A viral insulin-like peptide inhibits IGF-1 receptor phosphorylation and regulates IGF1R gene expression

Abstract

Objective: The insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time.

Methods: We chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs' effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain.

Results: VILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure.

Conclusions: This study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP' binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.

Keywords: Biased signaling; IGF-1; IGF1 receptor; IGF1 receptor inhibition; Insulin; Iridoviridae; Viral insulin/IGF-1 like peptides (VILPs).

Figure 1

Comparison of primary and predicted structures of MFRV and LCDV-Sa-VILPs with human insulin and IGF-1. A. Sequence alignment of VILPs (as synthesized) with human insulin and IGF-1. The sequence of double chain (dc) VILPs and their comparison to human insulin is shown in the upper panel and the sequence of single chain (sc) VILPs and their comparison to human IGF-1 is shown in the lower panel. The underlined residues represent substitutions compared to the human ligands or differences between the sc and dc forms of the VILPs as synthesized. Note that in the natural sequence, Ala3 in LCDV-Sa is replaced with Cys. The residues important for receptor binding are highlighted as indicated in the figure legend. Cysteine residues are in red. B: 3D structures of human insulin (PDB:1MSO) and IGF-1 (PDB: 2GF1) and predicted 3D structure of scVILPs. A-chains/domains are in green, B-chains/domains are in blue, C-domains are in pink and D-domains are in orange. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 2

MFRV and LCDV-Sa-VILPs can bind to human IR and IGF1R and stimulate autophoshorylation of the receptors. A-C: Binding competition dose–response curves showing the ability of VILPs to compete with 125-I labeled human insulin for binding to IR-A (A) and IR-B (B), and with 125-I labeled human IGF-1 for binding to IGF1R (C). The experiments were performed using IM-9 cells for measurements on IR-A binding, while R-/IR-B and R⁺ cells were used for measurements on IR-B and IGF1R, respectively. A representative curve for each peptide to each receptor is shown. Each point represents the mean ± SEM of duplicates, and each experiment was repeated at least three times. D-F: Dose–response curves for ligand-induced autophosphorylation of IR-A and IGF1R. HEK293 cells overexpressing human IR-A (D) or IGF1R (E and F) were used. In F, ligands at indicated concentrations were co-incubated with 5 nM IGF-1. Each point represents the mean ± SEM of duplicates (n ≥ 2).

Figure 3

Quantification of insulin/IGF signaling via IGF1R stimulated by MFRV and LCDV-Sa-VILPs. R⁺ cells were treated with human IGF-1, VILP, or a combination of VILP and 10 nM IGF-1, and the phosphorylation levels of IGF-1R, Akt, and Erk1/2 were evaluated after 30 min. The data points represent the mean ± SEM of signals quantified from at least three independent experiments, expressed as a fold change relative to10 nM IGF-1 (grey dashed line). Each data point corresponds to the signal of the phosphorylated protein normalized to the total protein. Statistical significance was determined by unpaired two-tailed Student's t-test (∗P < 0.05, ∗∗<0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Figure 4

Western blot analysis of MFRV and LCDV-Sa stimulated insulin/IGF signaling on human IR-A, IR-B, and IGF1R. R-/IR-A, R-/IR-B or R⁺ cells were used for measurements on human IR-A, IR-B or IGF1R, respectively. Cells were stimulated with increasing concentrations of insulin (IR-A and IR-B) or IGF-1 (IGF1R), VILP and VILP in combination with 10 nM insulin/IGF-1 depending on the cell line. Phosphorylation of the receptor, Akt and Erk1/2, as well as the relative amounts of the total proteins, were observed in 30 min after stimulation. Representative western blots are shown, each experiment was repeated at least three times.

Figure 5

Transfection of R⁺cells with VILPs or IGF-1 reveals unique effects of MFRV-VILP on IGF1R protein levels. A: Representative western blot of IGF1R, Akt and Erk1/2 phosphorylation, as well as the relative amounts of total IGF1R in transfected R⁺ cells stimulated with 10 nM IGF-1 for the final 30 min of a 35 h transfection. B: Quantification of IGF1R phosphorylation, C: Quantification of Akt phosphorylation, D: Quantification of Erk1/2 phosphorylation, E: Quantification of total IGF1R. Each data point in the quantifications represents the mean ± SD of signals quantified from at least four independent experiments. Ordinary one-way ANOVA followed by Tukey's multiple comparison test was applied (∗P < 0.05; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). The statistics are shown as each group is compared to the following: (i) control group (empty vector), represented in grey; (ii) empty vector + 10 nM IGF-1, represented in black; (iii) MFRV vector, represented in pink; and (iv) MFRV vector + 10 nM IGF-1, represented in green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 6

MFRV-VILP transfection decreases IGF1R gene expression but does not affect IR. Cells were transfected with empty vector or vectors carrying MFRV-VILP, human IGF-1, or LCDV-Sa-VILP. Gene expression was analyzed at different time points post-transfection (0, 11, 26, and 35 h) under serum-starved conditions. A and E: R⁺ cells, B-D and F: AML12 cells. Gene expression data are presented as a percentage of β-actin and represented as mean ± SEM n = 3–4). Ordinary one-way ANOVA followed by Tukey's multiple comparison test was applied to determine the statistical significance of the differences between groups (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Figure 7

MFRV-VILP decreases IGF-1 stimulated proliferation in vitro while stimulates long-lasting blood glucose lowering effects in vivo. A: Proliferation assay of R⁺ cells transfected with MFRV-VILP and IGF-1. Cells were transfected with empty vector or vectors carrying MFRV-VILP or human IGF-1, serum-starved for 24 h post-transfection and incubated or not (control) with 10 nM IGF-1 for an additional 24-hour period starting 24 h post-starvation. Incorporation of BrdU was assessed to measure cell proliferation. Results are expressed as fold over empty vector. Data points from the same experiment are labeled in identical color. A paired two-tailed Student's t-test was performed (∗P < 0.05). The statistics are shown as each group is compared to the following: (i) control group (empty vector), represented in grey; (ii) empty vector + 10 nM IGF-1, represented in black; (iii) MFRV vector, represented in pink; and (iv) MFRV vector + 10 nM IGF-1, represented in green. B and C: Insulin tolerance test. C57BL/6 J mice were injected i.p. with human insulin, dcLCDV-Sa-VILP, scMFRV-VILP, or saline. The insulin concentration was 6 nmol/kg in both panels, whereas the concentration of dcLCDV-Sa-VILP was 0.3 μmol/kg (B) and the concentration of scMFRV-VILP was 60 nmol/kg (C). Blood glucose was measured within the range of 0–180 min. Data are mean ± S.E.M. Mixed-effects analysis followed by Dunnett's multiple comparisons test was applied, n = 4–11 per condition (∗P < 0.05; ∗∗P < 0.01, ∗∗P < 0.001). Comparisons were made to saline (∗), or human insulin (#). The area under the curve for each condition is shown on the right side of each panel. Ordinary one-way ANOVA followed by Tukey's multiple comparison test was applied for statistical analysis (∗P < 0.05; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 8

scMFRV- VILP-IGF1Rzip cryoEM reconstruction. a. cryoEM density after 3D-flexible refinement of MFRV-IGF1Rzip, colored by chain. b-f. Density associated with refined model: b. ɑCT. c. ɑCTʹ. d. FnIII-1ʹ. e. L1. f. MFRV-VILP. g. overlay of hIGF-1 (black) from hIGF1-mIGF1R (pdb: 6PYH) with MFRV-IGF1Rzip with zoomed and reoriented inlay. h. apo-IGF1R with domains labelled (from PDB: 5U8R).