Journal: bioRxiv

Article Title: Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

doi: 10.1101/2024.02.05.578974

Figure Lengend Snippet: (A) Functional enrichment analysis on significantly downregulated genes in SU-DIPG13 cells treated with 10uM Sulfopin for 12 hours, compared to DMSO. Enrichr algorithm was used to compare downregulated genes against the Molecular Signatures Database (MSigDB) hallmark geneset . Dashed line denotes adjusted p-value = 0.05. MYC targets are significantly enriched among the Sulfopin downregulated genes. (B) RT-qPCR analysis of selected MYC target genes in SU-DIPG13 cells treated with 10uM Sulfopin for 12 hours, compared to DMSO. Fold change between Sulfopin and DMSO treated cells was calculated and the mean ± SD of two technical repeats is shown. (C) H3K27me3 Cut&Run read coverage over MYC target genes (‘MYC Targets V1’ hallmark geneset , n=200), in SU-DIPG13 cells treated with 10uM Sulfopin for 8 days compared to DMSO. Sulfopin treatment increases H3K27me3 levels on the TSS of MYC target genes. (D) Functional enrichment analysis of the genes associated with Sulfopin-unique H3K27me3 peaks, in SU-DIPG13 cells treated as in C. Dashed line denotes adjusted p-value = 0.05. MYC target geneset (‘MYC Targets V1’ hallmark geneset ) is mildly enriched among these genes, with adjusted p-value of 0.077. (E) Cell viability, as measured by CellTiterGlo, of eight DMG cultures (H3.3K27M: SU-DIPG13, SU-DIPG6, SU-DIPG17, SU-DIPG25 and SU-DIPG50. H3.1K27M: SU-DIPG36, SU-DIPG38 and SU-DIPG21), treated with Sulfopin for eight days with pulse at day four, compared to DMSO. Mean±SD of two technical replicates is shown. Logarithmic scale is used for the x-axis. Sulfopin treatment led to a mild reduction in cell viability in all H3-K27M glioma cultures. (F) Cell viability, as measured by CellTiterGlo, of two isogenic DMG cell lines (SU-DIPG13 and BT245) in which the mutant histone was knocked-out (KO), treated with the indicated concentration of Sulfopin for 8 days, compared to DMSO. For each cell line and concentration, the fold change in viability between Sulfopin and DMSO treated cells is shown. For SU-DIPG13-mean ± SE of at least two independent experiments is shown. For BT245-mean± SD of three technical replicates is shown. H3-K27M glioma cells show higher sensitivity to Sulfopin treatment compared to the KO cells. *P < 0.05; **P < 0.01 (two-sample t-test over all technical replicates). Significance adjusted after Bonferroni correction.

Article Snippet: Treatment was given daily for 18 days, by intraperitoneal injection according to the following groups: Vorinostat 200mg/kg daily (LC, V-8477-SU-DIPG13P*; MCE, HY-10221-SU-DIPG13), Sulfopin 40 mg/kg (gift from Dr. Nir London’s Lab), combination 200 mg/kg Vorinostst and 40 mg/kg Sulfopin, control mice received solvent 15% DMSO in 50% PEG400 (Sigma) in 0.15M NaCl.

Techniques: Functional Assay, Quantitative RT-PCR, Mutagenesis, Concentration Assay

Journal: bioRxiv

Article Title: Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

doi: 10.1101/2024.02.05.578974

Figure Lengend Snippet: (A) Timeline demonstrating the treatment protocol for the combination of Sulfopin and Vorinostat. (B) Percentage of cell viability, as measured by CellTiterGlo of SU-DIPG13 cells treated with Sulfopin and Vorinostat at the indicated concentrations, compared to DMSO. (C) BLISS index measured as the ratio between the observed and the expected effect of the combination of Sulfopin and Vorinostat, for each pair of concentrations, in SU-DIPG13.Synergy: Bliss <1, Additive: Bliss=1, Antagonist: Bliss>1. (D) Cell viability as measured by CellTiterGlo, of eight DMG cultures treated with Sulfopin (0uM, 2.5uM, 5uM, 10uM, 20uM and 40uM) and Vorinostat (1uM), compared to DMSO. H3.3-K27M and H3.1-K27M cultures are indicated in blue and orange, respectively. Mean±SD of two technical replicates is shown. H3.3-K27M cells showed higher sensitivity to the combined treatment compared to H3.1-K27M cells. (E) The BLISS index of the combination of Sulfopin (10uM) and Vorinostat (1uM), in the indicated cultures. An additive effect was detected in all the H3.3-K27M cultures at this set of concentrations. (F) Pearson correlation coefficient matrix of BLISS index of the combined treatment (Sulfopin (10uM) and Vorinostat (1uM)) and mRNA levels of MYC and its target genes, in the eight DMG cultures tested. mRNA levels were measured by RT-qPCR (Fig. S2F-G). Blue and yellow colors indicate negative or positive correlation, respectively. Negative correlation was detected between the BLISS indexes and the expression levels of MYC and its target genes. (G) Unsupervised hierarchical clustering of expression levels of 620 significantly DE genes detected in SU-DIPG13 cells treated with either Sulfopin (10uM, 8 days), Vorinostat (1uM, 72 hours), the combination of Sulfopin and Vorinostat or DMSO. Gene expression rld values (log2 transformed and normalized) were standardized for each gene (row) across all samples. Color intensity corresponds to the standardized expression, low (blue) to high (red). Clusters 1 and 4 demonstrate additive transcriptional patterns associated with the combined treatment. (H) Top: Gene Set Enrichment Analysis (GSEA) on SU-DIPG13 treated with combination of 10uM Sulfopin and 1uM Vorinostat compared to DMSO, showing significant downregulation of mTORC1 signaling (‘HALLMARK_MTORC1_SIGNALING’ geneset ) in the combined treatment. NES: Normalized Enrichment Score. FDR: false discovery rate. Bottom: Expression levels of significantly DE genes detected in the combined treatment compared to DMSO that are part of the mTORC1 signaling geneset. MTOR gene was added manually to the heat-map. Heatmaps were generated as described in B. (I) Top: Gene Set Enrichment Analysis (GSEA) on SU-DIPG13 treated as in C, showing significant downregulation of the epigenetic BMI-1 pathway and the oncogenic cAMP pathway in the combined treatment (BMI1_DN.V1_UP; CAMP_UP.V1_UP; MSigDB C6 oncogenic signature , ). Bottom: Expression levels of significantly DE genes detected in the combined treatment compared to DMSO that are part of the BMI-1 and cAMP genesets. Heatmaps were generated as in described B. (J) Western blot of SU-DIPG13 treated either with Sulfopin (10uM, 8 days), Vorinostat (1uM, 72 hours), the combination of Sulfopin and Vorinostat or DMSO, using the indicated antibodies. β-tubulin is used as loading control.

Article Snippet: Treatment was given daily for 18 days, by intraperitoneal injection according to the following groups: Vorinostat 200mg/kg daily (LC, V-8477-SU-DIPG13P*; MCE, HY-10221-SU-DIPG13), Sulfopin 40 mg/kg (gift from Dr. Nir London’s Lab), combination 200 mg/kg Vorinostst and 40 mg/kg Sulfopin, control mice received solvent 15% DMSO in 50% PEG400 (Sigma) in 0.15M NaCl.

