pmch n1 bltp3a 1 1327  (Addgene inc)

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) AlphaFold prediction of full-length BLTP3A with ConSurf conservation scores (top left) for each a.a. or cross-section of surface rendering of BLTP3A channel (top right) highlighting hydrophobic residues (orange). Linear representation of BLTP3A: per residue ConSurf scores (top) and RBG organization (bottom). IDR predicted intrinsically disordered region (light gray), CH C-terminal helix (dark gray). ( B ) Alignment of the a.a. of motifs important for the indicated protein interactions of BLTP3B with corresponding sequences of BLTP3A. ConSurf conservation scores for each a.a. is indicated by color (same color scheme as in ( A )). ( C ) Western blot of lysates of wild-type mouse tissues for BLTP3A and vinculin as a loading control. ( D ) Western blot of control and edited (BLTP3A^V5) cell clones for BLTP3A, V5, and alpha-tubulin as a loading control. ( E ) Fluorescence images of endogenously edited (left) or parental control (middle) A549 cells with antibodies against LAMP1 (green) and V5 (magenta). Scale bar, 10 μm. The insets are a zoom of a small region of the cell. Scale bar, 1 μm. Right: Fluorescence of exogenous BLTP3A-GFP (green) in endogenously edited A549 cell. Scale bar, 5 μm. Insets: zoom of square region of cell showing co-localization of endogenous BLTP3A^V5 signal from immunolabeling with antibodies against V5 (magenta) and BLTP3A-GFP fluorescence (green). Scale bar, 1 μm. ( F ) Fluorescence image of wild-type RPE-1 cell immunostained with antibodies against LAMP1 (green) and DAPI (blue). Scale bar, 5 μm. ( G ) Fluorescence image of an RPE-1 cell expressing exogenous BLTP3A-mRFP (large field, inverted grays) and LAMP1-GFP (not shown). Scale bar, 5 μm. The area enclosed by a dotted rectangle is shown at right at high magnification with BLTP3A-mRFP in magenta and LAMP1-GFP in green (individual channels are shown as inverted grays). Red arrows indicate large BLTP3A accumulations not associated with lysosomes. Scale bar, 1 μm. ( H ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles, many of them tethered to the surface of lysosomes. Scale bar, 500 nm. ( I ) Distance between the membranes of lysosomes and tethered vesicles from EM micrographs. Mean = 10.8 nm; standard error of the mean = ±0.20 nm. ( J ) Left: Fluorescence image of an RPE-1 cell expressing exogenous BLTP3A-mRFP (inverted grays) and immunolabeled with antibodies against VAMP7 (shown in the high mag fields at right). Scale bar, 5 μm. Right: zooms of different RPE-1 cells expressing exogenous BLTP3A-mRFP (magenta) and immunolabeled (green) with antibodies against the following endogenous proteins, VAMP4 and ATG9A. Individual channels are shown as inverted grays. Merge of channels on bottom. Scale bar, 1 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Residue, Western Blot, Control, Clone Assay, Fluorescence, Immunolabeling, Expressing

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Live fluorescence images (inverted grays) of RPE-1 cells expressing either GFP-Rab45 (left), BLTP3A-mRFP (center), or both proteins together (only BLTP3A is shown) (right) as indicated. Scale bar, 5 μm. High-magnification scale bar, 2 μm. ( B ) Genomic sequence of the edited BLTP3A locus (insertion of the V5 epitope) in A549 cell. Blue, small Gly-Ser linkers; green, V5 epitope sequence. ( C ) AlphaFold prediction of BLTP3A. The site where the V5 epitope (V904) was inserted is indicated. The long disordered sequence and the C-terminal helix are shown in gray. ( D ) Left: Fluorescence image of an RPE-1 cell expressing exogenous BLTP3B-mRFP (inverted grays) and immunolabeled with antibodies against endogenous VAMP7 (shown at right in the high magnification of the squared region in the main field). Scale bar, 5 μm. Right: zooms of different RPE-1 cells expressing exogenous BLTP3B-mRFP (magenta) and immunolabeled with antibodies (green) against endogenous VAMP4 or ATG9A. Individual channels are shown as inverted grays. Merge of channels on bottom. Scale bar, 1 μm. ( E ) Fluorescence images of RPE-1 cells expressing the indicated BLTP3A-mRFP construct. Scale bar, 5 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Expressing, Sequencing, Immunolabeling, Construct

