The final walk with preptin

Abstract

Preptin, a 34-amino acid peptide derived from pro-IGF2, is believed to influence various physiological processes, including insulin secretion and the regulation of bone metabolism. Despite its recognized involvement, the precise physiological role of preptin remains enigmatic. To address this knowledge gap, we synthesized 16 analogs of preptin, spanning a spectrum from full-length forms to fragments, and conducted comprehensive comparative activity evaluations alongside native human, mouse and rat preptin. Our study aimed to elucidate the physiological role of preptin. Contrary to previous indications of broad biological activity, our thorough analyses across diverse cell types revealed no significant biological activity associated with preptin or its analogs. This suggests that the associations of preptin with various diseases or tissue-specific abundance fluctuations may be influenced by factors beyond preptin itself, such as higher levels of IGF2 or IGF2 proforms present in tissues. In conclusion, our findings challenge the conventional notion of preptin as an isolated biologically active molecule and underscore the complexity of its interactions within biological systems. Rather than acting independently, the observed effects of preptin may arise from experimental conditions, elevated preptin concentrations, or interactions with related molecules such as IGF2.

Fig 1. Preptins and preptin derivatives synthesized.

Sequences are shown in single-letter codes. C-terminal carboxamides are depicted as -amide. The position with non-standard amino acids, precursors for ring-closing olefin metathesis (RCM) or Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions are shown as X and numbered, and the respective cyclized segments also shown as ChemDraw structures. (R)-amino acids are shown in standard single-letter codes (or as X) but in italics. α-Amino isobutyric acid in 15 is shown as Z.

Fig 2. CD spectra of peptides 1–19 recorded in phosphate buffer.

Peptides were divided into three groups: Human preptin analogs (A), stapled human preptin analogs (B), mouse and rat preptin analogs (C).

Fig 3

Binding curves for (A) IGF2R, (B) IGF1R, (C) IR-A. Inhibition of binding of human [¹²⁵I]-monoiodotyrosyl-ligand to corresponding receptor by IGF2 (cyan), IGF1 (magenta), insulin (orange), peptide 1 (black), peptide 2 (red), peptide 3 (blue), peptide 4 (green). Representative binding curve for each hormone or analog is shown, n ≥ 3 independent experiments with 2 replicates.

Fig 4. Stimulation of phosphorylation of Akt by preptins (at 10^-8M).

Stimulation of phosphorylation of intracellular Akt proteins in murine fibroblasts with deleted Igf1r gene (R- cells) (A), in MC3T3-E1 preosteoblasts (B), in U-2 OS osteoblasts (C) and MIN6 pancreatic beta cells (D) by respective preptins or preptin analogs. Data are presented as means ± S.D., relative to the signal in non-stimulated cells. Significant differences between marked values determined using Ordinary one-way ANOVA (Dunnett´s multiple comparisons test), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5. Changes in intracellular calcium after stimulation with preptin and its analogs at the concentration 10⁻⁴ M.

Murine fibroblasts with deleted igf1r gene (R- cells) (A), MC3T3-E1 preosteoblasts (B), U-2 OS osteoblasts (C) and MIN6 pancreatic beta cells (D). Data are presented as means ± S.D., relative to the signal in non-stimulated cells. Significant differences between marked values determined, using Ordinary one-way ANOVA (Dunnett´s multiple comparisons test), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 6. Changes in cAMP levels after stimulation with preptin and its analogs at a concentration of 10⁻⁴ M.

cAMP-HEK cells (A), U-2 OS osteoblasts (B) and MIN6 pancreatic beta cells (C). Data are presented as means ± S.D., relative to the signal in non-stimulated cells. Significant differences between marked values, determined using Ordinary one-way ANOVA (Dunnett´s multiple comparisons test), ****p < 0.0001.

Fig 7. Stimulation of insulin secretion by various concentrations of native mouse preptin (peptide 14) on pancreatic MIN6 cells.

Fig 8

Differentiation of hBMSC-TERT osteoblasts after treatment with IGF1 (A), IGF2 (B), and preptin (C). The data were evaluated using quantification of alkaline phosphatase (ALP) activity, normalized to cell viability presented as a fold change (F.C.) over non-induced cells (day 7), n = 3 independent experiments with 6 replicates. Data are presented as mean ± SEM, Ordinary one-way ANOVA (Dunnett´s multiple comparisons test), *p < 0.05, **p < 0.01. A.U. means arbitrary units.

Fig 9. Staining of hBMSC-TERT differentiated into osteoblasts with alizarin S.

Staining of hBMSC-TERT differentiated into osteoblasts for 10 days in vitro with alizarin S. Cells were treated with increasing concentrations of IGF1 (A), IGF2 (B) and rat preptin 19 (C). The upper panels in all cases show undifferentiated osteoblasts treated with the appropriate concentration of the compound; the lower panels always show differentiated osteoblasts treated with the adjacent concentration of the compound.