Journal: Cell Communication and Signaling : CCS

Article Title: SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

doi: 10.1186/s12964-024-01850-0

Figure Lengend Snippet: AP2M1’s Role in TRPV1’s SUMOylation-Dependent Membrane Localization. (A) Exploration of the AP2M1 protein-protein interaction network using the STRING database. CoIP-MS analysis was performed on protein lysates from MGC-803 cells stably expressing Flag-hTRPV1 WT and Flag-hTRPV1 K823R , identifying differential binding partners in TRPV1 K823R versus TRPV1 WT cell lines. (B) SUMOylation contributed to AP2M1’s modulation of TRPV1 membrane expression in GC xenografts. Tissue lysates from GC xenografts of WT and K823R groups underwent Co-IP using Flag beads, followed by IB with anti-AP2M1 and anti-Flag antibodies (biological replicates ≥ 3). (C) In vivo SUMOylation’s involvement in AP2M1-mediated changes in TRPV1 membrane expression. Tissue lysates from spontaneous gastric tumorigenesis in WT and KI mice were analyzed through Co-IP with either control IgG or anti-AP2M1 antibody, followed by IB with anti-AP2M1 and anti-TRPV1 (biological replicates ≥ 3). (D) Disrupting TRPV1-AP2M1 interaction enhanced TRPV1’s membrane presence in MGC-803 cells. Membrane proteins from stably transfected MGC-803 cells were biotinylated, isolated with streptavidin-agarose, and assessed by IB on 12% SDS-polyacrylamide gels using anti-Flag and on 8% SDS-polyacrylamide gels using anti-TfR antibodies (biological replicates ≥ 3). (E) AP2M1 knockdown eliminated TRPV1-AP2M1 association in MGC-803 cells. Following co-transfection with AP2M1 siRNA and either Flag-hTRPV1 WT or Flag-hTRPV1 K823R , cell lysates were subjected to precipitation with Flag beads and analyzed by Western blot to detect protein interactions (biological replicates ≥ 3). (F) AP2M1 knockdown enhanced TRPV1’s membrane localization in MGC-803 cells. Membrane proteins from cells co-transfected with AP2M1 siRNA and either Flag-hTRPV1 WT or Flag-hTRPV1 K823R were isolated using the biotin-avidin method and quantified by IB using anti-Flag or anti-TfR following biotinylation and purification with streptavidin-agarose (biological replicates ≥ 3)

Article Snippet: Additionally, we utilized the HA-AP2M1 plasmid (catalog number HG16144-NY) sourced from Sino Biological Inc., Shanghai, China.

Techniques: Membrane, Stable Transfection, Expressing, Binding Assay, Co-Immunoprecipitation Assay, In Vivo, Control, Transfection, Isolation, Knockdown, Cotransfection, Western Blot, Avidin-Biotin Assay, Purification

Journal: Cell Communication and Signaling : CCS

Article Title: SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

doi: 10.1186/s12964-024-01850-0

Figure Lengend Snippet: Impact of Disrupting AP2M1-TRPV1 Interaction on GC Cell Migration and Proliferation. (A) Mapping the TRPV1 interaction domain on AP2M1. Four AP2M1 truncation mutants were generated: HA-AP2M1 1–261 , HA-AP2M1 262–435 , HA-AP2M1 1–165 , and HA-AP2M1 166–261 . MGC-803 cells were transiently co-transfected with Flag-hTRPV1 WT and these mutants along with the HA-AP2M1 plasmid. IP used an anti-Flag antibody, followed by IB with anti-HA and anti-Flag to detect interactions. Input levels were verified by IB using anti-Flag and anti-HA antibodies (biological replicates ≥ 3). (B) Depiction of HA-AP2M1 WT deletions, including segments Δ166–175, Δ176–185, Δ186–195, Δ196–205, Δ206–216, Δ217–226, Δ227–237, Δ238–248, and Δ249–261, to identify critical interaction regions. (C) The 176–185 region on AP2M1 was crucial for anchoring TRPV1. Co-transfection of MGC-803 cells with Flag-hTRPV1 WT and HA-AP2M1 WT or mutants, followed by Co-IP using Flag beads and subsequent HA probing, identified this segment as key for TRPV1 binding (biological replicates ≥ 3). (D) AP2M1’s interaction with TRPV1 enhanced TRPV1’s membrane localization via SUMOylation. Co-expression of Flag-hTRPV1 WT or Flag-hTRPV1 K823R with either HA-AP2M1 WT or HA-AP2M1 Δ176–185 in AP2M1 -deficient MGC-803 cells allowed for membrane TRPV1 level assessment through the biotin-avidin method. IB analysis followed, targeting biotinylated membrane proteins with anti-Flag or anti-TfR (biological replicates ≥ 3). (E) Interrupting the TRPV1-AP2M1 bond reduced MGC-803 cell migration as shown by transwell migration assays. Migration counts are presented as mean ± SEM from ≥ 3 biological replicates; **** p < 0.0001, ns = no significant, analyzed by one-way ANOVA with Tukey’s test. Scale bar: 200 μm. (F) Colony formation assays revealed that disrupting TRPV1-AP2M1 interaction decreased the clonogenic potential of MGC-803 cells. Colony counts are summarized as mean ± SEM from ≥ 3 biological replicates; **** p < 0.0001, ns = no significant, via one-way ANOVA with Tukey’s test

