pcdna 3.2/v5-dest snap29-v5  (Addgene inc)

Journal: bioRxiv

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulate autophagy in Legionella infection

doi: 10.1101/2025.05.19.654886

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 3 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 3 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: 5µm. Scale bar in inset: 2µm. 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 25 th and 75 th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles, outliers are represented by dots. N = 52, 56 cells taken from 3 experimental replicates. p value was calculated using 2 tailed, type 3 Student’s t-test, ***p = 7.43E-8. Scale bar: 5µm. 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 3 independent experiments. p value was calculated using 2 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: 5µm.

Article Snippet: FLAG-STX17 was a gift from Noboru Mizushima (Addgene #45911; RRID Addgene_45911) and pcDNA 3.2/V5-DEST SNAP29-V5 was a gift from Anne Brunet (Addgene #69821; RRID Addgene_69821).

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

Journal: bioRxiv

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulate autophagy in Legionella infection

doi: 10.1101/2025.05.19.654886

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 indicated autophagy markers 1 h after infection. The data are means ± SEM of 50 cells representing three experiments. which were analyzed per sample to quantitate recruitment of FIP200 and ATG14L to bacteria. p value was calculated using 2 tailed type 3 Student’s t-test, ***p=5.83E-7(FIP200), ***p=1.92E-12(ATG14L), Scale bar: 5µm. 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 3 independent experiments. p value was calculated using 2 tailed, type 3 Student’s t-test, ***p =8.13E-16. Scale bar: 5µm. 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 2 tailed type 3 Student’s t-test, **p =0.00526 (WT, control vs STX17siRNA, 24h), **p =0.00815 (WT, control vs STX17siRNA, 48h), 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 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 6plex 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 2 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 represents means ± SD of 3 independent experiments. p value was calculated using 2 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: FLAG-STX17 was a gift from Noboru Mizushima (Addgene #45911; RRID Addgene_45911) and pcDNA 3.2/V5-DEST SNAP29-V5 was a gift from Anne Brunet (Addgene #69821; RRID Addgene_69821).

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

Journal: bioRxiv

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulate autophagy in Legionella infection

doi: 10.1101/2025.05.19.654886

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 100ug protein lysate from HEK293T 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 3 independent experiments p value was calculated using 2 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 represents means ± SD of 3 independent experiments. p value was calculated using 2 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 represents means ± SD of 3 independent experiments. p value was calculated using 2 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 represents means ± SD of 3 independent experiments. p value was calculated using 2 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: FLAG-STX17 was a gift from Noboru Mizushima (Addgene #45911; RRID Addgene_45911) and pcDNA 3.2/V5-DEST SNAP29-V5 was a gift from Anne Brunet (Addgene #69821; RRID Addgene_69821).

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