Journal: bioRxiv

Article Title: Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

doi: 10.1101/2020.09.29.319145

Figure Lengend Snippet: ( A ) SNAP25 sub-family proteins and their isoforms (CLUSTAL 2.1 ). ( B ) Co-crystal structure (PDB: 1XTG, ) of LC/A (white) and SNAP25 (dark gray). Eight substitutions in LC/A drive SNAP23 specificity (teal) through substrate control loops (pink) alongside prior substitutions (light gray). ( C ) Platform for the directed evolution of SNAP23 substrate specificity. 1. Random or site-directed mutagenesis (e.g., site-saturation); 2. QC by DNA sequencing; 3. High-throughput protein production; 4. Measure V 0 23 and V 0 for substrate specificity; 5. Confirmation screens. The most specific and consistent variant from the DARET assay entered the next round of directed evolution. ( D ) Sequence alignment of substrates used for screening (UniProt: P60880, O00161). The SNAP binding exosites in LC/A (residue numbers above) and cleavage site (scissors) are shown. The gradient of color indicates homology from identical (white, *), to strongly similar (light gray,:), weakly similar (teal, .), or dissimilar (dark teal).

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals, NBC1-18347) with 400 nM of either wild type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37°C for 1 hour in PBS, pH 7.2 (ThermoFisher, 20012-043).

Techniques: Control, Mutagenesis, DNA Sequencing, High Throughput Screening Assay, Variant Assay, Sequencing, Binding Assay, Residue

Journal: bioRxiv

Article Title: Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

doi: 10.1101/2020.09.29.319145

Figure Lengend Snippet: As shown by the specificity index (V 0 23 /V 0 25 ) at 2 μM substrate, qmLC/A in cell lysate is highly specific for SNAP25. However, diluting the cell lysate increases the SNAP23 specificity of qmLC/A. Each point represents a different technical or biological replicate at the indicated final lysate dilution. The average value and standard deviation are shown as numbers above each dilution with the total number of data points in parentheses. Horizontal lines indicate average value and vertical lines indicate one standard deviation. The four dilutions yielded significantly different specificity indices as analyzed by an unpaired t-test in GraphPad Prism ( p = 0.001).

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals, NBC1-18347) with 400 nM of either wild type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37°C for 1 hour in PBS, pH 7.2 (ThermoFisher, 20012-043).

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

doi: 10.1101/2020.09.29.319145

Figure Lengend Snippet: Cartoon representing the DARET assay for SNAP cleavage by LC/A variants. CTD, C-terminal domain, corresponds to residues 137 to 211 of SNAP25 or residues 134 to 206 of SNAP23.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals, NBC1-18347) with 400 nM of either wild type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37°C for 1 hour in PBS, pH 7.2 (ThermoFisher, 20012-043).

Techniques:

Journal: bioRxiv

Article Title: Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

doi: 10.1101/2020.09.29.319145

Figure Lengend Snippet: In Round 8 of directed evolution, diluted lysates (1:1800) containing qmLC/A, omLC/A, or the most SNAP23 specific variant from Round 7 (R7) were tested for SNAP cleavage in “salt-free” (50 mM HEPES pH 7.4, 0.05% Tween) or salt (50 mM KH 2 PO 4 pH 7.4) buffered conditions. ( A ) The qmLC/A variant is highly specific for SNAP25 in both no salt and salt conditions. ( B ) The R7 LC/A variant is specific for SNAP23 in no salt conditions but becomes specific for SNAP25 in salt. ( C ) The omLC/A variant is specific for SNAP23 in no salt and salt conditions. Each line depicts the mean of three technical replicates after subtracting a no-enzyme blank.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals, NBC1-18347) with 400 nM of either wild type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37°C for 1 hour in PBS, pH 7.2 (ThermoFisher, 20012-043).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

doi: 10.1101/2020.09.29.319145

Figure Lengend Snippet: ( A ) Residues occupying substrate control loops 20 and 50 are highlighted for wtLC/A (top) and omLC/A (bottom, computational modeling). ( B ) Putative modeled protein-protein interactions between SNAP23 sidechains (navy) and omLC/A substrate control loop residues (teal) (LigPlot + software). Hydrophobic interactions are depicted in purple, and putative polar contacts are shown in blue.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals, NBC1-18347) with 400 nM of either wild type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37°C for 1 hour in PBS, pH 7.2 (ThermoFisher, 20012-043).