Article Snippet: Additionally, we utilized the HA-AP2M1 plasmid (catalog number HG16144-NY) sourced from Sino Biological Inc., Shanghai, China.

Techniques: Migration, Generated, Transfection, Plasmid Preparation, Cotransfection, Co-Immunoprecipitation Assay, Binding Assay, Membrane, Expressing, Avidin-Biotin Assay

Journal: Cell Communication and Signaling : CCS

Article Title: SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

doi: 10.1186/s12964-024-01850-0

Figure Lengend Snippet: The TAT-AP2M1-176–185 Peptide Mitigates GC Pathogenicity by Modulating TRPV1 Localization and Function. (A) Design of TAT fusion peptides, including the hAP2M1 fragment 176–185 sequence and a scrambled sequence as a control, to investigate their effects on TRPV1-AP2M1 interactions. (B) Application of the TAT-AP2M1-176–185 peptide disrupted AP2M1’s binding to TRPV1 in MGC-803 cells expressing Flag-hTRPV1 WT or Flag-hTRPV1 K823R . Cells were treated with 20 µM of either TAT-AP2M1-176–185 or TAT-Scramble for 3 h, followed by Co-IP with an anti-Flag antibody and IB for AP2M1 detection (biological replicates ≥ 3). (C) Enhanced membrane expression of TRPV1 in MGC-803 cells post TAT-AP2M1-176–185 treatment, as measured by biotin-avidin purification and IB using anti-Flag or anti-TfR. Biological replicates ≥ 3. (D) Interruption of the TRPV1-AP2M1 interaction reduced MGC-803 cell migration, as shown by transwell migration assays. Data are presented as mean ± SEM from ≥ 3 biological replicates; **** p < 0.0001, with no significant (ns) difference noted in certain comparisons, analyzed by one-way ANOVA with Tukey’s test. Scale bar: 200 μm. (E) Disruption of TRPV1-AP2M1 binding also decreased the colony-forming ability of MGC-803 cells. Results are summarized as mean ± SEM from ≥ 3 biological replicates; **** p < 0.0001, ns = no significant, one-way ANOVA with Tukey’s test. (F) Schematic of xenograft experiments where BALB/c nude mice were subcutaneously (S.C.) injected with MGC-803 cells and subsequently intraperitoneally injected with TAT-AP2M1-176–185 or TAT-Scramble after one week to assess effects on tumor growth and pathology. (G) Mice treated with TAT-AP2M1-176–185 exhibited slower tumor growth in GC xenografts compared to those receiving TAT-Scramble (biological replicates ≥ 3). (H) Quantification of tumor size revealed significant reductions in tumors treated with TAT-AP2M1-176–185 compared to TAT-Scramble, demonstrating the peptide’s efficacy in reducing tumor growth. Tumor dimensions were used to calculate volume (L × W^2)/2, with data presented as mean ± SEM for 9–10 mice; ** p < 0.01, **** p < 0.0001, analyzed by two-way ANOVA with Tukey’s test. (I) Tumor weights at the study endpoint were significantly lower in the TAT-AP2M1-176–185 treatment group than in the TAT-Scramble group, further evidencing the peptide’s impact. Data from (G) are summarized, with mean ± SEM for 9–10 mice; *** p < 0.001, ns = no significant, by one-way ANOVA with Tukey’s test. (J) TAT-AP2M1-176–185 effectively abolished AP2M1’s binding to TRPV1 in xenograft tumors, as shown by Co-IP and IB analyses post-treatment (biological replicates ≥ 3). (K) Enhanced membrane localization of TRPV1 in GC tumors treated with TAT-AP2M1-176–185 compared to TAT-Scramble, as determined by membrane protein extraction and IB (biological replicates ≥ 3)

Article Snippet: Additionally, we utilized the HA-AP2M1 plasmid (catalog number HG16144-NY) sourced from Sino Biological Inc., Shanghai, China.