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images of RPE-1 cells expressing exogenous BLTP3A-mRFP (shown in inverted grays) and (not shown) GFP-tagged wild-type Rab7 (left) or dominant negative (DN) Rab7 (T22N) (right). Scale bar, 5 μm. ( B ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell expressing GFP-tagged dominant negative Rab7. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles. Scale bar, 500 nm. ( C ) BLTP3 chimeras design. Left: Surface representation of the predicted RBG core of BLTP3A. Red and blue indicate positive and negative charges, respectively, and gray indicates hydrophobic surfaces. Right: Surface representation (top) and ribbon representation (bottom) of the “untwisted” protein showing individual RBG motifs. Bottom: Cartoon of chimeras consisting of BLTP3A (dark orange) and BLTP3B (light orange) RBG motifs. ( D ) High-magnification live fluorescence images of RPE-1 cells expressing the indicated BLTP3-mRFP constructs (magenta) and LAMP1-GFP (green). Individual channels are shown as inverted grays. Scale bar, 1 μm. ( E ) Ribbon representation of the AlphaFold prediction of a.a. 1–336 of BLTP3A. Blue indicates loops connecting adjoining RBG motifs, and gray indicates the first beta-strand of the third RBG motif. ( F ) Fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-1-336-mRFP and either (not shown) GFP-Rab7 (left), or GFP-Rab7 T22N (right). Scale bar, 5 μm. A zoom of an area of the cell at left (dotted square) expressing BLTP3A-1-336-mRFP (magenta) is also shown, along with the Rab7 fluorescence (green), demonstrating the localization of BLTP3A-1-336-mRFP around the entire profile of lysosomes. Individual channels are shown as inverted grays. Scale bar, 2 μm. ( G ) Cartoon depicting the proposed association of BLTP3A vesicle clusters with the surface of lysosomes and the dependence of this association on Rab7.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Expressing, Dominant Negative Mutation, Construct

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Top: Linear representation of BLTP3A RBG organization and C-terminal truncations indicated by arrows. Bottom left: AlphaFold-based structure of the C terminus of BLTP3A channel. Individual residues are colored by conservation scores as Fig. . Truncations are indicated by arrows. Bottom right: Fluorescence images (inverted grays) of RPE-1 cells expressing the indicated BLTP3A-mRFP constructs along with dominant negative Rab7 (not shown) demonstrating that the property of BLTP3A to bind and cluster vesicles is dependent on its region comprised between a.a. 1327 and 1364. Note that the construct 1–1327, shows a focal accumulation next to the nucleus, which is VAMP7 negative, likely reflecting its pool bound to Rab45 (see Fig. ). Scale bar, 5 μm. Zoomed images (dotted squares) are shown below the main field along with VAMP7 fluorescence. Scale bar, 1 μm. ( B ) Western blots of cell extracts (inputs) of control and edited A549 cells, and of material immunoisolated from these extracts by anti-V5 magnetic beads. Immunolabeling for BLTP3A, V5 (endogenously tagged BLTP3A), and for GAPDH as a loading control, are shown. ( C ) Scatter plot of mass spectrometry-identified proteins in immunoisolated material from either control or endogenously edited BLTP3A^V5 A549 cells using anti-V5 magnetic beads ( N = 3, biological replicates). Proteins significantly enriched in material immunoisolated form edited cells compared to wild-type cells are plotted in the right-top quadrant. Proteins of note are labeled in magenta.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Expressing, Construct, Dominant Negative Mutation, Western Blot, Control, Magnetic Beads, Immunolabeling, Mass Spectrometry, Labeling

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3A-GFP and mApple-IST1 before and after addition of LLOMe. Arrowheads (magenta) point to BLTP3A accumulations shed from lysosomes upon addition of LLOMe. Scale bar, 5 μm. ( B ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker NPC1-GFP (green). Fluorescence of individual channels is shown in inverted grays. Scale bar, 2 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Marker