Techniques: Control, Protein-Protein interactions, Software

Journal: bioRxiv

Article Title: Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

doi: 10.1101/2020.09.29.319145

Figure Lengend Snippet: In Round 6 of directed evolution, 34 variants from the alpha exosite K337NDT LC/A library were tested for ( A ) SNAP25 or ( B ) SNAP23 cleavage in the DARET assay. Each line represents the diluted lysate (1:1800) of a single well of a 96 deep-well plate.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals, NBC1-18347) with 400 nM of either wild type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37°C for 1 hour in PBS, pH 7.2 (ThermoFisher, 20012-043).

Techniques:

Journal: bioRxiv

Article Title: Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

doi: 10.1101/2020.09.29.319145

Figure Lengend Snippet: ( A ) Rates of SNAP cleavage by batch-expressed, purified qmLC/A and ( B ) omLC/A at the indicated DARET substrate concentrations (n=3). ( C ) Deconvoluted ESI-MS of recombinant, full-length SNAP23 (fl-S23) treated with DTT and iodoacetamide to carbamidomethylate its cysteines (6x CAM). The mass spectrum of intact fl-S23 incubated with buffer (top, black) is compared to that of fl-S23 incubated with omLC/A (bottom, teal). Intact fl-S23 ( 1 ) and cleaved fl-S23 (cl. fl-S23, 2 ) peaks are labeled. Additional marked peaks correspond to the masses of peaks 1 and 2 plus one additional CAM (+57 Da), likely resulting from overalkylation by iodoacetamide . ( D ) Recombinant omBoNT/A was purified by IMAC followed by anion exchange (AEX) chromatography. The omBoNT/A is ≈95% nicked upon DTT reduction as demonstrated by the presence of the HC/A and omLC/A bands. ( E ) In vitro cleavage of recombinant fl-S23 by two independent preparations of omBoNT/A (1 and 2) visualized with a C-terminal anti-SNAP23 antibody; before proteolysis, omBoNT/A was reduced with TCEP. The untreated, wtLC/A, and wtLC/E lanes provide negative controls.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals, NBC1-18347) with 400 nM of either wild type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37°C for 1 hour in PBS, pH 7.2 (ThermoFisher, 20012-043).

Techniques: Purification, Recombinant, Incubation, Labeling, Chromatography, In Vitro

Journal: bioRxiv

Article Title: Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

doi: 10.1101/2020.09.29.319145

Figure Lengend Snippet: ( A ) Design of SNAP29 DARET substrate for fluorescence polarization assays of proteolytic cleavage, featuring the 73 C-terminal residues of SNAP29. ( B ) The omLC/A variant exhibits no SNAP29 cleavage in the DARET assay. SNAP29 is the closest related SNAP family member to SNAP25 and SNAP23. Trypsin provides a positive control for SNAP29 cleavage. Each solid line depicts the mean of three technical replicates (50 nM enzyme, 300 nM substrate). The dotted lines and the shaded area depict the standard deviation around the mean; however, the standard deviation for each condition tested is too small to be visible on the graph.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals, NBC1-18347) with 400 nM of either wild type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37°C for 1 hour in PBS, pH 7.2 (ThermoFisher, 20012-043).