Techniques: Sequencing, Control, Binding Assay, Expressing, Co-Immunoprecipitation Assay, Membrane, Avidin-Biotin Assay, Purification, Migration, Disruption, Injection, Protein Extraction

Journal: Cell Communication and Signaling : CCS

Article Title: SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

doi: 10.1186/s12964-024-01850-0

Figure Lengend Snippet: TRPV1 SUMOylation Suppresses GC Cell Proliferation and Migration by Activating the TRPV1-Ca 2+ -AMPK Pathway. (A) RNA sequencing (RNA-seq) performed on RNA extracted from MGC-803 cells stably expressing Flag-hTRPV1 WT and Flag-hTRPV1 K823R identified gene sets associated with the negative regulation of the AMPK signaling pathway. Gene set enrichment analysis (GSEA) indicated a differential expression favoring Flag-hTRPV1 K823R over Flag-hTRPV1 WT , with the normalized enrichment score (NES) and p-value shown. (B) Expression of phosphorylated AMPK (p-AMPK) relative to total AMPK in GC tissues from WT and K823R xenograft tumors was analyzed by Western blot. Quantification showed increased p-AMPK in WT compared to K823R (biological replicates ≥ 3); *** p < 0.001 by one-way ANOVA with Tukey’s test. (C) Normal stomach tissues from WT and KI mice were analyzed for p-AMPK/AMPK expression. No significant difference was observed; n ≥ 3 biological replications; ns = no significant difference by the two-tailed Student’s t -test. (D) GC tissues from spontaneous gastric tumorigenesis in WT and KI mice showed higher p-AMPK levels in WT; n ≥ 3 biological replications, ** p < 0.01 by the two-tailed Student’s t -test. (E) Disruption of TRPV1-AP2M1 interaction by deleting the N-terminal of TRPV1 (ΔN mutants) in MGC-803 cells resulted in increased AMPK activation; data present the mean ± SEM of n ≥ 3 biological replications; *** p < 0.001, **** p < 0.0001 by one-way ANOVA with Tukey’s test. (F) Similarly, co-transfection with HA-AP2M1 WT and HA-AP2M1 Δ176–185 demonstrated that disrupting TRPV1-AP2M1 binding enhanced AMPK activation; data present the mean ± SEM of n ≥ 3 biological replications; ** p < 0.01 by one-way ANOVA with Tukey’s test. (G) Treatment with TAT-AP2M1-176–185 peptide significantly increased AMPK activation in MGC-803 cells expressing TRPV1 variants, compared to the TAT-Scramble control; data present the mean ± SEM of n ≥ 3 biological replications; **** p < 0.0001 by one-way ANOVA with Tukey’s test. (H) In xenograft tumors from WT and K823R groups treated with TAT-AP2M1-176–185 or TAT-Scramble, p-AMPK levels were markedly higher in the TAT-AP2M1-176–185 treated group; data present the mean ± SEM of n ≥ 3 biological replications; **** p < 0.0001 by one-way ANOVA with Tukey’s test

Article Snippet: Additionally, we utilized the HA-AP2M1 plasmid (catalog number HG16144-NY) sourced from Sino Biological Inc., Shanghai, China.

Techniques: Migration, RNA Sequencing Assay, Stable Transfection, Expressing, Western Blot, Two Tailed Test, Disruption, Activation Assay, Cotransfection, Binding Assay, Control