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker LAMP1-GFP (green) expressed in wild-type A549 cells before and after addition of LLOMe. Fluorescence of individual channels is shown in inverted grays. Scale bar, 5 μm. ( B ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker LAMP1-GFP (green) expressed in VPS13C KO A549 cells before and after addition of LLOMe. Fluorescence of individual channels is shown in inverted grays. Scale bar, 5 μm. ( C ) Live fluorescence images (inverted grays) of RPE-1 cells expressing exogenous GFP-LRRK1 K746G (left) and BLTP3A-mRFP (right). A partial association of BLTP3A-mRFP and GFP-LRRK1 K746G was observed. Scale bar, 10 μm. ( D ) Western blot of lysate of RPE-1 cells expressing exogenous RFP-LRRK1 K746G or RFP-LRRK1 D1409A for RFP (to detect LRRK1 fusions), Rab7, phospho-Rab7 S72, and alpha-tubulin as a loading control. Individual lanes are biological replicates.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Marker, Expressing, Western Blot, Control

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Alignment of the region of BLTP3A orthologues from different species centered on the a.a. region required for mATG8 binding in human BLTP3A. The alignment shows a high degree of conservation of the key residues of the LC3-interacting region (LIR) motif among several chordates and also observed in flies. ( B ) Live fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-mRFP (top) or GFP-LC3B (bottom) in either fed (left) or starved (right) conditions. Arrows (magenta) indicate GFP-LC3B positive foci. Scale bar, 5 μm. ( C ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3A-mRFP and GFP-LC3B before and after addition of LLOMe. Arrowheads point to lysosomes where BLTP3A and LC3B decorate the entire lysosome profile upon addition of LLOMe. Scale bar, 5 μm. ( D ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3AΔLIR-mRFP and GFP-LC3B before and after addition of LLOMe. Arrowheads point to lysosomes where LC3B, but not BLTP3A lacking a LIR motif, decorates the entire lysosome profile upon addition of LLOMe. Scale bar, 5 μm. ( E ) Correlative fluorescence-FIB-SEM microscopy of GFP-LC3B and BLTP3A-mRFP-positive lysosomes in an RPE-1 cell 15 min after LLOMe addition. Left: Fluorescence image of an RPE-1 cell expressing GFP-LC3B (green), BLTP3A-mRFP (magenta), and mito-BFP (not shown), 15 min after addition of LLOMe. Scale bar, 5 μm. Region of cell for FIB-SEM marked by dotted line. Middle: High magnification of the RPE-1 cell at left (blue square) showing both the BLTP3A-mRFP (magenta) and the GFP-LC3B (green) channels. Individual channels are shown as inverted grays. Merged channel also shows mito-BFP (blue) used for aligning EM and fluorescence images. Scale bar, 2 μm. Right: EM reconstruction (scale bar, 1 μm) corresponding to the blue square from left. Lysosome, dark green; ER, yellow; mitochondria, blue; small vesicles, magenta.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Binding Assay, Fluorescence, Expressing, Microscopy

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) AlphaFold3 multimer prediction of full-length MAP1LC3B (green) and a.a. 1110–1150 of BLTP3A (magenta). Arrows indicate key residues of the LIR motif of BLTP3A. ( B ) AlphaFold3 multimer predictions of mATG8 proteins and a.a. 1110–1150 of BLTP3A with and without the LIR motif (ΔLIR). ( C ) Time-series of live fluorescence images (inverted grays) of BLTP3A-mRFP and GFP-LC3B before and after addition of GPN. Arrowheads point to lysosomes where BLTP3A and LC3B decorate the entire profile upon addition of GPN. Time, seconds. Scale bar, 5 μm. ( D ) Genomic sequence of the edited BLTP3A locus in A549 cell. Blue, gRNA; green, PAM; red, indel mutations. ( E ) Quantification of relative LAMP1 expression from western blots ( N = 3, biological replicates) of Fig. . Error bars indicate the standard error of the mean (SEM).

