the reaction inputs and immunoblotted with indicated antibodies. The data represent means ± SD of three independent experiments. P value was calculated using two-tailed type 3 Students t test. ** P = 0.0208 (ATG14L), ** P = 0.023 (VAMP8), *** P = 0.00046 (SNAP29). ( G ) Schematic showing the effect of PR-Ub of STX17 and SNAP29 on bacterial vacuole formation. PR-Ub of STX17 enhances interactions with ATG14L which results in the recruitment of ER membranes to the bacterial vacuole.PR-Ub of SNAP29 prevents fusion of the STX17-positive vacuoles with the VAMP8-positive lysosomes. .

Article Snippet: SNAP29-ser mutant , Lab generated using SNAP29 coding sequence of SNAP29 from addgene #69821 , .

Techniques: Residue, Purification, Incubation, In Vitro, Modification, Affinity Purification, Immunoprecipitation, Protein-Protein interactions, Two Tailed Test, Western Blot