Journal: Science Advances

Article Title: Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target

doi: 10.1126/sciadv.aba7910

Figure Lengend Snippet: ( A ) ACA blocked the initiation of pdmH1N1 virus replication cycle. Cell lysate were collected for virus mRNA (blue lines), vRNA (magenta lines), and cRNA (black lines) quantification as indicated. Student’s t test for each RNA type and corresponding time point. ( B ) ACA inhibited nuclear import of the IAV vRNA. Synchronized H1N1 infection were performed on MDCK cells (50 MOI). Cells were fixed at the indicated time points and hybridized with RNA probes against the IAV negative-stranded NP vRNA (red) and stained for DNA (blue), examined by confocal microscopy. Images are representative of three independent experiments. Scale bars, 10 μm. ( C and D ) Click chemistry/WaterLOGSY/protein ID (CWID) platform for identification of drug-binding targets. (C) Click-chemistry: chemical structure of azido-ACA showing the location of azido group (green circle) on ACA. Cellular distribution of azido-ACA is shown (green), whereas ACA was used as a negative control due to the lack of phosphine-reactive azido group. Scale bars, 50 μm. (D) WaterLOGSY-guided cellular fractionation was subjected to analysis for ACA-featured NMR spectra. “*” and “***” indicate mild and strong binding signals, respectively. The native polyacrylamide gel electrophoresis gel photo shows the selected cell fraction as detected by a fluorescent image analyzer. Red arrow indicates the specific azido-ACA–binding fragment. ( E ) Mutagenesis analysis of AP2M1 to rescue pdmH1N1 virus replication against ACA. Full-length AP2M1 (full), longin-like domain (LLD), MHD, and mutant AP2M1 were transfected to MDCK cells before virus infection and ACA treatment. One-way ANOVA. ** P < 0.01; n.s, not significant. ( F ) Partial sequence alignment of human, mouse, and dog AP2M1 is shown. N217 and K410, the key residues for ACA binding, are highlighted with a box. The predicted interaction surfaces on AP2M1 (red) are shown, while ACA (green) is displayed in stick and mesh representation.

Article Snippet: An AP2M1 human gene knockout kit (OriGene, KN401377) was used to establish the CRISPR knockout cell lines according to the manufacturer’s protocol.

Techniques: Virus, Infection, Staining, Confocal Microscopy, Binding Assay, Negative Control, Cell Fractionation, Polyacrylamide Gel Electrophoresis, Mutagenesis, Transfection, Sequencing

Journal: Science Advances

Article Title: Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target

doi: 10.1126/sciadv.aba7910

Figure Lengend Snippet: ( A ) A summary of virus protein YxxØ motif interacting with host AP2M1 proteins. ( B ) Competitive ELISA showing the blockade of AP2M1 and biotin-YxxØ peptide after ACA addition. The low binding affinity mutant D176A was taken as a control. One-way ANOVA. * P < 0.05 and ** P < 0.01. ( C ) A known AP2M1-YxxØ blocker Tyrphostin A23 exhibited broad-spectrum antiviral activity. Shown are the antiviral effects against four different viruses as indicated. One-way ANOVA when compared with the 0 μM group (0.1% DMSO). ( D ) CRISPR knockout of AP2M1 reduced pdmH1N1, EV-A71, ZIKV, and MERS-CoV replication. Viral load in the cell lysate ( n = 3) was evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Student’s t test. ( E ) AP2M1 −/− and WT 293T cells were treated with CHX before virus infection (10 MOI). Nuclear (Nuc) and cytoplasmic (Cyto) fractions were separated and detected at 2 hpi by Western blotting. ( F ) A549 cells transfected with GFP influenza–NP and mCherry-AP2M1 were incubated with DMSO or ACA for 24 hours. Live cell imaging was performed, and motile AP2M1/NP puncta were tracked (movies S1 and S2). Shown is the average velocity of trackable puncta within the overall distance traveled. Student’s t test. ( G ) AP2M1 facilitates the viral protein localization. Synchronized infections were used throughout the experiments. Colocalization was quantified using ImageJ (JACoP) colocalization software and Manders’ colocalization coefficients (MCCs). Bar charts indicate mean MCC values represented as percent colocalization (the fraction of green intensity that coincides with blue intensity in the case of IAV-NP/nucleus and the fraction of green intensity that coincides with red intensity in the case of EV-A71-2C/ER and ZIKV-NS3/ER) ± SD (error bars, n = 10 to 15). Scale bars, 10 μm. *** P < 0.001 by Student’s t test.

Article Snippet: An AP2M1 human gene knockout kit (OriGene, KN401377) was used to establish the CRISPR knockout cell lines according to the manufacturer’s protocol.