Techniques: Fluorescence, Variant Assay, Positive Control, Standard Deviation

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A SNAP29, SNAP23, STX3, SEC22B, and FKBP5 co-immunoprecipitation (SKA2 IP) and whole cell extract (WCE) in hippocampus (HIP), prefrontal cortex (PFC) and amygdala (AMY) samples of mice ( n = 8). B HIS pull down assay (replicated in 3 independent in vitro experiments). DDK(Flag)-tagged SNAP23, SNAP29, Syntaxin3 or Syntaxin4 was incubated with purified magnetic beads-HIS-tagged SKA2 or magnetic beads-HIS protein alone. After incubation, bead bound proteins were eluted at room temperature (RT) or at 95 °C and subjected to western blot analysis using antibodies against HIS and FLAG. Input lane contains HIS alone (left) or HIS-tagged SKA2 (right). C – M SIM-A9 cells transfected with SKA2, FKBP5 or their respective controls, were harvested 24 h later. After immunoprecipitation (IP) of protein complexes, input and co-IP proteins were quantified by western blotting. C , F , I , K Representative blots of ( D , E , G , H , J , L , M ). Graphs display quantification of SNAP29/SEC22B, STX3/SEC22B, SKA2/SNAP29, FKBP5/SEC22B protein association after SEC22B or SNAP29 IP (unpaired two tailed t-test: ( D ) t 6 = 8.945, p < 0.0001, ( E ) t 6 = 12.94, p < 0.0001, ( G ) t 6 = 6.056, p = 0.0009, ( H ) t 6 = 5.554, p = 0.0014; one-way ANOVA: ( J ) F 2, 9 = 17.28, p = 0.0008, Tukey’s post hoc test: ctrl vs. FKBP5-OE, p = 0.0743, ctrl vs. FKBP5-KO, p = 0.0218, FKBP5-OE vs. FKBP5-KO, p = 0.0006; unpaired two tailed t-test: ( L ) t 6 = 10.27, p < 0.0001, ( M ) t 6 = 8.140, p = 0.0002; n = mean derived from four independent in vitro experiments). * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: Purified Syntaxin3-DDK (Origene, TP300658), SNAP29-DDK (Origene, TP302179), Syntaxin4 (Origene, TP300347), SNAP23-DDK (Origene, TP301596) or correspondingly SEC22B-HIS (Origene, AR50533PU-S) (100 ng) was used for the binding reaction.

Techniques: Immunoprecipitation, Pull Down Assay, In Vitro, Incubation, Purification, Magnetic Beads, Western Blot, Transfection, Co-Immunoprecipitation Assay, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A , B IL-1β release measured via ELISA from supernatants of SIM-A9 cells 24 h after manipulation of SKA2 and/or FKBP5 expression, and following overnight LPS (100 ng/mL) and treatment with LLOMe (0.25 mM) for 3 h (unpaired two tailed t-test: (A) t 4 = 11.99, p = 0.0003; one-way ANOVA: B F 3, 8 = 158.6, p < 0.0001; Tukey’s post hoc test: ctrl vs. SKA2-OE, p = 0.0384, ctrl vs. FKBP5-OE, p < 0.0001, SKA2-OE vs. FKBP5-OE, p < 0.0001, FKBP5-OE vs. SKA2 + FKBP5 OE, p < 0.0001; n = mean derived from three independent in vitro experiments). C Schematic overview of the SA pathway with SKA2 and FKBP5. The cargo receptor TRIM16, together with SEC22B, transfers molecular cargo (e.g., IL-1β) to the autophagy-related LC3B-positive membrane carriers. SEC22B, now acting as an R-SNARE on the delimiting membrane facing the cytosol, carries out fusion at the plasma membrane in conjunction with the Q bc -SNAREs, SNAP23 and SNAP29 (SNAP23/29), and one of the plasma membrane Q a -SNAREs, STX3 or STX4 (STX3/4), thus delivering IL-1β to the extracellular milieu, where it exerts its biological functions. FKBP5 acts as a positive regulator of SA by enhancing TRIM16-SEC22B complex formation as well as autophagosome-plasma membrane fusion via the SNARE-protein complex assembly. In contrast, SKA2 inhibits the SNARE-protein complex formation during vesicle-plasma membrane fusion, thereby acting as gatekeeper of SA. D , E Schematic overview of in vivo microdialysis and the experimental design and timeline; each sample was collected over 30 min indicated by the light gray lines. Quantifications of IL-1β, determined by capillary-based immunoblotting from in vivo medioprefrontal cortex microdialysis of C57Bl/6NCrl mice injected intraperitoneally with ULK1 inhibitor (ULK1i, an autophagy inhibitor) or saline ( F ; repeated measures two-way ANOVA, time × treatment interaction: F 5, 30 = 7.064, p = 0.0002; Šidák’s multiple comparisons post hoc test, post-FS-1: p = 0.0084; n = 4 mice per group) as well as of wild type (WT) and global Fkbp5 knockout mice ( G ; repeated measures two-way ANOVA, time × genotype interaction: F 5, 30 = 34.15, p < 0.0001; Šidák’s multiple comparisons post hoc test: FS: p = 0.009, post-FS-1: p = 0.0163, post-FS-2: p = 0.0294; n = 4 mice per group). FS foot shock. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: Purified Syntaxin3-DDK (Origene, TP300658), SNAP29-DDK (Origene, TP302179), Syntaxin4 (Origene, TP300347), SNAP23-DDK (Origene, TP301596) or correspondingly SEC22B-HIS (Origene, AR50533PU-S) (100 ng) was used for the binding reaction.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Derivative Assay, In Vitro, Membrane, In Vivo, Western Blot, Injection, Saline, Knock-Out

