G3BP1 Succinylation at K413 is Critical for Cardiac Function by Modulating PI3K‐AKT‐mTOR Signal Axis

Figures

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  FIGURE 1. Decreased Suc levels on G3bp1 in two mice models developed cardiomyopathy and HF. (A) Schematic representation of Mybpc3 knockout (KO) mice generation using the CRISPR/Cas9 system. Sanger sequencing confirmed a 10922‐bp deletion spanning from exon 2 to exon 22. (B–D) Echocardiography data from Mybpc3 KO and wild‐type (WT) mice, showing core cardiac parameters including ejection fraction (EF), fractional shortening (FS), and left ventricular posterior wall thickness (LVPW). (n = 4 mice per group, each mouse assayed in triplicate). The data are presented as the mean ± SEM. Two‐tailed Student's t‐test: ns, not significant, ** p < 0.01, *** p < 0.001. s, systolic; d, diastolic. (E,F) Immunoblot analysis of anti‐succinyllysine in heart tissue of Mybpc3 KO (E) and TAC‐operated (F) mice using corresponding pan‐antibodies by western blots. WT and sham‐operated mice served as the control (Ctrl) group, respectively. Representative images from three independent experiments were displayed. (G) The MS/MS spectrum of the K411 Suc modified site in G3bp1. (H,I) Suc levels on G3bp1 in heart tissues of Mybpc3 KO and TAC‐operated mice, when compared with WT and sham mice, respectively, through IP…

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  FIGURE 2. Suc at K411 is a critical regulatory site for G3BP1 activity in mice hearts. (A,B) The abundance (A) and specificity (B) of AAV9‐mediated G3bp1 transgene expression were evaluated using western blot analysis of mouse hearts 4 weeks after injection. For each western blot, representative images from three independent experiments were displayed. (C,D) Morphological observations (C) and Masson‐stained sections (vertical and horizontal) (D) of mouse heart injected with AAV9‐Ctrl, ‐G3bp1WT, and ‐G3bp1K411R. Scale bar, 1.25 mm. (E) Representative image of myocardial fibrosis was observed in mice injected with AAV‐G3bp1WT when compared with AAV‐G3bp1K411R and Ctrl. Fibrosis areas are boxed to show the enlarged region. Scale bar, 200 µm (upper), 50 µm (bottom). (F) The ratio of heart weight to body weight (HW/BW) (n = 10 per group) and quantitative analysis of fibrotic scar from Masson's trichrome staining (n = 6 per group). Mice injected with AAV‐empty vector served as Ctrl group. The data are presented as the mean ± SEM; one‐way ANOVA test, * p < 0.05, ** p < 0.01,*** p < 0.001. (G) Representative images of Sirius red, WGA, and TUNEL staining in mice heart injected with AAV‐Ctrl, ‐G3bp1…

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  FIGURE 3. G3bp1 KI (p.E411G) mice displayed reduced cardiac function. (A) Schematic of G3bp1 A‐to‐G single base KI mouse model generated using CRISPR/Cas9 sgRNA. (B) The mutation in G3bp1 (c.1232A>G, mouse K411 corresponds to human K413) in the KI mouse was confirmed by Sanger sequencing. The c.1232 site is indicated by a black arrow. (C) Representative images of Sirius red, WGA, and TUNEL staining in WT and G3bp1 KI/KI mice hearts. Cardiomyocyte specificity was confirmed by co‐staining with cTnT and α‐actinin, respectively. Scale bar, 200, 20, and 50 µm from up to bottom. (D) Quantification of TUNEL‐positive cardiomyocytes in (C). Data are presented as the mean ± SEM; two‐tailed Student's t test, n = 8. ** p < 0.01. (E) Masson's trichrome staining identified fibrosis in G3bp1KI/KI mice heart when compared with WT and quantitative analysis of fibrotic scar (n = 8 per group). The data are presented as the mean ± SEM; two‐tailed Student's t‐test, ** p < 0.01. (F) Immunoblot analysis of classical biomarkers for cardiac hypertrophy and fibrosis, including Col3a1, Anp, Bnp, and α‐Sma in WT and G3bp1 KI/KI mice models. Gpadh and Tubulin served as loading controls. A representative image from…

