Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: A workflow of APEX2-mediated proximity labeling and generation of inducible HEK293 cell lines expressing MAAP2-APEX2 and APEX2 (A) Diagram of workflow for APEX2-mediated proximity labeling. 293 iMAAP2−APEX2 or 293 iAPEX2 cells were infected with AAV2 ΔMAAP and transfected with pHelper. At 16 hpi, doxycycline (Dox) was added to induce expression of MAAP2-APEX2 or APEX2, and infected with AAV2 ΔMAAP . Upon addition of biotin-phenol (BP) and H 2 O 2 , APEX2 catalyzes the oxidation of BP to biotin-phenoxyl radicals within ∼20 nm in diameter, thereby resulting in the biotinylation of MAAP2-associated proteins (APs) in 1 min. The reaction is then quenched and followed by a pulldown of MAAP2-APs using streptavidin magnetic beads. MAAP2-APs are identified by quantitative mass spectrometry (qMS). (B) MAAP2-APEX2 and APEX2 only expressing lentiviruses. TripZ-APEX2 and TripZ-MAAP2-APEX2 lentiviruses express APEX2 only (serves as negative control) and MAAP2-APEX2, respectively. APEX2 and MAAP2-APEX2 were expressed with a V5-tag at the N terminus of APEX2 as indicated. (C) Dox-inducible cell lines. 293 iAPEX2 and 293 iMAAP2−APEX2 cells were seeded on wells of a six-well plate, Dox was added to induce expression of APEX2 (∼29 kDa with the linker and V5 tag) or MAAP2-APEX2 (∼43 kDa with the linker and V5 tag). Cell lysates of mock (−) and Dox-added (+) 293 iAPEX2 or 293 iMAAP2−APEX2 cells were immunoblotted for V5 tag. β-actin is shown as a loading control.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Labeling, Expressing, Infection, Transfection, Magnetic Beads, Mass Spectrometry, Negative Control, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: Identification of MAAP2-associating host proteins by APEX2-mediated proximity labeling (A) Optimized biotinylation for sampling of quantitative mass spectrometry (qMS). 293 iMAAP2−APEX2 and 293 iAPEX2 cells in a T25 flask were infected with AAV2 ΔMAAP at an MOI of 5,000 vgc/cell. At 16 hpi, Dox was added. At 2 dpi, APEX2-mediated biotinylation was carried out as described in . Cells were incubated with biotin-phenol for 30 min at 37°C. H 2 O 2 was added and incubated for 1 min at room temperature to biotinylate proximal proteins. The reaction was then quenched. The cells were harvested and lysed in RIPA buffer. The supernatant of the lysates was then incubated with streptavidin beads. Ten percent of the washed beads were separated on SDS-PAGE for Coomassie staining and western blotting (WB), respectively. The western blot was probed with Alexa Fluor 680-conjugated streptavidin (Thermo Fisher). (B) Analysis of the identified proteins by qMS. The remaining 90% of the washed beads were subjected to on-bead digestion and liquid chromatography-tandem mass spectrometry analysis. Three repeats were carried out. The qMS data listed in Table S1 were analyzed. The volcano plot shows statistical significance (−log 10 , p value) vs. the magnitude of change (log 2 , fold-change) for differentially interacted proteins between MAAP2-APEX2-and APEX2-expressing cells. Pink dots indicate enriched proteins by >2-fold and green dots indicate depleted proteins by >2-fold. The t test (unpaired, two-tailed) was employed for determination of statistical significance ( p value). The horizontal dash line indicates p < 0.05. (C) Gene Ontology (GO) annotation of top enriched proteins. In the volcano plot (B), proteins clustered in the upper/right corner as divided by the blue dash lines, which were enriched by >16 times and had significance ( p value) of >0.01 in the MAAP2-APEX2-expressing cells, were analyzed by GO. Proteins are shown in circles with colors indicating their functions. The size of the circles represents the enrichment score (fold-change in log 2 ).

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Labeling, Sampling, Mass Spectrometry, Infection, Incubation, SDS Page, Staining, Western Blot, Liquid Chromatography, Expressing, Two Tailed Test

