Journal: Cell reports

Article Title: PTK7 is a positive allosteric modulator of GPR133 signaling in glioblastoma

doi: 10.1016/j.celrep.2023.112679

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Ligand candidate cDNAs were obtained from the following commercially available plasmids: PTK7 (Addgene, Cat#65250), PCDHGC3 (SinoBiological, Cat#HG22064-UT), TFRC (Addgene, Cat#69610), NECTIN2 (SinoBiological, Cat#HG10005-G), CADM4 (SinoBiological, Cat#HG16033-G), MCAM (SinoBiological, Cat#HG10115-M), PLXDC2 (SinoBiological, Cat#HG23536-U).

Techniques: Recombinant, Magnetic Beads, Blocking Assay, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Derivative Assay, Plasmid Preparation, Software

Journal: Signal transduction and targeted therapy

Article Title: Palmitoylation regulates myelination by modulating the ZDHHC3-Cadm4 axis in the central nervous system.

doi: 10.1038/s41392-024-01971-5

Figure Lengend Snippet: Fig. 1 Cadm4 is specifically palmitoylated at cysteine-347. a Cadm4 expressed in N2a cells or from mouse brain was analyzed for protein palmitoylation by Acyl-RAC assay, and quantified. HA+, with hydroxylamine, HA−, without hydroxylamine. b, c N2a cells expressing Cadm4- Flag was incubated with 2-BP for various dose and time, and were evaluated for the level of palm-Cadm4, and quantified (n = 3 biological replicates, one-way ANOVA; *p ≤0.05, **p ≤0.01, ****p ≤0.0001, n.s. for not significant). d Protein sequences of Cadm4 from various species were aligned for analyzing cysteine conservation. e Cadm4 or its mutants (C347A and C339A/C347A) were expressed in N2a cells and subjected for Acyl-RAC assay, and quantified (two-tailed t-test, n = 3, ****p ≤0.0001). Cadm4 and its mutants (C347A and C339A/C347A) expressed in N2a cells (f) or endogenously expressed Cadm4 (g) were processed with mPEG-labeling assay. The mPEG labeling causes the band shift, red arrows pointed. Data are represented as mean ± SEM

Article Snippet: For staining, following antibodies were used: Cadm4 (R&D Systems, MAB41641, 1:100); APC (Abcam, ab16794; 1:200); Iba1 (Abclonal, A19776; 1:100), GFAP (Abcam, ab7260; 1:100), MBP (Abcam, ab40390; 1:100) and NF (Abcam, ab7794; 1:100), Alexa Fluor-488 (Invitrogen, A11006; 1:200), Alexa Fluor-594 (Invitrogen, A11037; 1:200) and Alexa Fluor-647 (Invitrogen, A21236; 1:200).

Techniques: Expressing, Incubation, Two Tailed Test, Labeling, Electrophoretic Mobility Shift Assay

Journal: Signal transduction and targeted therapy

Article Title: Palmitoylation regulates myelination by modulating the ZDHHC3-Cadm4 axis in the central nervous system.

doi: 10.1038/s41392-024-01971-5

Figure Lengend Snippet: Fig. 2 Palmitoylation regulates Cadm4 for dynamic PM localization. a N2a cells expressing either Cadm4 or Cadm4-C347A were fixed for immunofluorescence analysis, Na/K-ATPase is a marker for PM. b Intensity of the red and green fluorescence was profiled along the slash. c N2a cells expressing Cadm4 or Cadm4-C347A were treated with DMSO or 2-BP for preparing plasma membrane fractions, which were evaluated by WB and quantified (one-way ANOVA followed by Bonferroni post hoc test, n = 3, **p = 0.009; ***p = 0.001). d N2a cells expressing Cadm4 or Cadm4-C347A were treated with DMSO or Dynasore for preparing plasma membrane fractions, which were evaluated by WB and quantified (two-tailed t-test, n = 3, **p ≤0.01). Live-cell imaging of N2a cells expressing Cadm4-GFP/Cadm4-C3471-GFP with RFP- Rab5 (e), for colocalization analysis (f). Data are represented as mean ± SEM

Article Snippet: For staining, following antibodies were used: Cadm4 (R&D Systems, MAB41641, 1:100); APC (Abcam, ab16794; 1:200); Iba1 (Abclonal, A19776; 1:100), GFAP (Abcam, ab7260; 1:100), MBP (Abcam, ab40390; 1:100) and NF (Abcam, ab7794; 1:100), Alexa Fluor-488 (Invitrogen, A11006; 1:200), Alexa Fluor-594 (Invitrogen, A11037; 1:200) and Alexa Fluor-647 (Invitrogen, A21236; 1:200).