Journal: Advanced Science

Article Title: Delivering Antisense Oligonucleotides across the Blood‐Brain Barrier by Tumor Cell‐Derived Small Apoptotic Bodies

doi: 10.1002/advs.202004929

Figure Lengend Snippet: sCABs are transcytosed by BMECs to cross the BBB. a) TEM images showing a phagosome containing a sCAB in the BMEC of a mouse 10 min after an i.v. sCABs injection. The right panel shows a magnified view of the phagophore selected by the red square and arrow. Scale bar: 500 nm. b) TEM images showing the structure of TJs after sCABs passed through microvessels 15 min after an i.v. sCABs injection. The TJs in the red squares are shown at high magnifications on the right. The red arrow depicts sCABs that passed through BMECs. Scale bar: 500 nm. c) Representative fluorescence images showing the intact structure of TJs after sCABs treatment containing Cy5‐ASO. The right panels show magnified views of the area selected by the white square. Scale bar: 25 µm. d) Representative fluorescence microscopy images showing sCABs containing Cy5‐ASO phagocytized by b. End3 cells in the BBB model. Scale bar: 25 µm. e) Representative fluorescence images showing Cy5‐ASO delivered by sCABs but not the naked one was phagocytized by microglial cells in the BBB model 24 h after the incubation. The morphology of microglial cells as imaged with differentia linterference contrast (DIC). Scale bar: 25 µm. f) Fluorescence images showing GFP‐labeled sCABs containing Cy5‐ASO phagocytized by microglia 24 h after the incubation. The right panels show magnifications of the area selected by the white square. Scale bar: 10 µm. g–k) Representative fluorescence images showing the colocalization of sCABs containing Cy5‐ASO with special protein markers ((g) labeled with clathrin, (h) with caveolin‐1, (i) with EEA‐1, (h) with Rab11 involved in endocytosis, and (k) with Snap23). The colocalization of clathrin and caveolin‐1 was examined at 5 min after sCABs incubation, EEA‐1 and Rab11 at 10 min, and Snap23 at 20 min. The right panels show magnifications of the areas selected by the white square. Scale bar: 25 µm. Images are representative of three independent experiments.

Article Snippet: Moreover, Snap23 (anti‐Snap23 antibody, BA2805, Boster, China), a membrane receptor involved in the interaction of endosomes with the basolateral membrane, was examined at 20 min.