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: STX7 or SNAP23 colocalizes with MAAP2 in both wtAAV2-infected cells and rAAV2-producing cells (A and D) wtAAV2 infection. HEK293 cells were mock-infected or infected with wtAAV2 followed by pHelper transfection. At 2 dpi, the cells were co-immunostained for MAAP2 and SNAP23 (A) or MAAP2 and STX7 (C). (B and E) rAAV2 production. HEK293 cells were mock or transfected with pR2C2, pHelper, and prAAV2. At 2 dpt, the transfected cells were co-immunostained for MAAP2 and SNAP23 (B) or MAAP2 and STX7 (D). SNAP23 (B) or STX7 (D) cells were co-immunostained with a secondary antibody conjugated with a far-red dye. Co-immunostained cells were observed under a Leica STED microscope with a 100× objective lens. Images captured in the far-red wavelength were pseudo-colored in red. The colors of confocal images correspond to blue for DAPI, green for MAAP, and red for SNAP23 or STX7 as indicated. Scale bar, 5 μm. Representative confocal images are shown. (C and F) Quantification of colocalization. Pearson’s correlation coefficients were measured for colocalization of MAAP2 with SNAP23 (C) or STX7 (F) using NIH ImageJ.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Infection, Transfection, Microscopy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: SNAP23 or STX7 interacts with MAAP2 in cells but not in vitro (A and B) Co-IP. HEK293 cells were transfected with pCI-MAAP2 Flag or pCI-empty. At 2 dpt, cells were harvested and lysed. 90% of the lysates were used for immunoprecipitation with anti-Flag-conjugated magnetic beads. Western blotting was performed for detection of MAAP2 Flag and SNAP23, respectively (A), and for detection of MAAP2 Flag and STX7, respectively (B). β-actin is shown as a loading control. 10% of the lysates were loaded as the whole cell lysate (WCL). (C and D) In vitro pulldown assay. Approximately 2 μg of the purified GST-MAAP2 protein and negative control GST protein were employed as baits to pull down ∼2 μg prey proteins; purified SNAP23 (C) or STX7 (D) using glutathione agaroses. Western blotting was performed for detection of GST-MAAP2 and control GST using anti-GST (C and D), for the detection of SNAP23 using anti-SNAP23 (C), and for the detection of STX7 using anti-STX7 (D). ∼200 ng of the bait and pray proteins were loaded as inputs. Asterisks indicate the major detected GST-MAAP and GST.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: In Vitro, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Control, Purification, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: A proposed model of the role of SNARE proteins in AAV egress AAV capsids produced in the nucleus are egressed through the nuclear pore complex (NPC), the endoplasmic reticulum (ER), and the Golgi apparatus, where a portion of the vectors are associated with EVs (marked with exosome marker CD63), on which MAAP2 is attached. , These EV-associated or -carried AAVs are routed to late endosomes (Rab7+) or MVB through the egress pathway of EVs as exosomes to secrete EV-AAVs. A portion of the late endosomes/MVBs are captured or recognized by v-SNARE proteins (e.g., STX7). STX7 interacts with t-SNARE proteins, e.g., SNAP23, for the fusion of late endosomes with lysosome, where SNAP23 makes up the heterodimeric t-SNAREs required for lysosome exocytosis, leading to the formation of lysosome/endosome hybrid for degradation of the virions. On the other hand, the EV-associated AAV can reach early endosomes (Rab5+) and then traffic to recycling endosomes (Rab11+), where they can be released as microvesicles. Knockout of SNARE expression can block the formation of a lysosome/endosome hybrid, resulting in less virion degradation and more virion release/secretion out of the plasma membrane.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Produced, Marker, Knock-Out, Expressing, Blocking Assay, Membrane

Journal: Molecular Biology of the Cell

Article Title: Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

doi: 10.1091/mbc.E13-10-0592

Figure Lengend Snippet: The K 2P channel TASK-1 interacts with the SNARE protein syntaxin-8. (A) The topology of TASK-1 and stx8. (B) Membrane yeast two-hybrid screen with TASK-1 or TASK-3 as bait and stx8 or mutants thereof as prey. The Q179A mutant of stx8 cannot assemble with other SNARE proteins; in the Δ100–140 mutant the linker between the Hc domain and the SNARE domain was excised. (C) RT-PCR analysis of TASK-1 and endosomal SNARE proteins in human brain and in A549 cells. Asterisks represent nonspecific PCR products. (D) Coimmunoprecipitation of stx8 and TASK-1 endogenously expressed in A549 cells. The complex containing TASK-1 was precipitated from cell lysate with a TASK-1–specific antibody from Alomone (APC-024), and a Western blot of the precipitate was probed with TASK-1, stx8, and stx7 antibodies (left); the cell lysate (input) was used as positive control. Coimmunoprecipitation with an unrelated immunoglobulin G antibody (Santa Cruz Biotechnology) was used as a negative control (right).

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with either mouse anti-myc antibody (1:1000; Santa Cruz Biotechnology), rabbit anti-GFP antibody (1:1000; Abcam, Cambridge, United Kingdom), anti–TASK-1 (1:1000; Alomone), anti-stx8 (1:1000; BD Biosciences, Heidelberg, Germany), or anti-stx7 (1:1000; Acris Antibodies, Herford, Germany).