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  FIGURE 4. G3BP1 interacts with insulin‐degrading enzyme (IDE) in the human heart. (A) Interactome mapping of G3BP1 in the human heart using GST pull‐down coupled with mass spectrometry (MS). Purified recombinant GST‐G3BP1 was incubated with human embryonic heart tissue lysates to perform GST pull‐down and then visualized by Silver staining before MS analysis. (B) 41 Interacting proteins with unique peptides >2. (C) Protein–protein interaction (PPI) network of all 106 interacting proteins visualized using the STRING database and networkD3 R package. The central nodes of network were displayed. (D)Gene Ontology (GO) enrichment analysis on 106 interacting proteins for G3BP1 (Fisher's exact test; p < 0.05). (E) KEGG enrichment analysis on 106 interacting proteins for G3BP1 (Fisher's exact test; p < 0.05). (F) Representative images of Co‐IP validation for G3BP1 and IDE in vitro. Anti‐Flag immunoprecipitation assays were performed in HEK293T cells, followed by immunoblotting with the indicated antibodies. Representative images from three independent experiments were displayed. (G) Overexpression of IDE decreases phosphorylation of Akt at Ser473 in 293T cells. Quantification data of pAKT(S473)…

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  FIGURE 5. G3bp1 de‐Suc at K411 induces Ras‐related GTP binding A (Rraga) expression. (A) Summary of 4D‐LFQ (label‐free quantitation) intensity of global quantitative proteomic analysis of nine mouse heart tissues (n = 3 per group) injected with the three corresponding AAV viruses as indicated. (B) Score plots of principal component analysis (PCA) of AAV‐Ctrl, ‐G3bp1WT, and ‐G3bp1K411R proteome. (C) Overview of significantly differentially expressed proteins (DEPs) in three comparison groups (fold‐change>1.5, p < 0.05). (D–F) Volcano plots showing the DEPs in three comparison groups, including AAV‐G3bp1WT versus AAV‐Ctrl (D); AAV‐G3bp1K411R versus AAV‐Ctrl (E); AAV‐G3bp1WT versus AAV‐G3bp1K411R (F). Up‐ and down‐regulated proteins are highlighted in red and blue, respectively. (G) G3bp1 expression was induced by 1.65‐ and 1.67‐folds in mice heart injected with AAV‐G3bp1WT and ‐G3bp1K411R, respectively, when compared with AAV‐Ctrl according to proteomic analysis. Fold‐change = 0.984 (G3BP1 WT vs K411R). The data are presented as the mean±SEM, n = 3. (H) AAV injection‐mediated G3bp1 expression in mice heart was validated by western blot (n = 3). For each Western blot, a representative image…

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  FIGURE 6. G3bp1 de‐Suc at K411 and G3bp1 knockdown promote Raga expression and mTORC1 activation. (A) Bubble plot of GO enrichment analysis on 29 DEPs from the comparison‌ between G3bp1K411R and G3bp1WT group. (B) Western blot confirmed the elevated expression of RragA and activation of AKT (Ser473) and mTOR downstream targets 4E‐BP1 and p70‐S6K in mice hearts injected with AAV‐ G3bp1K411R when compared with AAV‐ G3bp1WT, a representative image from three independent experiments was displayed. (C) The downstream activities of serine/threonine kinases including AKT and AMPK, were evaluated using phospho‐(Ser/Thr) kinase substrate antibody kit. A representative image from three independent experiments is displayed. (D) Protein levels of G3BP1, Raga, and phosphorylated p70‐S6K‐pT389 and 4EBP1‐pT37/46 were analyzed by western blot in G3bp1KI/KI and WT mice heart. A representative image from three independent experiments was displayed (left). Quantification data of G3BP1 expression when normalized against beta‐Tubulin (right). ** p < 0.01 (two‐tailed Student's t test, n = 6). (E) Representative image showing decreased protein level of G3bp1 in G3bp1KI/KI mice by immunofluorescence assay. Scale…

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  FIGURE 7. G3BP1 de novo mutation (p.E411G) from DCM patient reduces G3BP1 Suc level and IDE binding. (A) Pedigrees of the family (#094), illustrating the affected individuals and the segregation of the G3BP1 c.1232A>G mutation. (B) Sanger sequencing confirmed de novo missense mutations in G3BP1. (C) Sequence alignment of G3BP1 E411G in different species. (D) Diagram of multiple nonsynonymous mutations that locates or adjacent to the G3BP1 K413suc site from the GnomAD database v4.1.1 and literature. (E) Exogenous expression of G3BP1 E411G and K413R led to decreased G3BP1 Suc levels when compared with G3BP1 WT. 293T cells were transfected with Flag‐tagged WT and point mutants of G3BP1, and empty vector (Ctrl), and cell lysates were then immunoprecipitated with an anti‐Flag antibody, followed by immunoblotting with a pan Suc antibody. For each Western blot, a representative image from three independent experiments was displayed. (F) Co‐IP assay indicated that G3BP1 K413R and E411G interacted significantly less with IDE than did WT in 293T cells. (G) Co‐IP assay indicated that G3BP1 K413R and E411G interacted significantly less with TSC1 than did WT in 293T cells. A representative image from…