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Transformation Assay, Generated, Western Blot, Control

Journal: bioRxiv

Article Title: Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

doi: 10.1101/2024.02.05.578974

Figure Lengend Snippet: (A) Scheme of the single-molecule imaging experimental setup : cell-derived mono-nucleosomes are anchored in a spatially distributed manner on polyethylene glycol (PEG)-coated surface. Captured nucleosomes are incubated with fluorescently labeled antibodies directed against the H3K27ac modification. Total internal reflection fluorescence (TIRF) microscopy is utilized to record the position and modification state of each nucleosome. Time series images are taken to allow detection of maximal binding events. (B) Single-molecule imaging quantification of the percentage of H3K27ac nucleosomes, in SU-DIPG13 cells treated with either Sulfopin (10uM, 8 days), Vorinostat (1uM, 72 hours), or the combination of Sulfopin and Vorinostat, normalized to DMSO. Mean fold ± SE of at least two independent experiments is shown. H3K27ac global levels are lower in the combined treatment compared to cells treated solely with Vorinostat. *P < 0.05 (two sample t-test). (C) SU-DIPG13 cells were treated as in B, and analyzed by western blot using the indicated antibodies. (D) Left panel: Heatmap shows H3K27ac read coverage around the TSS (+/-5Kb) of the significantly DE genes shown in , in SU-DIPG13 cells treated with the combination of 10uM Sulfopin and 1uM Vorinostat versus DMSO. Average coverage is shown on top. Color intensity corresponds to the standardized expression. Clusters 1-4 are indicated. Right panel: The log2 ratio of H3K27ac read coverage in SU-DIPG13 cells treated with the combination of 10uM Sulfopin and 1uM Vorinostat vs. DMSO was calculated. Heatmap shows the ratio around the TSS (+/-5Kb) of the significantly DE genes shown in , and average coverage is shown on top. Color intensity corresponds to the ratio between samples, low (red) to high (blue). Clusters 1-4 are indicated, with cluster 1 presenting the strongest local decrease in H3K27ac following the combined treatment compared to DMSO. (E) IGV tracks of MTOR and SLC7A5 gene promoters, showing H3K27ac coverage in SU-DIPG13 cells treated as indicated. (F) Functional enrichment analysis of the genes linked to enhancers (top targets of high confident enhancers) marked with H3K27ac exclusively in SU-DIPG13 cells treated with Vorinostat, and not in the combined treatment. gProfiler algorithm was used to calculate enrichment against the KEGG pathways DB . Dashed line denotes adjusted p-value = 0.05. Genes associated with Vorinostat-unique enhancers are enriched for oncogenic signaling pathways. (G) IGV track of AKT3 and SEC13 linked enhancers, showing H3K27ac coverage in SU-DIPG13 cells treated with 1uM Vorinostat or the combination of 10uM Sulfopin and 1uM Vorinostat. (H) Normalized expression levels of AKT3 and SEC13 genes in SU-DIPG13 cells treated as in G. Mean ± SD of three technical repeats is shown.*P < 0.05 (two-sample t-test).

Article Snippet: Treatment was given daily for 18 days, by intraperitoneal injection according to the following groups: Vorinostat 200mg/kg daily (LC, V-8477-SU-DIPG13P*; MCE, HY-10221-SU-DIPG13), Sulfopin 40 mg/kg (gift from Dr. Nir London’s Lab), combination 200 mg/kg Vorinostst and 40 mg/kg Sulfopin, control mice received solvent 15% DMSO in 50% PEG400 (Sigma) in 0.15M NaCl.

Techniques: Imaging, Derivative Assay, Incubation, Labeling, Modification, Fluorescence, Microscopy, Binding Assay, Western Blot, Expressing, Functional Assay, Protein-Protein interactions

Journal: bioRxiv

Article Title: Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

doi: 10.1101/2024.02.05.578974

Figure Lengend Snippet: (A-B) SU-DIPG13P* cells were injected to the pons of immunodeficient mice to form tumors. Ten days post injection, mice were treated for 18 days with either DMSO, Sulfopin, Vorinostat, or the combination of Sulfopin and Vorinostat. (A) In-vivo bioluminescent imaging of DMG xenografts following 18 days of treatment. The heat map superimposed over the mouse head represents the degree of photon emission by DMG cells expressing firefly luciferase. (B) DMG xenograft tumor growth as measured by change in bioluminescent photon emission following 15 days of treatment with either DMSO (n=8), Sulfopin (n=4), Vorinostat (n=5) or the combination of Sulfopin and Vorinostat (n=6). Data points represent the fold-change in maximum photon flux between day 3 and day 18 under treatment for each mouse. *P < 0.05 (two-tailed Mann-Whitney U-test). (C-D) Immunofluorescent staining of brain sections from mice treated with DMSO (n=4) or the combination of Sulfopin and Vorinostat (n=4). (C) Representative fluorescence images of H3-K27M (red) and mTOR (green). (D) Percentage of mTOR positive cells out of the total H3-K27M-positive cells. H3-K27M positive cells show lower levels of mTOR following the combined treatment compared to DMSO. *P < 0.05 (two-tailed t-test).

Article Snippet: Treatment was given daily for 18 days, by intraperitoneal injection according to the following groups: Vorinostat 200mg/kg daily (LC, V-8477-SU-DIPG13P*; MCE, HY-10221-SU-DIPG13), Sulfopin 40 mg/kg (gift from Dr. Nir London’s Lab), combination 200 mg/kg Vorinostst and 40 mg/kg Sulfopin, control mice received solvent 15% DMSO in 50% PEG400 (Sigma) in 0.15M NaCl.

Techniques: Injection, In Vivo, Imaging, Expressing, Luciferase, Two Tailed Test, MANN-WHITNEY, Staining, Fluorescence

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein–Protein Interaction Data *

doi: 10.1074/mcp.M115.050773

Figure Lengend Snippet: RRP analysis of Pin1 interactome. (A) Graphical presentation of analyzed Pin1 interactome based on: Protein Interaction Network analysis (2). (B) Phase contrast microscopy images of PC-3 cells transfected with indicated siRNAs on 384-well plate. (C) Graphical presentation of correlation of cell viability effects of individual siRNAs in PC-3 and PNT2 cells. (D) RRP ranking of top ten proteins with most similar function to Pin1. FunCoup similarity index (min. 0, max. 1) indicates for very high similarity in function with Pin1 for most RRP top ranked proteins. NA, not applicable due to lack of sufficient database information. Validation indicates either physical or functional validation for indicated interaction. N.d., not determined.

Article Snippet: The antibodies against PIN1 (H-123:SC-15340), c-Jun, MYC, FTSJ1 (B-2:SC-390355), DDX24 (C-12:SC-104863), FNBP3 ( d -20:SC-68080), PME-1 (B-12: SC-25278), Lamin A/C, (H-110 SC-20681; H-110), rabbit anti goat IgG-HRP (SC-2922), and normal rabbit IgG (SC-2027) were purchased from Santa Cruz Biotechnology.

Techniques: Microscopy, Transfection, Functional Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein–Protein Interaction Data *

doi: 10.1074/mcp.M115.050773

Figure Lengend Snippet: Validation of CSKN2B and PTOV1 as functional Pin1 interacting proteins. (A) Western blot analysis of phosphorylated c-Jun expression in PC-3 cells depleted of either Pin1, PTOV, or CSKN2B. (B) Western blot analysis of Pin1 expression in PC-3 cells transfected either with Pin1, PTOV, or CSKN2B siRNAs.