Techniques: Two Hybrid Screening, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Negative Control

Journal: Molecular Biology of the Cell

Article Title: Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

doi: 10.1091/mbc.E13-10-0592

Figure Lengend Snippet: Coexpression of TASK-1 with stx8 or stx7 in CHO cells and Xenopus oocytes. (A) Current–voltage relation of rTASK-1 expressed in CHO cells. The currents were measured using voltage ramps from −120 to +40 mV at pH 7.4 (black curve) and 6.0 (red curve). (B) TASK-1 current–voltage relation measured in the same batches of CHO cells 48 h after transfection of TASK-1 alone (black curve) and after cotransfection of TASK-1 with stx8 (green curve); mean values ± SEM of n = 28 cells. (C) Mean outward currents ± SEM measured in CHO cells at 0 mV after transfection with rat TASK‑1 alone (black) and after cotransfection of TASK-1 with stx8 (green) or stx7 (orange). (D) Typical current–voltage relation measured 48 h after injection of human TASK-1 cRNA (black curve) and after coinjection of TASK-1 and stx8 cRNA. For experiments with human TASK-1 we used the NQ TASK-1 mutant, which displays a higher current amplitude ( Zuzarte et al. , 2009 ; Materials and Methods ). (E) Mean outward currents ± SEM measured in Xenopus oocytes at 0 mV after injection of hTASK‑1 or hTASK-3 cRNA alone (black) or together with 1.5, 3, or 6 ng stx8 cRNA per oocyte as indicated. (F) Mean outward currents ± SEM measured in Xenopus oocytes at 0 mV measured after injection of hTASK‑1 cRNA alone (black) or together with 6 ng cRNA encoding stx8, VAMP8, vti1b, or stx7. (G) Mean surface expression of HA-tagged hTASK-1 channels (measured in relative light units [RLUs]) in Xenopus oocytes after injection of TASK-1 cRNA alone or together with 6 ng of stx8 or stx8 Q179A . (H) Mean hTASK-1 current measured in Xenopus oocytes after injection of hTASK-1 cRNA alone or together with stx8 or stx8 Q179A . In all bar graphs the number of oocytes or CHO cells from which the data were obtained is indicated in brackets. Note that in the series of experiments shown in E, F, and H, coinjection of 6 ng of stx8 cRNA caused a reduction of TASK-1 current to values between 13 and 23% of control, illustrating that there was a certain degree of variability among different batches of oocytes. For this reason, TASK-1 (and other) currents with and without coinjection of a second cRNA were always compared in the same batch of oocytes (measured on the same day); normalized current amplitudes of at least three different batches are combined in the bar graphs.

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with either mouse anti-myc antibody (1:1000; Santa Cruz Biotechnology), rabbit anti-GFP antibody (1:1000; Abcam, Cambridge, United Kingdom), anti–TASK-1 (1:1000; Alomone), anti-stx8 (1:1000; BD Biosciences, Heidelberg, Germany), or anti-stx7 (1:1000; Acris Antibodies, Herford, Germany).

Techniques: Transfection, Cotransfection, Injection, Mutagenesis, Expressing

Journal: Molecular Biology of the Cell

Article Title: Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

doi: 10.1091/mbc.E13-10-0592

Figure Lengend Snippet: Dissection of the interacting regions of stx8 and TASK-1. (A) Topology of stx8, stx7, and the stx8/stx7 chimeras. (B) TASK-1 currents measured in Xenopus oocytes expressing TASK-1 and stx8 or stx8/stx7 chimeras. (C) Normalized hTASK-1 currents measured in Xenopus oocytes expressing hTASK-1 and stx8 or deletion mutants of stx8. (D) Normalized currents measured in Xenopus oocytes expressing TASK-3/TASK-1 or TASK-1/TASK-3 chimeras alone or together with stx8 or stx8Q179A. (E) Schematic drawing of TASK-3/TASK-1 and TASK-1/TASK-3 chimeras.

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with either mouse anti-myc antibody (1:1000; Santa Cruz Biotechnology), rabbit anti-GFP antibody (1:1000; Abcam, Cambridge, United Kingdom), anti–TASK-1 (1:1000; Alomone), anti-stx8 (1:1000; BD Biosciences, Heidelberg, Germany), or anti-stx7 (1:1000; Acris Antibodies, Herford, Germany).