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Sequencing, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Brightfield image of RPE-1 cells incubated with MSU crystals (200 μg/mL) for 2 h. Arrowheads indicate MSU crystals. Scale bars, 5 μm. ( B ) Live fluorescence images of RPE-1 cells expressing BLTP3A-mRFP and LAMP1-GFP (shown as inverted greys) and incubated with MSU crystals for 2 h. Scale bar, 5 μm. Zoomed images (dotted rectangles) of individual channels are shown to the right. Scale bars, 2 μm. Arrowheads point to the surface of crystal-containing vacuoles positive for BLTP3A-mRFP and LAMP1-GFP. ( C ) Live fluorescence images of RPE-1 cells co-expressing either BLTP3A-mRFP (top row) or BLTP3AΔLIR-mRFP (bottom row) with GFP-LC3B and incubated with MSU crystals for 2 h. Individual channels are shown as inverted greys. Scale bar, 10 μm. Zoomed images (dotted rectangles) of individual channels are shown to the right. Scale bar, 2 μm. Arrowheads point to the surface of the crystal-containing vacuoles positive for GFP-LC3B to highlight the presence of BLTP3A-mRFP (top) and the absence of BLTP3AΔLIR-mRFP (bottom). ( D ) Time-series of live fluorescence images (inverted grays) of BLTP3A-mRFP and GFP-LC3B in RPE-1 cells incubated with MSU crystals. Scale bar, 2 μm. Arrowheads point to the surface of crystal-containing vacuoles.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Incubation, Fluorescence, Expressing

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Western blots of cell extracts (inputs) of control and edited A549 cells (BLTP3A KO clones) for the proteins indicated. ( B ) Fluorescence images of parental control (left) or BLTP3A KO (right) A549 cells with antibodies against LAMP1 (green). Scale bar, 20 μm. ( C ) Fluorescence images of parental control (top row) or BLTP3A KO (bottom row) A549 cells with antibodies against galectin-3 (inverted greys). Cells were treated with vehicle control (left column) or 1 mM LLOMe (right column). Scale bar, 10 μm. ( D ) Quantification of galectin-3 spots per cell from field ( C ) ( N = 3, biological replicates). Error bars report the standard error of the mean (SEM). ** P < 0.01. Mean number of galectin-3 spots per cell and number of cells counted per condition indicated.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Western Blot, Control, Clone Assay, Fluorescence