Article Snippet: The antibodies against PIN1 (H-123:SC-15340), c-Jun, MYC, FTSJ1 (B-2:SC-390355), DDX24 (C-12:SC-104863), FNBP3 ( d -20:SC-68080), PME-1 (B-12: SC-25278), Lamin A/C, (H-110 SC-20681; H-110), rabbit anti goat IgG-HRP (SC-2922), and normal rabbit IgG (SC-2027) were purchased from Santa Cruz Biotechnology.

Techniques: Functional Assay, Western Blot, Expressing, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 2. Pin1 binding to the AF-1 is enhanced by ERK2 phosphorylation. Overlays of 2D [1H,15N]-HSQC NMR spectra of (A) 15N-labeled AF-1, (B) 15N-labeled pAF-1, and (C) 15N-labeled pAF-1 P76A mutant without or with 1 molar equivalent of human Pin1 reveal larger chemical shift perturbations (CSPs) in the phosphorylated state indicating stronger binding of Pin1 to phosphorylated AF-1. Peaks belonging to regions of particular interest noted by blue and green labeling are discussed further in the results. (D) AlphaFold3 model of Pin1 interaction with pAF-1 containing phosphorylated S112 (pS112) and T75 (pT75) residues with NMR CSPs from 15N-labeled pAF-1 ± 1x Pin1 (2B) shows the Pin1-binding epitope on pAF-1 extends to regions beyond the pS112 region. Spheres represent residue alpha carbons colored by CSP magnitude [white (CSP = 0) to magenta (CSP = 0.03)] or noting peaks that disappear (black). CSP plots comparing addition of Pin1 full-length, WW domain, or PPIase domain at 1 molar equivalent to (E) 15N-labeled AF-1, (F) 15N-labeled pAF-1, and (G) 15N-labeled pAF-1 P76A mutant reveal that each Pin1 domain binds weakly to AF-1, but binding of full-length Pin1 elicits larger CSPs in targeted regions of pAF-1 and pAF-1 P76A mutant.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Phospho-proteomics, Labeling, Mutagenesis, Residue

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 4. AF-1 peptides delineating contributions of specific AF-1 regions in binding to Pin1. (A) Sequence and relative locations of peptides corresponding to AF-1 regions that include the S112 phosphorylation site with unphosphorylated S112 (green) or phosphorylated S112 (teal); T75 phosphorylation site with unphosphorylated T75 (purple) or phosphorylated T75 (coral); and the PFWP motif (mustard). Overlays of 2D [1H,15N]-TROSY HSQC NMR spectra of 15N-labeled Pin1 with (B) S112, (C) T75, (D) pS112, (E) pT75, and (F) W39 peptides titrated up to 5 molar equivalents. Shown alongside spectra are AlphaFold3 models of Pin1 colored by magnitude of CSP [pale blue/white (CSP = 0) to magenta (CSP = 0.1)] elicited by 2X titration of respective peptide shown in complex with Pin1. The WW domain is colored pale blue, and the PPIase domain is white. Peptides in models are colored corresponding to their identity as indicated in 5A. (G) CSP plots comparing addition of unphosphorylated or phosphorylated peptides at 5 molar equivalents to 15N-labeled Pin1. CSP profiles reveal Pin1 interaction patterns unique to each AF-1 peptide, with phosphorylated AF-1 peptides driving enhanced interaction to the WW domain.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Sequencing, Phospho-proteomics, Labeling, Titration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 6. Approaches to studying Pin1 binding and catalysis. Previous structural studies of Pin1 isomerization depended heavily on the use of short peptide sequences (~20 amino acids). In addition to short peptides, our studies using a larger protein domain with multiple potential canonical and noncanonical Pin1 sites uncovered a multivalent binding interaction that catalyzes a noncanonical W-P motif with functional cellular relevance.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Functional Assay

Journal: Current biology : CB

Article Title: FERONIA Receptor Kinase Contributes to Plant Immunity by Suppressing Jasmonic Acid Signaling in Arabidopsis thaliana.

doi: 10.1016/j.cub.2018.07.078

Figure Lengend Snippet: Figure 1. FER Receptor Kinase Functions Upstream of MYC2 to Regulate JA Signaling (A) Gene expression comparisons among fer-DEs and stress hormone-DEs. Color represents log10 p values from the indicated overlaps calculated from Fisher’s exact test by GeneOverlap. The number of genes in each intersection is indicated. (B–E) The upregulation of several JA-induced genes, RESPONSIVE TO DESICCATION 26 (RD26, B), TYROSINE AMINO TRANSFERASE (TAT, C), VEGETATIVE STORAGE PROTEIN 1 (VSP1, D) and PLANT DEFENSIN 1.2 (PDF1.2, E), in fer and their JA induction were confirmed by qPCR. SE was calculated based on 3 sets of samples per treatment, and Student’s t test was used to calculate the statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, and ns, not significant in Student’s t test. (F) Venn diagram showing overlaps between coronatine-induced (COR Up) or coronatine-repressed genes (COR Down) and genes up- (fer Up) or downregulated (fer Down) in fer. The coronatine-regulated genes were previously published [28], and genes differentially expressed in fer were determined by RNA-seq. (G) Pseudomonas syringae tomato DC3000 accumulated more in fer. The bacteria were infiltrated into 5-week-old plants, and leaf discs were collected at different days after infiltration (dai). Bacterial accumulation was measured by colony-forming units (CFUs) per leaf disc. Average and SD were calculated from three replicates. The experiments were repeated more than 5 times with similar results [29]. (H and I) Loss-of-function myc2 mutant suppresses fer phenotype in growth as shown with four-week-old plants of WT, myc2, fer, and fer myc2 double mutants, bar in (H) represents 2 cm and quantification of the 5th leaf petioles length (average and SE were based on n = 15; I).

Article Snippet: Tomato DC3000 Cor- Dr. Ping He N/A Chemicals, Peptides, and Recombinant Proteins MG132 Sigma-Aldrich Cat#M7449 Cycloheximide Sigma-Aldrich Cat#C4859 Complete protease inhibitor cocktail tablets Sigma-Aldrich Cat#11836170001 Trizol Thermo Fisher Cat#15596018 RNeasy Kit QIAGEN Cat#74904 Phos-tag reagent Wako Cat#ALL-107 Linsmaier and Skoog Caisson Laboratories Cat#LPS03-1LT Dynabeads Protein A Thermo Fisher Cat#00354691 Jasmonic Acid Sigma-Aldrich Cat#J2500 Glutathione HiCap Matrix QIAGEN Cat#30900 Amylose Resin New England Biolabs Cat#E8021S Anti-FLAG M2 Magnetic Beads Sigma-Aldrich Cat#M8823-1ML Glu-C Thermo Fisher Cat#90054 Trypsin Roche Cat#03708969001 Ponceau S Sigma-Aldrich Cat#P7170-1L K252a Sigma-Aldrich Cat#K2015-200ul Halt protease and phosphatase inhibitor cocktail Thermo Fisher Cat#78440 Alkaline phosphatatse Sigma-Aldrich Cat#P7923-2KU Deposited Data Raw data files for RNA sequencing NCBI Sequence Read Archive SRA: PRJNA215313 (https://www.ncbi.nlm. nih.gov/biosample?LinkName=bioproject_ biosample_all&from_uid=2318102.)