Techniques: Injection, Fluorescence, Microscopy, Incubation, Labeling

Journal: Journal of Ovarian Research

Article Title: SNAP23 promotes the malignant process of ovarian cancer

doi: 10.1186/s13048-016-0289-9

Figure Lengend Snippet: SNAP23 is highly expressed in OC tissues and cells. a . Immunohistochemistry analysis in The Human Protein Atlas revealed that normal ovarian tissues nearly express SNAP23, but most ovarian tumor tissues are positive for SNAP23. Protein was expressed at high levels in tumor tissues. b and c . The results of qRT-PCR and western blot assays showed that SNAP23 mRNA and protein are hyper-expressed in most ovarian cancer cell lines. d and e . Both of two designed siRNAs showed favorable inhibition, and siRNA1 had a better efficiency

Article Snippet: By analyzing the Human Protein Atlas, we compared the protein expression of SNAP23, SNAP25, SNAP29 and SNAP47 in ovarian normal and tumor tissues.

Techniques: Immunohistochemistry, Quantitative RT-PCR, Western Blot, Inhibition

Journal: Journal of Ovarian Research

Article Title: SNAP23 promotes the malignant process of ovarian cancer

doi: 10.1186/s13048-016-0289-9

Figure Lengend Snippet: Knockdown of SNAP23 alters OC cell line proliferation, migration and invasion in vitro. a . Both of the designed siRNAs showed favorable inhibitory efficiency, and siRNA1 had a better efficiency than siRNA2. b . Depletion of SNAP23 undermined both SK and A2780 cells ( p < 0.05). c . Colony numbers of SK and A2780 cells transfected with si-SNAP23 are less than those transfected with si-NC ( p < 0.001). d . Migratory and invasion rates of SK and A2780 cells transfected with si-SNAP23 are decreased compared with NC group. e . Si-SNAP23 impaired migration as compared with NC group in wound healing assay ( p < 0.001)

Article Snippet: By analyzing the Human Protein Atlas, we compared the protein expression of SNAP23, SNAP25, SNAP29 and SNAP47 in ovarian normal and tumor tissues.

Techniques: Knockdown, Migration, In Vitro, Transfection, Wound Healing Assay

Journal: Journal of Ovarian Research

Article Title: SNAP23 promotes the malignant process of ovarian cancer

doi: 10.1186/s13048-016-0289-9

Figure Lengend Snippet: SNAP23 depletion increase OC cells apoptosis without influencing on cell cycle in vitro. a and b . SK cells transfected with si-SNAP23 led to apoptosis increase compared to si-NC. c and d . Silencing SNAP23 of SK cells showed no influence on cell cycle stage

Article Snippet: By analyzing the Human Protein Atlas, we compared the protein expression of SNAP23, SNAP25, SNAP29 and SNAP47 in ovarian normal and tumor tissues.

Techniques: In Vitro, Transfection

Journal: Journal of Ovarian Research

Article Title: SNAP23 promotes the malignant process of ovarian cancer

doi: 10.1186/s13048-016-0289-9

Figure Lengend Snippet: SNAP23 associated with a poor PFS and may influence apoptotic and metabolic processes in OC. a . KM-ploter analysis revealed that high expression of SNAP23 was associated with a poor progress-free-survival of OC patients (1187 patients were involved, and the median PFS of SNAP23 high expression were 17.0 months, while SNAP23 low expression 19.3 months). b and c . GO enrichment analysis indicated that SNAP23 expression was highly correlated with genes enriched in the metabolic process and biological regulation. d . We hypothesized that SNAP23 might influence cancer process via inhibiting apoptosis and promoting metabolic process

Article Snippet: By analyzing the Human Protein Atlas, we compared the protein expression of SNAP23, SNAP25, SNAP29 and SNAP47 in ovarian normal and tumor tissues.

Techniques: Expressing