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images (inverted greys) of BLTP3A KO A549 cells expressing indicated RFP protein (top row) with antibodies against galectin-3 (bottom row). Dotted magenta line indicates cell boundary. Cells were treated with vehicle control (left column) or 1 mM LLOMe (right three columns). Scale bar, 10 μm. ( B ) Quantification of galectin-3 spots per cell from field ( A ) ( N = 3, biological replicates.). Error bars report the standard error of the mean (SEM). *** P < 0.001; ** P < 0.01; * P < 0.05; n.s., not significant. Mean number of galectin-3 spots per cell and number of cells counted per condition indicated.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Expressing, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: CNTNAP2 is cleaved by α-secretase and furin. a , b ADAM10 and ADAM17 cleaved mature CNTNAP2 (mCNT) in HEK cells. HEK cells were co-transfected with CNTNAP2 and empty vector, ADAM10 or ADAM17 plasmids, and harvested 24 h after transfection for Western Blot analysis. Non-transfected HEK (NT HEK) was used as a negative control. CTFα1 and CTFα2 were increased/generated by ADAM10 and ADAM17. n = 4 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. c , d ADAM10 is the major α-secretase that generates CTFα1. CNTNAP2 was co-transfected with GFP into HEK, ADAM10-knockout (KO), or ADAM17-KO HEK cells. The CTFα1/mCNT ratio was reduced in ADAM10-KO cells while increased in ADAM17-KO cells. n = 3 independent experiments, unpaired t -test, ** p < 0.01. e , f Furin cleaved mature CNTNAP2 in HEK cells. HEK cells were co-transfected with CNTNAP2 and vector or furin. CTFf at 55 kDa was increased by furin. n = 3 independent experiments, unpaired t -test, * p < 0.05. g CNTNAP2 on the cell membrane was cleaved in HEK and N2a cells. CNTNAP2 was co-transfected with vector (myc), ADAM10, ADAM17, or furin plasmids into HEK and N2a cells. Cells were fixed 24 h after transfection, and immunocytochemistry (ICC) was performed. CNTNAP2 was detected by an anti-CNTNAP2 antibody targeting the N-terminal 1001–1042 aa outside the cell membrane. CNTNAP2 was mainly expressed on the cell membrane and was reduced by co-transfected plasmids. Images were acquired using Zeiss Apotome to reflect the morphology. Scale bar represents 50 μm. h Quantification of ICC in HEK and N2a. CNTNAP2 mean intensity was measured using the corresponding conventional fluorescence images in Supplementary Fig. . n = 10 cells from 2 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: Transfection, Plasmid Preparation, Western Blot, Negative Control, Generated, Knock-Out, Membrane, Immunocytochemistry, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Sequential cleavages of furin, α- and γ-secretase. a Western blot of control (Vector), ADAM10 overexpressing, and ADAM17 overexpressing HEK cells. Exposure 1 was used to analyze CTFf in the ADAM17 overexpressing cells and C96 in the ADAM10 overexpressing cells; exposure 2 was used for assessing C79 and C96 in the ADAM17 overexpressing cells. TAPI-1 (10 μM) is an ADAM17-specific inhibitor; FI-2 (20 μM) stands for Furin inhibitor-2. b Quantification of the dynamic changes of mature CNTNAP2 and CTF levels in the ADAM10/ADAM17 transfected cells upon inhibitor treatments in ( a ). n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. P values stand for comparisons with the Control group unless noted by the brackets. c , d Dynamic changes of CTFs in the furin transfected cells, showing the furin-ADAM17 cleavages. n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. P values stand for comparisons with the Control group unless noted by the brackets. e Western Blot of control (Vector), ADAM10 overexpressing, and ADAM17 overexpressing HEK cells. GSI is γ-secretase inhibitor L-685,458 (20 nM). f Quantification of the sequential cleavage by α- and γ-secretase in ( e ). n = 3 independent experiments, unpaired t -test, * p < 0.05, ** p < 0.01. g Schematic of CNTNAP2 processing. Mature CNTNAP2 undergoes sequential cleavages by furin and α-, γ-secretase. Furin cleaves CNTNAP2 to generate CTFf, which is further processed by α-secretase. α-secretase ADAM10 and ADAM17 share the same cutting sites but have different site preferences. ADAM10 primarily cleaves at I79 to produce the predominant C79, and ADAM17 preferably cleaves at L96 to produce the weaker C96. C96 is subsequently processed by α-secretase into C79. γ-secretase further cleaves C79 at L53 within the transmembrane domain to generate CICD. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: Western Blot, Control, Plasmid Preparation, Transfection