Techniques: Expressing, Marker, Clinical Proteomics, Membrane, Two Tailed Test, Live Cell Imaging

Journal: Signal transduction and targeted therapy

Article Title: Palmitoylation regulates myelination by modulating the ZDHHC3-Cadm4 axis in the central nervous system.

doi: 10.1038/s41392-024-01971-5

Figure Lengend Snippet: Fig. 3 Internalized depalmitoylated Cadm4 is targeted for protein degradation. a Cadm4 or Cadm4-C347A was expressed in N2a cells, treated with DMSO or 2-BP (50 µM) and fixed for immunofluorescence analysis, Lamp1 is a marker for lysosome. b The colocalization of Cadm4/Lamp1 was quantified (one-way ANOVA followed by Bonferroni post hoc test, n = 5, ***p < 0.001). N2a cells expressing Cadm4 or Cadm4-C347A were incubated with CHX (cycloheximide, 100 µM) for various length of time, lysates of which were subjected for WB analysis (c) and the levels of Cadm4 were quantified (d). **p = 0.007, paired t-test, n = 3. N2a cells expressing Cadm4 treated with DMSO or 2-BP (50 µM) were incubated with CHX for different periods, analyzed by WB (e) and the levels of Cadm4 were quantified (f). **p = 0.018, paired t-test, n = 3. N2a cells expressing Cadm4 or Cadm4-C347A were incubated with or without CHX, CHQ (Chloroquine, 50 µM), 3-MA (Methyladenine, 3 mM) or Mg132 (25 µM), and subjected for WB analysis (g), and quantified (h). Data are represented as mean ± SEM

Article Snippet: For staining, following antibodies were used: Cadm4 (R&D Systems, MAB41641, 1:100); APC (Abcam, ab16794; 1:200); Iba1 (Abclonal, A19776; 1:100), GFAP (Abcam, ab7260; 1:100), MBP (Abcam, ab40390; 1:100) and NF (Abcam, ab7794; 1:100), Alexa Fluor-488 (Invitrogen, A11006; 1:200), Alexa Fluor-594 (Invitrogen, A11037; 1:200) and Alexa Fluor-647 (Invitrogen, A21236; 1:200).

Techniques: Marker, Expressing, Incubation

Journal: Signal transduction and targeted therapy

Article Title: Palmitoylation regulates myelination by modulating the ZDHHC3-Cadm4 axis in the central nervous system.

doi: 10.1038/s41392-024-01971-5

Figure Lengend Snippet: Fig. 4 Blocking Cadm4 palmitoylation results in severe defects in CNS myelination. Brain lysates from WT and Cadm4-KI mouse were evaluated for the level of Cadm4 and palm-Cadm4 (a), and quantified (b, two-tailed t-test, n = 3, **p ≤0.01, ***p ≤0.001). c, d Corpus callosum was isolated from prefixed brains of WT and Cadm4-KI mouse and examined for myelination under electron microscopy. Abnormal myelination is pointed out by red arrows: loss of myelination, demyelination, thickened myelin sheath/hypermyelination, myelination contains multiple axons (c); myelinated axons per field of view (****p < 0.0001, two-tailed t-test, n = 26–28), % of abnormal myelination (****p < 0.0001, two-tailed t-test, n = 32–35), and g-ratio (****p < 0.0001, two-tailed t-test, n = 372–415) were quantified from 4–6 mice (d). e Lysates of corpus collosum from WT and Cadm4-KI mice were evaluated for MBP level by WB, and quantified (two-tailed t-test, n = 3, **p ≤0.01). Mice brains were processed for transparency with X-clarity and stained for MBP, NF (neurofilament) and DAPI for imaging (f), % of myelination and MBP fluorescence intensity were quantified (g, two-tailed t-test, n = 6, ***p ≤0.001, ****p < 0.0001). Frozen coronal sections of corpus callosum were stained with Cadm4, APC and DAPI for imaging (h), the fluorescence intensity of Cadm4 and APC was quantified (i, two-tailed t-test, n = 6, ****p < 0.0001). j PM fractions were prepared from corpus callosum of WT and Cadm4-KI mice and analyzed for the levels of Cadms and Mag by WB and quantified (two-tailed t-test, n = 3, **p ≤0.01). Immunoprecipitations were performed with the brain lysates of WT and Cadm4-KI mouse by using Cadm4 antibody, the IP eluent was analyzed for Cadm4 binding proteins (k), and quantified (l, two-tailed t-test, n = 3, **p ≤0.01). Data are represented as mean ± SEM

Article Snippet: For staining, following antibodies were used: Cadm4 (R&D Systems, MAB41641, 1:100); APC (Abcam, ab16794; 1:200); Iba1 (Abclonal, A19776; 1:100), GFAP (Abcam, ab7260; 1:100), MBP (Abcam, ab40390; 1:100) and NF (Abcam, ab7794; 1:100), Alexa Fluor-488 (Invitrogen, A11006; 1:200), Alexa Fluor-594 (Invitrogen, A11037; 1:200) and Alexa Fluor-647 (Invitrogen, A21236; 1:200).