Techniques: Gene Expression, RNA Sequencing, Bacteria, Mutagenesis

Journal: Current biology : CB

Article Title: FERONIA Receptor Kinase Contributes to Plant Immunity by Suppressing Jasmonic Acid Signaling in Arabidopsis thaliana.

doi: 10.1016/j.cub.2018.07.078

Figure Lengend Snippet: Figure 3. FER Phosphorylation of MYC2 De- stabilizes MYC2 (A) FER phosphorylation of MYC2NM (N and middle domains) is reduced when 12 phosphorylation sites are mutated to alanine, indicated as MYC2NMA12. Phosphorylated MYC2NM and FERK autophos- phorylation revealed by kinase assay is indi- cated. The bottom panel indicates MYC2NM and MYC2NMA12 used in the assay. (B) MYC2A12 phosphorylation is reduced in vivo. MYC2-FLAG and MYC2A12-FLAG were immuno- precipitated from transgenic plants, and the IP product was treated with phosphatase and resolved on a Phos-tag gel. There is a shift in the MYC2- FLAG, and the shift in MYC2A12-FLAG is minimal. (C and D) MYC2A12 has prolonged half-life than that of WT MYC2. Ten-day-old transgenic plants MYC2-FLAGox (C) or MYC2 A12-FLAGox (D) were incubated with CHX for indicated times and were collected and flash frozen. Total proteins were ex- tracted and resolved on SDS-PAGE. MYC2-FLAG and MYC2A12-FLAG were detected by anti-FLAG. Ponceau S staining is used as loading control. Quantification was carried out using ImageJ. (E) Both MYC2-FLAGox and MYC2A12-FLAGox are hypersensitive to JA in the root growth assay, indi- cating that MYC2A12 is functional (average and SD were calculated based on n = 14–18 plants). Sta- tistical significance was calculated using Tukey HSD test and p values less than 0.05 were consid- ered significant. (F) MYC2 protein accumulates in fer mutant. Five- week-old WT, fer, and myc2 plants were infiltrated with Pst DC3000 for indicated times. Total or nu- clear proteins were prepared from each sample, and the accumulation of MYC2 and FER was de- tected with anti-MYC2 or anti-FER. Anti-Histone H3 and Ponceau S staining were used as loading controls. Quantification of MYC2 was carried out using ImageJ. See also Figures S2G, S2H, and S3 and Tables S1, S2, and S3.

Article Snippet: Tomato DC3000 Cor- Dr. Ping He N/A Chemicals, Peptides, and Recombinant Proteins MG132 Sigma-Aldrich Cat#M7449 Cycloheximide Sigma-Aldrich Cat#C4859 Complete protease inhibitor cocktail tablets Sigma-Aldrich Cat#11836170001 Trizol Thermo Fisher Cat#15596018 RNeasy Kit QIAGEN Cat#74904 Phos-tag reagent Wako Cat#ALL-107 Linsmaier and Skoog Caisson Laboratories Cat#LPS03-1LT Dynabeads Protein A Thermo Fisher Cat#00354691 Jasmonic Acid Sigma-Aldrich Cat#J2500 Glutathione HiCap Matrix QIAGEN Cat#30900 Amylose Resin New England Biolabs Cat#E8021S Anti-FLAG M2 Magnetic Beads Sigma-Aldrich Cat#M8823-1ML Glu-C Thermo Fisher Cat#90054 Trypsin Roche Cat#03708969001 Ponceau S Sigma-Aldrich Cat#P7170-1L K252a Sigma-Aldrich Cat#K2015-200ul Halt protease and phosphatase inhibitor cocktail Thermo Fisher Cat#78440 Alkaline phosphatatse Sigma-Aldrich Cat#P7923-2KU Deposited Data Raw data files for RNA sequencing NCBI Sequence Read Archive SRA: PRJNA215313 (https://www.ncbi.nlm. nih.gov/biosample?LinkName=bioproject_ biosample_all&from_uid=2318102.)

Techniques: Phospho-proteomics, Kinase Assay, In Vivo, Transgenic Assay, Incubation, SDS Page, Staining, Control, Growth Assay, Functional Assay, Mutagenesis

Journal: Current biology : CB

Article Title: FERONIA Receptor Kinase Contributes to Plant Immunity by Suppressing Jasmonic Acid Signaling in Arabidopsis thaliana.

doi: 10.1016/j.cub.2018.07.078

Figure Lengend Snippet: Figure 4. FER Regulation of MYC2 and Modulation by RALF23 (A) Four-week-old plants show growth phenotypes of WT, RALF23ox, fer, and RALF23ox fer. Scale bar represents 2 cm. (B) RALF23ox plants are more sensitive to JA inhibition of root growth; average and SD were calculated from 14–18 seedlings. Statistical significance was calculated using Tukey HSD test, and p values less than 0.05 were considered significant (n = 14–18). (C) RALF23ox plants are more susceptible to bacterial infection. The experiment is described in Figure 1G. Average and SD were calculated from 3 replicates. The statistical significance is evaluated by Student’s t test; ***p < 0.001 (n = 3). The experiment was repeated three times with similar results. (D) RALF23ox plants accumulate more MYC2 in response to pst DC3000 than WT control. Four-week-old plants with the indicated genotypes were infiltrated with Pst DC3000 for 2 days, and nuclear protein was prepared and blotted with anti-MYC2 or anti-histone H3. (E–H) Relative JA target gene expression analysis in RALF23ox shows that RALF23 plays an important role in JA signaling: RD26/UBQ5 (B), TAT/UBQ5 (C), VSP1 induction by 50 mM JA (JA50/CK, D) and PDF1.2 induction by 50 mM JA (JA50/CK, E). RNA was prepared from 10-day-old WT or RALF23ox seedlings with or without 50 mM JA, and qPCR was performed with the indicated genes. SE was calculated based on 3 sets of samples per treatment, and Student’s t test was used to calculate the statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. (I) Similar to the effect of RALF23 on MYC2 in RALF23ox, short-term RALF23 treatment also promotes MYC2 stability. FER-GFP, MYC2-FLAG, and S1P-YFP were co-expressed with RALF23 or vector only for 48 hr. Total protein was extracted from leaf discs and resolved on SDS-PAGE. All three leaves assayed showed elevated MYC2 level. (J) A working model for FER and RALF23 regulation of MYC2 during pst DC3000 infection. FER phosphorylates and inhibits MYC2 to positively contribute to plant defense. RALF23 peptide, the processing of which is increased by bacterial infection, functions to inhibit FER receptor signaling hence negatively contributes to bacterial defense. See also Figures S3A, S3D, and S4 and Table S3.

Article Snippet: Tomato DC3000 Cor- Dr. Ping He N/A Chemicals, Peptides, and Recombinant Proteins MG132 Sigma-Aldrich Cat#M7449 Cycloheximide Sigma-Aldrich Cat#C4859 Complete protease inhibitor cocktail tablets Sigma-Aldrich Cat#11836170001 Trizol Thermo Fisher Cat#15596018 RNeasy Kit QIAGEN Cat#74904 Phos-tag reagent Wako Cat#ALL-107 Linsmaier and Skoog Caisson Laboratories Cat#LPS03-1LT Dynabeads Protein A Thermo Fisher Cat#00354691 Jasmonic Acid Sigma-Aldrich Cat#J2500 Glutathione HiCap Matrix QIAGEN Cat#30900 Amylose Resin New England Biolabs Cat#E8021S Anti-FLAG M2 Magnetic Beads Sigma-Aldrich Cat#M8823-1ML Glu-C Thermo Fisher Cat#90054 Trypsin Roche Cat#03708969001 Ponceau S Sigma-Aldrich Cat#P7170-1L K252a Sigma-Aldrich Cat#K2015-200ul Halt protease and phosphatase inhibitor cocktail Thermo Fisher Cat#78440 Alkaline phosphatatse Sigma-Aldrich Cat#P7923-2KU Deposited Data Raw data files for RNA sequencing NCBI Sequence Read Archive SRA: PRJNA215313 (https://www.ncbi.nlm. nih.gov/biosample?LinkName=bioproject_ biosample_all&from_uid=2318102.)