Techniques: Virus, Competitive ELISA, Binding Assay, Mutagenesis, Control, Activity Assay, CRISPR, Knock-Out, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Western Blot, Transfection, Incubation, Live Cell Imaging, Software

Journal: Science Advances

Article Title: Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target

doi: 10.1126/sciadv.aba7910

Figure Lengend Snippet: ( A ) Effects of virus NP-YxxØ substitutions on virus growth and replication. Recombinant viruses were subject to multiple-cycle replication assays in A549 cells . N.D indicates failed rescue in three independent experiments. ( B ) Effects of virus NP-YxxØ substitutions on IAV NP nucleus import. A549 cells were treated with CHX before infection with WT and mutant viruses (10 MOI). Nuclear (Nuc) and cytoplasmic (Cyto) fractions were separated at 2 hpi for Western blotting to quantify the NP amount. Human β-actin and Lamin A were used for normalization of Cyto and Nuc, respectively. ( C ) Disruption of host AP2M1/ virus NP binding abolished the replication and transcriptional activity of influenza A polymerase. Shown are the relative polymerase activities with AP2M1 knockout (magenta bars) or NP mutants on YxxØ sites (blue bars). AP2M1 KD efficiency and NP overexpression were detected by Western blotting. Student’s t test . ( D to F ) Growth of WT and mutant (Y296A) GFP virus in a mouse model. BALB/c mice were intranasally infected with 10 5 or 10 4 PFU of the indicated viruses. Shown are the (D) survival rate and (E) body weight change. (F) Three mice from each 10 5 PFU-infected group were euthanized on 1, 3, and 5 dpi for analysis of in vivo dynamics after GFP virus infection. ( G ) Proposed model for host AP2M1-mediated intracellular trafficking of different viruses. Various viral proteins as indicated are commonly recruited by the mu subunit of host membrane trafficking AP2 adaptor complex (i.e., AP2M1 or μ2) through recognizing the viral YxxØ motif, while ACA disrupts these distinct steps of the viral life cycle.

Article Snippet: An AP2M1 human gene knockout kit (OriGene, KN401377) was used to establish the CRISPR knockout cell lines according to the manufacturer’s protocol.

Techniques: Virus, Recombinant, Infection, Mutagenesis, Western Blot, Disruption, Binding Assay, Activity Assay, Knock-Out, Over Expression, In Vivo, Membrane

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet: Live TIRF Imaging of CCPs in AP2 KO MEF Cells (A) MEFs from conditional AP2μ fl/fl mice ( Figure S2 A) were treated in vitro with CRE recombinase, as indicated, followed by immunoblotting (IB) as shown. The lower band in the AP2μ IB is nonspecific; the specific AP2μ band is indicated by an arrow. In all subsequent experiments, AP2μ fl/fl MEFs were either left untreated or treated with CRE for 14 days-two rounds (henceforth referred as AP2-WT and AP2-KO, respectively). (B) AP2-WT and AP2-KO MEFs were analyzed for mRNA levels of Ap2m1 and Cltc using qRT-PCR. mRNA levels are reported relative to untreated controls and normalized to the 18S gene. Error bars are calculated on technical replicates (n = 3). (C) Cumulative frequency distribution of the initial MSD of clathrin-coated structures in MEF AP2-WT and AP2-KO cells imaged by TIRF. Clathrin events with initial MSD larger than 0.01 μm 2 (dotted line) were excluded in the plots displaying fluorescence intensity cohorts (D). (D) Automated analysis of clathrin-coated structure formation at the plasma membrane from 12 cells and ∼439 clathrin traces from MEF KO cells. (E) Representative TIRF microscopy time series acquired every 2 s from the bottom surface of MEF AP2-KO cells, stably expressing CLTA-TagRFP together with AP2σ-EGFP. The TIRF snapshots (left) were recorded at 224 and 138 s, and the corresponding right panels are kymographs from the complete time series. The yellow tracings display the path used to generate the kymographs. The green channels in the kymographs were shifted upward by 5 pixels. Endocytic “clathrin-only” structures are present (e.g., pits 1 and 2).

Article Snippet: Inventoried Taqman assay, Ap2m1 , Applied Biosystems , Mm01702796_g1.

Techniques: Imaging, In Vitro, Western Blot, Quantitative RT-PCR, Fluorescence, Clinical Proteomics, Membrane, Microscopy, Stable Transfection, Expressing

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet:

Article Snippet: Inventoried Taqman assay, Ap2m1 , Applied Biosystems , Mm01702796_g1.

Techniques: Transduction, Recombinant, Electron Microscopy, Reverse Transcription, TaqMan Assay, cDNA Synthesis, Control, Plasmid Preparation, Software