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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: Western Blot, Plasmid Preparation, Transfection

Journal: Scientific Reports

Article Title: Furin inhibitor protects against neuronal cell death induced by activated NMDA receptors

doi: 10.1038/s41598-018-23567-0

Figure Lengend Snippet: ( a ) Effect of NMDA treatment on cell viability. Cortical neurons were incubated with NMDA at the indicated concentrations for 15 minutes. After 24 h of NMDA treatment, cell viability was determined by performing the XTT dye-reduction assay. The absorbance at 450 nm was measured, and the relative cell viability was expressed as the percentage of the absorbance at 450 nm of each treatment group against that of the untreated control group. Results are the means ± SE (n = 5 independent experiments). *Indicates a significant difference from the NMDA-untreated group ( p < 0.05). ( b ) Effect of NMDA receptor antagonist on NMDA-induced cell injury. Cell viability in cultures of 0 µM and 30 µM NMDA-treated cells without (white bars) or with (black bars) 10 µM MK-801. The relative cell viability was expressed as the percentage of the absorbance at 450 nm of each treatment group against that of the untreated control group. Results are the means ± SE (n = 3 independent experiments). *Indicates a significant difference from the NMDA-untreated group ( p < 0.05); and # , a significant difference from the NMDA-treated and MK801-untreated group ( p < 0.05). ( c ) Cortical neurons were labeled with Fluo-8 acetoxymethyl ester for 30 min, and then 30 µM NMDA was added with or without 10 µM MK-801. Fluorescence ratio images are displayed in pseudocolor as indicated by the color bar. Pseudocolor represents changes in fluorescence ratios between 0 ( blue ) and 2 ( red ) corresponding to 1 ( green ), which is defined as the basal fluorescence intensity before NMDA stimulation. Representative ratio images of cortical neuron cultures incubated with 0 µM NMDA (Control), 30 µM NMDA (NMDA) or 30 µM NMDA and 10 µM MK-801 (NMDA + MK-801) are shown. Scale bar represents 100 µm. In the right panel, changes in Fluo-8 fluorescence are expressed as Δ F / F 0, where F 0 is basal fluorescence intensity before NMDA stimulation. Results are the means ± SE (n = 5 independent experiments).

Article Snippet: Furin inhibitor 2 (Cat. No. SCP0148) and MK-801 (Cat. No. M107) were obtained from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Incubation, Labeling, 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: 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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: Western Blot, 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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: In Vitro, Incubation, Mass Spectrometry, Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Migration, Construct, Electrophoresis, 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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: Binding Assay, Expressing, Transfection, Plasmid Preparation, Variant Assay, 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: 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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: Western Blot, 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: 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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: Western Blot, 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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: In Vitro, Incubation, Mass Spectrometry, Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Migration, Construct, Electrophoresis, 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: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

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