Techniques: Inhibition, Infection, Control, Targeted Gene Expression, Plasmid Preparation, SDS Page

Journal: Cancer cell

Article Title: Cathepsin S Regulates Antigen Processing and T Cell Activity in Non-Hodgkin Lymphoma.

doi: 10.1016/j.ccell.2020.03.016

Figure Lengend Snippet: Figure 1. Functional Characterization of CTSS Y132D Mutation in FL (A) Distribution of CTSS mutations in 299 FL patients. (B) The total number of FL and DLBCL patients and the frequency of the Y132D mutation in the reported studies. (C) Stacked barplot indicating the percentage of patients with CTSS WT and mutated (Y132D/N). (D) Schematic representation of tyrosine (Y) 132 amino acid localization on CTSS binding site, and tridimensional structure of the proteins, colored according to the amino acid electrostatic potential. (E) Western blot of recombinant of CTSS-His WT and mutated CTSS-His Y132D using an anti-His-tag antibody. M, marker. (F) Quantification of western blot signals of CTSS mature protein normalized to the pro-CTSS signal (n = 3). (G) Graphical representation of CD74 sequence between amino acids 89 and 121 and fluorescence resonance energy transfer (FRET) experimental design. The CTSS substrate is highlighted in blue, the arrow indicates the exact cleavage site, and the CLIP sequence is shown in green. (H) Representative FRET experiment with recombinant CTSS-His-WT and CTSS-His-Y132D. Signal Intensity normalized to the background (n = 3). (I) Quantification FRET emission normalized to CTSS WT signals (n = 6). The p value was calculated using a paired t test. (F, H, and I) Data are presented as mean ± standard deviation. See also Figure S1 and Tables S1, S2, and S3.

Article Snippet: After blocking in 5% milk (Applichem, cat #A0830) in PBS-0.1%Tween (Fisher scientific, cat #BP337), membranes were incubated overnight in 5% milk in PBS-Tween with the following primary antibodies: goat anti-human Cathepsin S (RD, cat #AF1183, 1:2000), Cathepsin B (D1C7Y) XP Rabbit (Cell signaling mAb #31718), rabbit anti-mouse Cathepsin S (Sino Biological, cat #50769-R054, 1:1000), mouse anti-human CD74 (PIN.1, StressMarq, cat #SMC-116, diluted 1:1000), rat-anti-mouse CD74 (clone ln-1, BD biosciences, cat #555317, 1:1000), rabbit anti-his-Tag (Cell Signaling, cat #D3I10, dilute 1:1000), mouse anti-human alpha-Tubulin (clone B-5-1-2, Sigma Aldrich, cat#T6072, diluted 1:10000), anti-beta actin (clone 8H10D10, ThermoFisher Scientific, cat# MA5-15452, 1:1000).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Western Blot, Recombinant, Marker, Sequencing, Förster Resonance Energy Transfer, Standard Deviation

Journal: Cell communication and signaling : CCS

Article Title: Pin1 promotes human Ca V 2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.

doi: 10.1186/s12964-024-01960-9

Figure Lengend Snippet: Fig. 1 Interaction and colocalization of CaV2.1 and Pin1. A Representative immunoblots showing the association of Myc-tagged rat Pin1 (Myc-Pin1) with the GST fusion protein comprising human CaV2.1 distal carboxy-terminal fragment (GST-CaV2.1-C-ter), but not the GST protein per se. Lysates from HEK293T cells overexpressing Myc-Pin1 were subject to GST pull-down assay, followed by immunoblotting with the anti-Myc antibody (α-Myc). GST and GST-CaV2.1 fusion proteins were detected with the anti-GST antibody (α-GST). Input represents ~ 1% of total cell lysate volume. Arrowheads denote the location of GST or GST-CaV2.1 fusion protein bands. Molecular weight markers (in kDa) are labeled to the left. Arrowheads denote the corresponding GST fragments. B Representative immunoblots demonstrating the coimmunoprecipitation of Myc-Pin1 with the human long carboxy-terminal CaV2.1 in HEK293T cells. Coexpression with the Myc vector was used as the control. HEK293T cell lysates were immunoprecipitated (IP) with α-Myc, followed by immunoblotting with α-Myc or the anti-human CaV2.1 carboxy-terminal antibody (α-CaV2.1-CT). The apparent molecular weight of human CaV2.1 long- isoform is ~ 280–290 kDa (arrowhead). Corresponding expression level of human CaV2.1 and Myc-Pin1 in the lysates is shown in the Input lane, which represents ~ 10% of the total protein used for immunoprecipitation. C Representative immunoblots displaying the interaction of endogenous CaV2.1 (arrow) and Pin1 (arrowhead) in the rat brain. Endogenous rat CaV2.1 in the brain comprises a major isoform with an apparent molecular weight ~ 190 kDa, as well as a minor isoform of ~ 220 kDa. Rat forebrain lysates were immunoprecipitated with the anti-rat CaV2.1 antibody (α-CaV2.1) or the rabbit IgG. D Representative immunoblots depicting the colocalization of endogenous CaV2.1 and Pin1 at presynaptic and postsynaptic compartments in the rat brain. Following subcellular fractionation, rat brain lysates were separated into homogenate (H), soluble (S1), crude membrane (P2), synaptosomal (SPM), and two postsynaptic density (PSD I, PSD II) fractions, with synaptophysin and PSD95 serving as the presynaptic and the postsynaptic markers, respectively. The labels 40 µg and 20 µg denote the amount of total protein loaded in each lane. E Representative immunoblots illustrating corresponding protein level of endogenous CaV2.1 and Pin1 in cultured rat cortical neurons with the indicated days in vitro (DIV). F Representative confocal images of endogenous CaV2.1 (left panels) and Pin1 (right panels) immunofluorescent signals in rat DIV10 cortical neurons, showing colocalization of CaV2.1 or Pin1 (green) with synaptophysin-puncta (red) along neurites, as denoted by arrowheads and further highlighted by yellow puncta in the merge images. The boxed regions in the images shown in the upper rows are magnified for detailed inspection in the corresponding lower rows. Scale bars, 50 μm (upper rows) and 12.5 μm (lower rows). See Supplementary Figure S1 for additional confocal images exemplifying the colocalization of CaV2.1/Pin1 with MAP2, tau, or PSD-95. G Quantification of puncta density (Puncta/100 µm) and puncta colocalization ratio (Fraction of colocalization) in rat DIV10 cortical neurons. (i) Puncta densi ties per 100-µm neurite: 57.17 ± 9.32 (CaV2.1), 59.39 ± 12.50 (Pin1), 53.19 ± 10.53 (synaptophysin), and 49.69 ± 12.09 (PSD95). (ii-iii) The fraction of CaV2.1 and Pin1 puncta colocalized with synaptophysin is ~ 64.80 ± 13.80% and 64.78 ± 10.71%, respectively. The fraction of CaV2.1 and Pin1 puncta colocalized with PSD95 is ~ 50.48 ± 7.93% and 55.08 ± 11.61%, respectively. (iv-v) The fraction of synaptophysin puncta colocalized with CaV2.1 and Pin1 is ~ 60.97 ± 8.77% and 64.51 ± 11.65%, respectively. The fraction of PSD95 puncta colocalized with CaV2.1 and Pin1 is ~ 66.12 ± 8.79% and 66.15 ± 12.08%, respectively. The data were compiled from 17–25 individual neurites associated with 3–6 different cortical neurons

