Fig 1: LLPS of CNPY3 drives oncogenic function by promoting MDM2 phosphorylation. (A) Schematic representation of the domain architecture of human CNPY3 and the deletion mutant (CNPY3-Del) used in this study. (B-C) Representative confocal microscopy images showed that purified GFP-tagged CNPY3 is concentration-dependent (B) and enhanced by higher salt concentrations (C). (D-E) Quantification of CNPY3 droplet formation demonstrates its dependence on both protein concentration (D) and salt concentrations (E). (F) Representative images showed that treatment with the crowding agent 10% PEG8000 (Biosharp, 0159) promotes the formation of liquid droplets by purified GFP-CNPY3. (G) Representative images demonstrated that treatment with 5% 1,6-hexanediol (MACKLIN, H810887) effectively disrupts GFP-CNPY3 liquid droplets. (H-I) Quantification of CNPY3 droplet formation after PEG8000 (H) and 1,6-hexanediol treatment (I). (J) Representative confocal images of HCT116 and RKO cells treated with 1,6-hexanediol. (K-L) Representative images (K) and recovery curves (L) from a fluorescence recovery after photobleaching (FRAP) experiment show rapid and substantial fluorescence recovery within the photobleached CNPY3-GFP droplet. (M) Representative confocal images of HCT116 and RKO cells transfected with CNPY3-GFP and CNPY3-Del plasmids. (N) Representative imagines (left) and semi-quantitative analysis (right) of pSMDM2 in HCT116 cells with overexpression CNPY3-GFP or CNPY3-Del plasmids, compared with control plasmids. (O) Western blot showed pSMDM2 and p53 expression after overexpression of CNPY3 or CNPY3-Del plasmids in HCT116 (left) and RKO cells (right), using β-tubulin as a control. (P) Histogram of CTG luminescence in HCT116 (left panel) and RKO cells (right panel) with vector, CNPY3 and CNPY3-Del plasmid overexpression. (Q) Colony assay and quantitative analysis of HCT116 cells with vector, CNPY3 and CNPY3-Del plasmid overexpression. Note: two-tailed *P < 0.05 and ns by a Tukey's multiple comparisons test (N, P, Q); ns, not significant; pSMDM2, phosphorylated MDM2-Ser166; data are presented as mean ± SD.
Fig 2: CNPY3 promotes p53 ubiquitin-proteasome degradation. (A-B) Act D-chase assays (Sigma, A4262; 5 μM) (left) and semi-quantitative statistical results (right) were used to evaluate CNPY3 accumulation in HCT116 cells with CNPY3 overexpression (A) and CNPY3 knockdown (B), using β-tubulin as a control. (C-D) CHX-chase assays (MedChemExpress, HY-12320; 100ug/ml) (left) and semi-quantitative statistical results (right) were used to evaluate CNPY3 degradation in HCT116 cells with CNPY3 overexpression (C) and CNPY3 knockdown (D), using β-tubulin as a control. (E-F) The p53 protein levels of CNPY3 overexpressing HCT116 (E) and RKO cells (F) with or without MG132 treatment (Selleck, S2619; 20 μM, 5 h), using β-tubulin as a control. (G-H) The p53 protein levels of CNPY3 knockdown HCT116 (G) and RKO cells (H) with or without MG132 treatment, using β-tubulin as a control. (I-J) Ubiquitination assays of endogenous p53 in the lysate from HA-ubi overexpressing HCT116 and RKO cells with CNPY3 knockdown (I) and CNPY3 overexpression (J). (K) Co-IP analysis of binding efficiency of p53 and MDM2 through p53-IP in HCT116 cells with CNPY3 knockdown and overexpression, compared with controls. (L-M) The protein levels of p53 and BAX in CNPY3 overexpression RKO (L) and HCT116 cells (M) after Nutlin-3 treatment, using β-tubulin as a control. (N) Ubiquitination assays of p53 in the lysate from HA-ubi overexpressing HCT116 cells with CNPY3 overexpression after Nultin-3 treatment. Note: two-tailed *P < 0.05 by a two-way ANOVA test (A, B, C, D); Act D, Actinomycin D; CHX, Cycloheximide; data are presented as mean ± SD.
Fig 3: SREBP2-CNPY3 axis promotes phosphorylation and nuclear translocation of MDM2. (A) Flow chart of liquid chromatography-tandem mass spectrometry analysis and candidate proteins identified by CNPY3. (B-C) Endogenous Co-IP was performed in HCT116 (B) and RKO cells (C) lysates using an anti-CNPY3 antibody or control IgG. (D) Exogenous Co-IP analysis of Flag-CNPY3 and HA-MDM2 in HCT116 cells using anti-Flag nanobody agarose beads. (E) Representative IF staining images of colocalization between CNPY3 and MDM2 (left) and IF co-localization analysis (right) in HCT116 and RKO cells. (F) GST pull-down assay confirmed the direct interaction between purified his-tagged MDM2 protein and purified GST-tagged CNPY3 protein in vitro. (G) Histogram showed that MDM2 mRNA levels remain unchanged in HCT116 and RKO cells upon either knockdown (left) or overexpression (right) of CNPY3, compared to their respective controls. (H) Western blot showed MDM2, pSMDM2, and CNPY3 expression after CNPY3 overexpression or knockdown in HCT116 (left) and RKO cells (right), using β-tubulin as a control. (I) Representative IF staining (left) and semi-quantitative statistics (right) exhibited MDM2 sublocalization after overexpression of CNPY3 in HCT116 and RKO cells. (J) Cytoplasmic and nuclear protein isolation and western blotting were performed in HCT116 (left) and RKO cells (right) overexpressing CNPY3 or control. (K-L) Western blot showed pSMDM2 and MDM2 in HCT116 and RKO cells with Fatostatin treatment (K) and overexpression of SREBP2 (L), using β-tubulin as a control. (M) Western blot showed pSMDM2 and CNPY3 in CNPY3 overexpression HCT116 cells after Fatostatin treatment, using β-tubulin as a control. Note: two-tailed *P < 0.05 and ns by an unpaired t test (G, I); ns, not significant; pSMDM2, phosphorylated MDM2-Ser166; data are presented as mean ± SD.
Fig 4: Mechanism schematic diagram. SREBP2-activated CNPY3 undergoes liquid-liquid phase separation to enhance MDM2 phosphorylation and nuclear translocation, thereby accelerating ubiquitination-mediated degradation of wild-type p53 and driving colorectal cancer progression.
Supplier Page from Sino Biological, Inc. for MDM2 (1-118) Protein