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Identification of α-secretase cleavage sites on CNTNAP2. a Peptide in vitro cleavage. Two peptides were incubated in the assay buffer with or without ADAM17 at 37 °C for 24 h and then were sent to Mass Spectrometry analysis. The extracted ion chromatogram showed two intact peptides (top) and cleaved complementary fragments (bottom). Peptide 1 (M108-G84) was cleaved by ADAM17 at 97H/L96. Peptide 2 (L96-N72) was cleaved at 80 A/79I. b N-terminal sequencing results identified the first 5 amino acids (aa) of CTFα1 as IRNGV and the first 5 aa of CTFα2 as LDSAS. The bottom sequence shows the last 108 to 72 aa of CNTNAP2 from the C-terminus and α-secretase cleavage sites at L96 and I79. c Protein ladders of the C-terminal CNTNAP2. C79 corresponded to the size of CTFα1, and C96 showed the same size as CTFα2. d , e Mutations at the α-secretase cleavage sites L96 and I79 affected CNTNAP2’s cleavage. Wildtype (WT) or mutant plasmids were co-transfected with empty vector (EV), ADAM10 (AD10), or ADAM17 (AD17) into HEK cells. Alterations in the migration rate were observed in constructs containing D98R. Two upper/lower blots show samples from the same experiment. The parallel blots were processed in the same electrophoresis chamber and scanned together simultaneously. n = 3 independent experiments, two-way ANOVA followed by Dunnett’s multiple comparisons test (compare ADAM10 and ADAM17 with Control for each plasmid), row factor = mutation, column factor = ADAM10/17 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: In Vitro, Incubation, Mass Spectrometry, Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Migration, Construct, Electrophoresis, Control, Over Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Pathogenic mutations affect CNTNAP2 processing. a Schematic of CNTNAP2 protein and locations of pathogenic mutations found in ASD patients. Mutations predicted deleterious or at conserved sites are noted in red. ADAM10/17-mediated α-cleavage affected by the mutations was summarized in the brackets (10 = ADAM10, 17 = ADAM17; slight affections are noted in purple, while severe impacts are marked in red). SP signal peptide, DISC discoidin-like domain, L1-4 four laminin A-like G domains, FBG fibrinogen-like domain, E1-2 two epidermal growth factor (EGF)-like domains, TM transmembrane domain; 4.1b, 4.1 binding domain; PDZb, PSD-95/Discs large/zona occludens-1 (PDZ) binding domain. Braces represent three lobes of CNTNAP2 protein. The figure is to scale. b , c CNTNAP2 mutations affected expression patterns of CNTNAP2 full-length and CTFs. CNTNAP2 plasmids were co-transfected with GFP into HEK cells. n = 3 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05; ** p < 0.01; **** p < 0.0001. d , e Altered α-secretase cleavage in CNTNAP2 mutations. CNTNAP2 plasmids were co-transfected with empty vector (EV), ADAM10 (AD10), or ADAM17 (AD17) plasmids into HEK cells. Mutations showed variant cleavage patterns. Three blots show samples from the same experiment. The blots were processed in parallel and scanned together simultaneously. n = 3 independent experiments, two-way ANOVA followed by Dunnett’s multiple comparisons test (compare ADAM10 and ADAM17 with Control for each plasmid), row factor = mutation, column factor = ADAM10/17 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: Binding Assay, Expressing, Transfection, Plasmid Preparation, Variant Assay, Control, Mutagenesis, Over Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Impairment of α-secretase cleavage resulted in repetitive and social behavior abnormalities in mice. a Schematic representation of CRISPR/Cas9 knock-in method used to generate mutant I1254T mice by mutating “ATA” (Ile) to “ACG” (Thr) at the major α-cleavage site of CNTNAP2. b Body weight record of WT and I1254T mice. The weight of both mice was recorded daily, and no significant difference was found between mutants and WT mice (WT, n = 15; I1254T, n = 15). c Isolation-induced ultrasonic vocalizations (UsVs) test. The numbers of distress calls from infants of WT and mutants were detected at the age of P3, P6 and P12 (WT, n = 13; I1254T, n = 13). d Juvenile play test. Time involved in social interaction, as well as repetitive behaviors like grooming in WT and I1254T mutant mice were measured at the age of P21 when interacting with unfamiliar mice ( n = 10 per group: male = 5, female = 5). e , f T maze and three chamber sociability tests performed at 7-week-old WT and mutants. I1254T mutation at the α-cleavage site increased repetitive behavior abnormalities in T maze ( e ) and decreased social interactions ( f ) in I1254T mutants, and C79 overexpression rescued the ASD-like behaviors ( n = 12 per group: male = 6, female = 6). g – i Social and repetitive behavior tests in Cntnap2 −/− mice. C79 overexpression rescued the social interactions ( g , i ) and repetitive behaviors ( h ) in Cntnap2 −/− mice ( n = 11: male = 6, female = 5 in WT and KO groups; n = 12: male = 6, female = 6 in KO + C79 group). Statistical significance was assessed by either one- or two-way ANOVA followed by Turkey or Bonferroni’s test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: CRISPR, Knock-In, Mutagenesis, Isolation, Over Expression