Article Snippet: Briefly, the cDNA fragments for human long-isoform CaV2.1 amino-terminal region (CaV2.1-N-ter; amino acids 1–99), cytoplasmic loop segment connecting transmembrane domains I and II (I-II loop) (CaV2.1-I-II; amino acids 361–487), II-III loop (CaV2.1-II-III; amino acids 715–1245), III-IV loop (CaV2.1-III-IV; amino acids 1512– 1567), proximal carboxy-terminal region (CaV2.1-pC-ter; amino acids 1816–2203), and distal carboxy-terminal region (CaV2.1-C-ter; amino acids 2204–2510), as well as human Pin1 (Addgene 19027), were subcloned into the pGEX vector (GE Healthcare Biosciences, Piscataway, NJ, USA) and expressed in the E. coli strain BL21.

Techniques: Western Blot, Pull Down Assay, Molecular Weight, Labeling, Plasmid Preparation, Control, Immunoprecipitation, Expressing, Fractionation, Membrane, Cell Culture, In Vitro

Journal: Cell communication and signaling : CCS

Article Title: Pin1 promotes human Ca V 2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.

doi: 10.1186/s12964-024-01960-9

Figure Lengend Snippet: Fig. 2 Reduction of CaV2.1 protein expression by Pin1. A (Left) Representative immunoblots showing the coimmunoprecipitation of Myc-tagged human long carboxy-terminal CaV2.1 (CaV2.1-6myc) with HA-tagged Pin1 R69L mutant (HA-Pin1-R69L) in HEK293T cells. Cells transfected with Myc vector (−) were used as control. Arrowhead denotes the primary CaV2.1 protein band. (Right) Representative immunoblots (top) and quantification (bottom) of the effect of coexpression (in the molar ratio 3:1) with Flag vector (−), Flag-tagged Pin1 (Flag-Pin1) or Flag-tagged Pin1-R69L mutant (Flag-Pin1-R69L) on CaV2.1 pro tein level in HEK293T cells. Tubulin expression was chosen as the loading control. Total CaV2.1 protein density was standardized as the ratio to the cognate tubulin signal, followed by normalization with respect to the corresponding vector control. Normalized CaV2.1 protein levels (n = 9–10): vector, 1.02 ± 0.20; Pin1, 0.38 ± 0.18; Pin1-R69L, 0.89 ± 0.35. Asterisks denote significant difference from the vector control (*, P < 0.05). B (Top panels) Representative immu noblots demonstrating the effect of Pin1 coexpression on protein level of Myc-tagged human CaV2.1 short-isoform (CaV2.1-Short-6myc; ~260–270 kDa), HA-tagged rat CaV1.2 (CaV1.2-HA; ~260 kDa), or Flag-tagged bovine CaV2.2 (CaV2.2-Flag; ~255–280 kDa) in HEK293T cells. (Bottom panels) Quantification of relative Ca2+ channel protein level (n = 4–6). CaV2.1-short: vector, 1.03 ± 0.18; Pin1, 0.48 ± 0.26. CaV1.2: vector, 1.01 ± 0.16; Pin1, 1.12 ± 0.28. CaV2.2: vec tor, 1.00 ± 0.14; Pin1, 0.98 ± 0.39. Asterisks denote significant difference from the vector control (*, P < 0.05). C-E (Top panels) Representative immunoblots displaying the effect of shRNA knockdown (shPin1-1, shPin1-2) (left) and ATRA-induced suppression (right) of endogenous Pin1 expression on protein level of overexpressed human CaV2.1 in HEK293T cells (C), or endogenous rat CaV2.1 in PC12 cells (D) and cultured cortical neurons (E). Cells were subject to treatment with 25 or 50 µM ATRA for 24 h. shGFP and DMSO were employed as infection and drug treatment controls, respectively. Tubulin and actin were used as loading controls. Arrows refer to the endogenous 190-kDa rat CaV2.1. (Middle panels) Quantification of relative Ca2+ channel protein levels (n = 4–10). Total CaV2.1 protein density was standardized as the ratio to the loading control, followed by normalization with respect to the corresponding shGFP or DMSO control. HEK293T cells: (left) shGFP (1.00 ± 0.07), shPin1-1 (1.91 ± 0.71), shPin1-2 (2.17 ± 0.44); (right) DMSO (1.00 ± 0.11), ATRA (1.91 ± 0.44). PC12 cells: (left) shGFP (1.00 ± 0.09), shPin1-1 (2.01 ± 0.75), shPin1-2 (2.44 ± 0.92); (right) DMSO (1.00 ± 0.13), ATRA (1.49 ± 0.24). Cortical neurons: (left) shGFP (1.04 ± 0.16), shPin1-1 (1.63 ± 0.28), shPin1-2 (1.75 ± 0.44); (right) DMSO (1.04 ± 0.16), 25 µM ATRA (1.63 ± 0.28), 50 µM ATRA (1.75 ± 0.44). (Bottom panels) Quantification of relative Pin1 protein level (n = 3–8). Asterisks denote significant difference from the cognate shGFP or DMSO control (*, P < 0.05)

Article Snippet: Briefly, the cDNA fragments for human long-isoform CaV2.1 amino-terminal region (CaV2.1-N-ter; amino acids 1–99), cytoplasmic loop segment connecting transmembrane domains I and II (I-II loop) (CaV2.1-I-II; amino acids 361–487), II-III loop (CaV2.1-II-III; amino acids 715–1245), III-IV loop (CaV2.1-III-IV; amino acids 1512– 1567), proximal carboxy-terminal region (CaV2.1-pC-ter; amino acids 1816–2203), and distal carboxy-terminal region (CaV2.1-C-ter; amino acids 2204–2510), as well as human Pin1 (Addgene 19027), were subcloned into the pGEX vector (GE Healthcare Biosciences, Piscataway, NJ, USA) and expressed in the E. coli strain BL21.