Techniques: Dissection, Expressing

Journal: Molecular Biology of the Cell

Article Title: Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

doi: 10.1091/mbc.E13-10-0592

Figure Lengend Snippet: Analysis of endocytosis of TASK-1 using an antibody uptake assay. (A) COS-7 cells expressing HA epitope–tagged TASK-1 (or TASK-1 mutants) and stx8 (or stx8 mutants) were incubated with an anti-HA antibody at 4°C to label the channels at the cell surface and then warmed to 37°C to initiate internalization. After incubation at 37°C for 30 min, the anti-HA–labeled channels that remained on the surface were detected with a secondary antibody labeled with Alexa Fluor 594 (red). Then the cells were permeabilized, and the internalized channels were detected with a different secondary antibody, labeled with Alexa Fluor 488 (green). The measurements were carried out 48 h after transfection of TASK-1, TASK-1 mutants, stx8, stx8 mutants, or stx7. Note that rTASK‑1 Y317A corresponds to hTASK-1 Y300A . (B) Statistical evaluation of the antibody uptake assay under different conditions. The ratio between the fluorescence of internalized channels (green) and channels at the cell surface (red) was calculated at 0 and 30 min after heating to 37°C; number of cells is indicated in brackets. Scale bars, 50 μm.

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with either mouse anti-myc antibody (1:1000; Santa Cruz Biotechnology), rabbit anti-GFP antibody (1:1000; Abcam, Cambridge, United Kingdom), anti–TASK-1 (1:1000; Alomone), anti-stx8 (1:1000; BD Biosciences, Heidelberg, Germany), or anti-stx7 (1:1000; Acris Antibodies, Herford, Germany).

Techniques: Expressing, Incubation, Labeling, Transfection, Fluorescence

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Localization of SNAREs on early endosomes by using immunocytochemistry. (a) Fluorescence micrographs showing endosomes labeled with dextran-Alexa 488 (green channel) immunostained for syntaxin 13 (Left) and use 1 (Right) (red channel). To determine colocalization, linescans were performed through the intensity centers of green endosomes (examples indicated by white lines). (b) Representative linescan analysis, obtained from the images shown in a, showing intensity profiles of green (endosomes) and red (antibody-staining) signals. (c) Colocalization between early endosomes and SNARE proteins as determined by linescan analysis and correlation (see Materials and Methods). A correlation coefficient of 1 represents complete colocalization, whereas a correlation coefficient of ≈0 represent independent distribution. Coint, simultaneous labeling with dextran-Alexa 488 and dextran-Alexa 594 (positive control); 2nd AB, omission of the primary antibody (negative control); syphy, synaptophysin; syx, syntaxin; eb, endobrevin/VAMP8; β-tub, β-tubulin; tfr, transferrin receptor. Values are means of two independent experiments with 60 analyzed endosomes each. Error bars indicate the range of values.

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Immunocytochemistry, Fluorescence, Labeling, Staining, Positive Control, Negative Control

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Effects of adding recombinant SNAREs on fusion of early endosomes. (a) Effects of Q-SNAREs considered to be involved in the fusion of early endosomes, including syntaxin 16 (syx 16), syntaxin 13 (syx 13), vti1a, and syntaxin 6 (syx 6). Values are means ± SEM of 7–10 independent experiments. (b) Effects of late endosomal and neuronal Q-SNAREs, including syntaxin 7 (syx 7), vti1b, syntaxin 8 (syx 8), syntaxin 1 (syx 1), and SNAP-25. Values are means ± SEM of 7–13 independent experiments. (c) Effects of R-SNAREs, including VAMP4, synaptobrevin/VAMP2 (syb), endobrevin/VAMP8 (eb), cellubrevin/VAMP3, Ti-VAMP/VAMP7, VAMP5, ykt6, and sec22. Values are means ± SEM of three to seven independent experiments. Individual Q-SNAREs were used at 12 μM, with R-SNAREs tested at 25 μM.

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Recombinant

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Proteoliposomes containing the Q-SNAREs syntaxin 13, syntaxin 6, and vti1a show no specificity for R-SNAREs in fusion. (a) Fusion with liposomes containing VAMP4, endobrevin, or synaptobrevin. Fusion was monitored by fluorescence dequenching due to dilution of labeled phospholipids with unlabeled phospholipids during fusion (see Materials and Methods) and normalized to maximal fluorescence measured after adding detergent at the end of the reaction. As control, Q-SNARE liposomes were preincubated for 1 h at room temperature with purified endobrevin or synaptobrevin lacking the transmembrane domain (soluble fragment, final concentration of 30 μM) before starting the fusion reaction. (b–d) Dose-dependent inhibition by soluble R-SNAREs of fusion between liposomes containing the Q-SNAREs syntaxin13, vti1a, and syntaxin 6 and liposomes containing the R-SNARE VAMP4. Measurements were performed at 37°C with an overall protein concentration of 3 μM in the liposomes. The soluble R-SNAREs were added simultaneously with the acceptor liposomes at the start of the reaction (concentrations represent final assay concentrations).

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Fluorescence, Labeling, Purification, Concentration Assay, Inhibition, Protein Concentration