Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Directed evolution overview. ( 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 25 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, space).

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

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

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Directed evolution of a SNAP23-specific LC/A. ( A ) Improvement in specificity index through directed evolution in salt-free buffer. The most specific variant from each round was assayed multiple times (replicates shown). The average specificity index (horizontal bars) and standard deviation (error bars) are shown. The final dilution of cell lysates for screening is indicated by color code. ( B ) Increased specificity index achieved through directed evolution in salt buffer. ( C ) Roadmap for directed evolution showing the fold increase in specificity indices over eight rounds (R1 to R8). As shown in Fig. S1, the substrate specificity of the qmLC/A variant is sensitive to screening dilution; therefore, each point represents the variant’s average specificity normalized to the qmLC/A average specificity at the corresponding screening dilution. Advancement to the next round (solid line) weighed specificity index, protein solubility, and stability.

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

Techniques: Variant Assay, Standard Deviation, Solubility

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Enzyme kinetics of LC/A variants.

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

Techniques:

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Characterization of omLC/A cleavage. ( 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 six cysteines (6 × 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 . The cleaved peptide was not directly observed, but inferred from the mass difference of peaks 1 and 2. The deconvolution error is +/- 2 Da. ( 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. ( F ) In cellulo cleavage of SNAP23 and SNAP25 in SiMA-H1 cells infected with adenovirus delivering DNA encoding mCherry/SNAP23. Cells were treated with omBoNT/A or wtBoNT/A or without toxin (ct). Proteins, full-length (fl) or cleaved (cl), were identified by Western blotting with anti-SNAP23, -SNAP25, or -mCherry antibodies (M, MW marker). Full-length images of these gels with multiple exposures where necessary are shown in Fig. S12 and S13).

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

Techniques: Purification, Recombinant, Incubation, Labeling, Chromatography, In Vitro, Infection, Western Blot, Marker

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: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Western Blot, Expressing, Labeling

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: EHD2 KD in 3T3-L1 adipocytes causes impaired SNARE protein assembly. PLA was used to assess pairwise interactions of proteins in 3T3-L1 adipocytes, fixed 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), and following 0 (Basal), 5- or 20-min stimulation with 100 nM insulin. Representative images with PLA signal in green and nuclei staining (DAPI) in blue are shown alongside corresponding quantification of PLA signal per cell normalized to basal control for the following protein pairs: (A) SNAP23 and VAMP2; (B) SNAP23 and Munc18c; (C) VAMP2 and Munc18c; and (D) Syntaxin4 and Munc18c. Mean ± SD of n = 3 independent experiments are shown. Quantification was performed in ImageJ, and statistical analysis was done using two-way ANOVA, Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar = 10 µm.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Staining

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: Reduced cholesterol and altered PM lipid content in EHD2 KO adipocytes. (A) Whole-cell and (B) PM samples were subjected to immunoblotting for Munc18c, Sx4, and SNAP23. All data are presented as percentage of WT levels (WT = 100%, dashed line), n = 3 biological replicates (A) and n = 3–6 (B). Data are displayed as mean ± SD, and unpaired two-sample t test was used for statistical analysis. Significance was determined according to ** p ≤ 0.01. (C) Cholesterol levels in FC, PM, and serum (S) from WT and EHD2 KO mice. Differences assessed by two-way ANOVA, involving an interaction (x) between genotype (g) and sample (t) with a posthoc Student’s t test. (D) Score plots from OPLS-DA calculated on membrane lipidomic data acquired in positive (top) and negative (below) electrospray ionization mode. t1, first predictive component; to[1], first orthogonal component. WT in white, EHD2 in gray. (E) Lipids showing significant differences between WT and EHD2 KO PMs (variable importance of projection (VIP >1). PE, PE_ep, PC, and SM. Enrichment was accessed using χ 2 statistics; PC_ep, q = 0.0013, PE, q = 0.011, PE_ep, q = 0.00081, SM, q = 0.00022. Significance was determined according to * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001, n = 3 biological replicates. NKA = Na + /K + -ATPase. All displayed results were obtained from inguinal adipocytes 2 wk of HFD.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Western Blot, Membrane

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