Techniques: Expressing, Western Blot, Mutagenesis, Transfection, Plasmid Preparation, Control, shRNA, Knockdown, Cell Culture, Infection

Journal: Cell communication and signaling : CCS

Article Title: Pin1 promotes human Ca V 2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.

doi: 10.1186/s12964-024-01960-9

Figure Lengend Snippet: Fig. 3 Attenuation of cell-surface and functional expression of CaV2.1 by Pin1. A Surface biotinylation experiments in HEK293T cells coexpressing CaV2.1- 6myc with Flag vector (−), Flag-Pin1, or Flag-Pin1-R69L. (Left) Representative immunoblots showing cell-surface expression of human CaV2.1. Lysates from biotinylated intact cells were either directly employed for immunoblotting analyses (Total), or subject to streptavidin pull-down prior to immunoblotting analyses (Surface). GAPDH was used as the loading control. CaV2.1-6myc, α2δ, and β4a were coexpressed in the molar ratio 1:2:1. Arrowheads denote the primary CaV2.1 protein band. (Right panels) Quantification of relative total protein level (total signal), surface protein level (surface signal), and membrane trafficking efficiency (surface/total ratio) of CaV2.1 (n = 4–5). Total protein density was standardized as the ratio of total CaV2.1 to GAPDH signals. Surface protein density was standardized as the ratio of surface CaV2.1 to cognate total GAPDH signals. Membrane trafficking efficiency was calculated as the ratio of surface protein density to standardized total protein density. All data were normalized with respect to the corresponding vector control. Total CaV2.1: vector, 1.00 ± 0.11; Pin1, 0.36 ± 0.21; Pin1-R69L, 0.96 ± 0.31. Surface CaV2.1: vector, 1.00 ± 0.09; Pin1, 0.36 ± 0.28; Pin1-R69L, 0.83 ± 0.38. CaV2.1 trafficking: vec tor, 1.00 ± 0.11; Pin1, 0.93 ± 0.48; Pin1-R69L, 0.99 ± 0.49. Asterisks denote significant difference from the vector control (*, P < 0.05). B Two-electrode voltage clamp experiments in Xenopus oocytes expressing CaV2.1 in the absence or presence of Pin1, or Pin1-R69L. (Left panels) Representative Ba2+ current traces through human CaV2.1 channel. From a holding potential of -90 mV, oocytes were subject to 70-ms test pulses ranging from − 80 to + 60 mV (in 10-mV in crements). Oocytes coinjected with CaV2.1 cRNA and water were used as the control. (Right) Quantification of relative peak Ba2+ current amplitude at + 20 mV (n = 33–47). Data were normalized with respect to the CaV2.1-water coinjection control (−). Normalized CaV2.1 current amplitude: control, 1.00 ± 0.48; Pin1, 0.32 ± 0.20; Pin1-R69L, 0.82 ± 0.48. Asterisks denote significant difference from the water control (*, P < 0.05). C (Left) Representative immunoblots comparing the effect of 24-hr treatment with DMSO (−) or 50 µM ATRA on cell-surface expression of human CaV2.1 in HEK293T cells. Arrowheads denote the primary CaV2.1 protein band. (Right Panels) Quantification of relative CaV2.1 protein level and membrane trafficking efficiency (n = 5). Total CaV2.1: DMSO, 1.00 ± 0.10; ATRA, 3.40 ± 1.07. Surface CaV2.1: DMSO, 1.03 ± 0.16; ATRA, 3.25 ± 1.65. CaV2.1 trafficking: DMSO, 1.00 ± 0.12; ATRA, 1.19 ± 0.33. Asterisks denote significant difference from the DMSO control (*, P < 0.05)

Article Snippet: Briefly, the cDNA fragments for human long-isoform CaV2.1 amino-terminal region (CaV2.1-N-ter; amino acids 1–99), cytoplasmic loop segment connecting transmembrane domains I and II (I-II loop) (CaV2.1-I-II; amino acids 361–487), II-III loop (CaV2.1-II-III; amino acids 715–1245), III-IV loop (CaV2.1-III-IV; amino acids 1512– 1567), proximal carboxy-terminal region (CaV2.1-pC-ter; amino acids 1816–2203), and distal carboxy-terminal region (CaV2.1-C-ter; amino acids 2204–2510), as well as human Pin1 (Addgene 19027), were subcloned into the pGEX vector (GE Healthcare Biosciences, Piscataway, NJ, USA) and expressed in the E. coli strain BL21.

Techniques: Functional Assay, Expressing, Plasmid Preparation, Western Blot, Control, Membrane

Journal: Cell communication and signaling : CCS

Article Title: Pin1 promotes human Ca V 2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.

doi: 10.1186/s12964-024-01960-9

Figure Lengend Snippet: Fig. 6 Requirement of Pin1-mediated isomerization for CaV2.1 degradation by RNF138. A Representative immunoblots showing the coimmunoprecipi tation of endogenous CaV2.1 (arrow), RNF138 (open triangle), and Pin1 (arrowhead) in the rat brain. Rat forebrain lysates were immunoprecipitated with α-rCaV2.1 or the rabbit IgG. B Representative immunoblots demonstrating the coimmunoprecipitation of RNF138 with CaV2.1 and Pin1 (left), but not CaV2.1 and the isomerase-inactive mutant Pin1-R69L (right). Lysates from HEK293T cells coexpressing Flag-RNF138 with Myc vector (−), CaV2.1-6myc, HA- Pin1, or HA-Pin1-R69L were immunoprecipitated with α-Myc, followed by immunoblotting with α-Myc, α-Flag, and α-HA. Arrowheads denote the primary CaV2.1 protein band. C Representative immunoblots depicting the deficient coimmunoprecipitation of RNF138 with CaV2.1-LCA. Lysates from HEK293T cells coexpressing Flag-RNF138 with Myc vector (−), CaV2.1-6myc, or CaV2.1-LCA-6myc were immunoprecipitated with α-Myc, followed by immunoblot ting with α-Myc and α-Flag. Arrowhead denotes the primary CaV2.1 protein band. D Lack of effect of RNF138 on CaV2.1-LCA protein expression in HEK293T cells. Representative immunoblots and quantification of the effect of RNF138 coexpression on CaV2.1 (left panels) or CaV2.1-LCA (right panels). Normalized CaV2.1 protein level (n = 6): vector, 1.00 ± 0.18; RNF138, 0.45 ± 0.18. Normalized CaV2.1-LCA protein level (n = 6): vector, 1.00 ± 0.14; RNF138, 1.08 ± 0.31. Asterisk denotes significant difference from the cognate vector control (*, P < 0.05). E Lack of effect of RNF138 on functional expression of CaV2.1-LCA in Xenopus oocytes. (Left panels) Representative Ba2+ current traces and quantification of the effect of RNF138 coexpression on CaV2.1 (left panels) or CaV2.1- LCA (right panels). Oocytes coinjected with CaV2.1/CaV2.1-LCA cRNA and water were used as the control. Normalized CaV2.1 current amplitude at + 20 mV (n = 22–24): control, 1.00 ± 0.28; RNF138, 0.30 ± 0.19. Normalized CaV2.1-LCA current amplitude at + 20 mV (n = 22–25): control, 1.00 ± 0.57; RNF138, 0.93 ± 0.50. Asterisk denotes significant difference from the cognate control (*, P < 0.05)

Article Snippet: Briefly, the cDNA fragments for human long-isoform CaV2.1 amino-terminal region (CaV2.1-N-ter; amino acids 1–99), cytoplasmic loop segment connecting transmembrane domains I and II (I-II loop) (CaV2.1-I-II; amino acids 361–487), II-III loop (CaV2.1-II-III; amino acids 715–1245), III-IV loop (CaV2.1-III-IV; amino acids 1512– 1567), proximal carboxy-terminal region (CaV2.1-pC-ter; amino acids 1816–2203), and distal carboxy-terminal region (CaV2.1-C-ter; amino acids 2204–2510), as well as human Pin1 (Addgene 19027), were subcloned into the pGEX vector (GE Healthcare Biosciences, Piscataway, NJ, USA) and expressed in the E. coli strain BL21.