Techniques: Blocking Assay, Two Tailed Test, Isolation, Electron Microscopy, Staining, Imaging, Binding Assay

Journal: Signal transduction and targeted therapy

Article Title: Palmitoylation regulates myelination by modulating the ZDHHC3-Cadm4 axis in the central nervous system.

doi: 10.1038/s41392-024-01971-5

Figure Lengend Snippet: Fig. 5 ZDHHC3 mediates Cadm4 palmitoylation and regulates its PM localization and protein stability. Cadm4-GFP was expressed in WT and ZDHHC3-KO cells, and fixed for immunofluorescence imaging (a), Na/K-ATPase is a marker for PM, the profile of the intensity of the red and green fluorescence was measured along the slash (b). c PM fractions were prepared from WT or ZDHHC3-KO cells expressing Cadm4 and subjected for WB analysis, and quantified (two-tailed t-test, n = 3, **p ≤0.01). Cadm4-GFP was expressed in WT and ZDHHC3-KO cells and fixed for immunofluorescence imaging (d), Lamp1 is a marker for lysosome; the colocalization of Cadm4/Lamp1 was analyzed (e, ****p < 0.0001, two-tailed t-test, n = 5). f WT or ZDHHC3-KO cells expressing Cadm4-Flag were incubated with CHX (100 µM) for different period, subjected for WB analysis and quantified (paired t-test, *p < 0.05). WT or ZDHHC3-KO cells expressing Cadm4-Flag were incubated with or without CHX, CHQ (50 µM) or 3-MA (3 mM) and subjected for WB analysis (g), and quantified (h, one-way ANOVA followed by Bonferroni post hoc test, n = 4, *p ≤0.05, **p ≤0.01, ***p < 0.001). Data are represented as mean ± SEM

Article Snippet: For staining, following antibodies were used: Cadm4 (R&D Systems, MAB41641, 1:100); APC (Abcam, ab16794; 1:200); Iba1 (Abclonal, A19776; 1:100), GFAP (Abcam, ab7260; 1:100), MBP (Abcam, ab40390; 1:100) and NF (Abcam, ab7794; 1:100), Alexa Fluor-488 (Invitrogen, A11006; 1:200), Alexa Fluor-594 (Invitrogen, A11037; 1:200) and Alexa Fluor-647 (Invitrogen, A21236; 1:200).

Techniques: Imaging, Marker, Expressing, Two Tailed Test, Incubation

Journal: Signal transduction and targeted therapy

Article Title: Palmitoylation regulates myelination by modulating the ZDHHC3-Cadm4 axis in the central nervous system.

doi: 10.1038/s41392-024-01971-5

Figure Lengend Snippet: Fig. 8 The proposed mechanism that ZDHHC3 mediated palmitoylation regulates CNS myelination via controlling stable PM localization and protein stability of Cadm4. Cadm4 is palmitoylated at cysteine-347 to sustain its stable presence on PM, where it binds with Cadm2/3 and Mag and together, they ensure the proper formation of CNS myelination. Upon the downregulation of palm-Cadm4, either by Cadm4-C347A mutation or the deletion of ZDHHC3, Cadm4 is prone to be internalized and targets for protein degradation, which impairs the interactions of surface proteins and downregulates the level of APC/MBP, the latter may hinder the maturation of oligodendrocytes, potentially through the WNT/β-Catenin signaling pathway, and thus contribute to myelin abnormalities, e.g., loss of myelination, demyelination, and hypermyelination and cognitive deficits in CNS. Most interestingly, ZDHHC3-Cadm4 signaling is tightly involved in neuroinflammation in mice models of demyelination, as downregulating palm-Cadm4 in Cadm4-KI/ZDHHC3-KO mice leads to immuno-activation and neuroinflammation and remarkably, LPS/EAE-induced neuroinflammation significantly downregulates levels of palm-Cadm4 and ZDHHC3, which subsequently modulates PM localization of Cadm4 and deteriorates oligodendrocytes maturation. Combined, the axis of ZDHHC3-Cadm4 palmitoylation is not only vital for CNS myelination but also may serve as conserved pathological mechanism for demyelination diseases related to neuroinflammation

Article Snippet: For staining, following antibodies were used: Cadm4 (R&D Systems, MAB41641, 1:100); APC (Abcam, ab16794; 1:200); Iba1 (Abclonal, A19776; 1:100), GFAP (Abcam, ab7260; 1:100), MBP (Abcam, ab40390; 1:100) and NF (Abcam, ab7794; 1:100), Alexa Fluor-488 (Invitrogen, A11006; 1:200), Alexa Fluor-594 (Invitrogen, A11037; 1:200) and Alexa Fluor-647 (Invitrogen, A21236; 1:200).

Techniques: Mutagenesis, Activation Assay