Techniques: Western Blot, Immunoprecipitation, Mutagenesis, Plasmid Preparation, Expressing, Control, Functional Assay

Journal: Cell communication and signaling : CCS

Article Title: Pin1 promotes human Ca V 2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.

doi: 10.1186/s12964-024-01960-9

Figure Lengend Snippet: Fig. 7 Proteostatic regulation of EA2-causing CaV2.1 nonsense and missense mutants by Pin1 in HEK293T cells. A Representative immunoblots and quantification of the effect of ATRA on EA2-causing CaV2.1 nonsense (R1281x, R1669x) and missense (F1406C, E1761K) mutants. Lysates from transfected cells were subject to treatment with DMSO or 50 µM ATRA for 24 h. Normalized CaV2.1-R1281x protein level (n = 6): DMSO, 1.00 ± 0.17; ATRA, 4.05 ± 1.19. Normalized CaV2.1-R1669x protein level (n = 6): DMSO, 1.00 ± 0.11, ATRA, 4.35 ± 1.45. Normalized CaV2.1-F1406C protein level (n = 6): DMSO, 1.00 ± 0.32; ATRA, 2.69 ± 1.05. Normalized CaV2.1-E1761K protein level (n = 5): DMSO, 1.00 ± 0.36; ATRA, 7.25 ± 2.24. Asterisks denote significant difference from the cognate DMSO control (*, P < 0.05). B Representative immunoblots and quantification of the effect of Pin1 and Pin1-R69L on CaV2.1-F1406C. Normalized CaV2.1-F1406C protein level (n = 7–8): vector, 1.00 ± 0.16; Pin1, 0.38 ± 0.26; Pin1-R69L, 1.05 ± 0.32. Asterisk denotes significant difference from the vector control (*, P < 0.05). C Representative immunoblots and quantification of the effect of shRNA knockdown of endogenous Pin1 on CaV2.1-F1406C. Normal ized CaV2.1-F1406C protein level (n = 4): shGFP, 1.00 ± 0.12; shPin1-1, 4.74 ± 2.00; shPin1-2, 3.58 ± 1.98. Normalized endogenous Pin1 protein level (n = 6–8): shGFP, 1.00 ± 0.13; shPin1-1, 0.65 ± 0.23; shPin1-2, 0.42 ± 0.19. Asterisks denote significant difference from the shGFP control (*, P < 0.05). D Representative immunoblots and quantification of the Pin1 coimmunoprecipitation efficiency of CaV2.1 and CaV2.1-F1406C. Lysates from cells coexpressing HA-Pin1 with CaV2.1 or CaV2.1-F1406C were subject to treatment with 10 µM MG132 for 24 h, followed by immunoprecipitation with α-Myc and immunoblotting analyses with α-Myc and α-HA. Arrowheads denote the primary CaV2.1 protein band. Normalized coimmunoprecipitated Pin1 protein level (n = 3): CaV2.1, 1.00 ± 0.04; Cav2.1-F1406C, 1.32 ± 0.10. Asterisk denotes significant difference from CaV2.1 (*, P < 0.05). E Representative immunoblots and quantification of the effect of ATRA on relative protein expression of CaV2.1 and CaV2.1-F1406C. Normalized protein level in response to DMSO (n = 7): CaV2.1, 1.00 ± 0.12; F1406C, 0.51 ± 0.28. Normalized protein level in response to ATRA (n = 13–14): CaV2.1, 1.00 ± 0.12; CaV2.1-F1406C, 1.01 ± 0.32. Asterisk denotes significant difference from CaV2.1 (*, P < 0.05)

Article Snippet: Briefly, the cDNA fragments for human long-isoform CaV2.1 amino-terminal region (CaV2.1-N-ter; amino acids 1–99), cytoplasmic loop segment connecting transmembrane domains I and II (I-II loop) (CaV2.1-I-II; amino acids 361–487), II-III loop (CaV2.1-II-III; amino acids 715–1245), III-IV loop (CaV2.1-III-IV; amino acids 1512– 1567), proximal carboxy-terminal region (CaV2.1-pC-ter; amino acids 1816–2203), and distal carboxy-terminal region (CaV2.1-C-ter; amino acids 2204–2510), as well as human Pin1 (Addgene 19027), were subcloned into the pGEX vector (GE Healthcare Biosciences, Piscataway, NJ, USA) and expressed in the E. coli strain BL21.

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, shRNA, Knockdown, Immunoprecipitation, Expressing

Journal: Cell communication and signaling : CCS

Article Title: Pin1 promotes human Ca V 2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.

doi: 10.1186/s12964-024-01960-9

Figure Lengend Snippet: Fig. 10 Schematic model of Pin1 regulation of human CaV2.1 proteostasis at the ER. A ER quality control of CaV2.1 is proposed to entail at least three sets of different protein folding checkpoints, each associated with a unique ER-associated degradation mechanism. Probably during translation or early pro tein folding stage, an upstream E3 ubiquitin ligase (E3 in cyan) might be recruited to promote proteasomal degradation of misfolded CaV2.1. Subsequent post-translational folding at the ER might involve both Pin1-dependent and -independent pathways. The current study supports the idea that, following phosphorylation-dependent, Pin1-catalyzed cis/trans isomerization, misfolded CaV2.1 is subject to polyubiquitination and proteasomal degradation me diated by the downstream E3 ligase RNF138. All-trans retinoic acid (ATRA), a therapeutic drug for leukemia, directly binds to and promotes degradation of Pin1, effectively preventing CaV2.1 degradation by RNF138. On the other hand, in the Pin1-independent protein folding pathway, a distinct downstream E3 ligase (E3 in green) is speculated to target misfolded CaV2.1 to the proteasome as well. B Dominant-negative effect of EA2-causing mutants is proposed to comprise both Pin1/RNF138-dependent and -independent ER quality control mechanisms. Through as yet unknown process (red question mark), misfolded EA2 missense mutants may interfere with protein folding of CaV2.1 WT, and thereby facilitate Pin1-catalyzed isomerization and consequently enhance RNF138-mediated polyubiquitination and proteasomal degradation of CaV2.1 WT. In contrast, perhaps via interaction with their amino-terminal regions (red question mark), misfolded EA2 nonsense mutants may disrupt either the upstream or the downstream Pin1/RNF138-independent protein folding steps of CaV2.1 WT, leading to RNF138-independent proteasomal degradation, as well as unfolded protein response, of CaV2.1 WT

Article Snippet: Briefly, the cDNA fragments for human long-isoform CaV2.1 amino-terminal region (CaV2.1-N-ter; amino acids 1–99), cytoplasmic loop segment connecting transmembrane domains I and II (I-II loop) (CaV2.1-I-II; amino acids 361–487), II-III loop (CaV2.1-II-III; amino acids 715–1245), III-IV loop (CaV2.1-III-IV; amino acids 1512– 1567), proximal carboxy-terminal region (CaV2.1-pC-ter; amino acids 1816–2203), and distal carboxy-terminal region (CaV2.1-C-ter; amino acids 2204–2510), as well as human Pin1 (Addgene 19027), were subcloned into the pGEX vector (GE Healthcare Biosciences, Piscataway, NJ, USA) and expressed in the E. coli strain BL21.

Techniques: Control, Ubiquitin Proteomics, Phospho-proteomics, Dominant Negative Mutation