Design of peptides to inhibit TANGO1-cTAGE5 heterodimerization.

A The organization and the domains of TANGO1 and cTAGE5. Each of these proteins contains two cytoplasmic coiled-coil domains. TANGO1 coiled-coil 2 (T1-CC2) is highlighted in green. cTAGE5 CC2 in magenta. B Alphafold2 prediction of the structure of the heterodimer of TANGO1 (green) and cTAGE5 (magenta). C Schematic indicating where the peptide sequences map to in TANGO1 and cTAGE5. D Two peptides were synthesized of 30 (P2) and 31 (P5) amino acids respectively, conjugated to a 12 amino-acid C-terminal lysosomal escape motif (shown in black text). E Alignment of human, mouse, and zebrafish sequences. F Representative western blot of U2OS cell lysates treated with P2, P5 or P2 + P5, probed for cTAGE5. β-tubulin was used as a loading control. G Representative Western blot of U2OS cell lysates treated with P2, P5 or P2 + P5, probed for TANGO1. Calnexin was used as a loading control. H Quantification of blots showing TANGO1 (green) and cTAGE5 (magenta) levels (mean +/- SD) in control or peptide-treated cells (normalized to control) *p < 0.05, Student’s t test comparing cTAGE5 (magenta line) or TANGO1 (green line) in control vs treated. N = 3 (TANGO1), N = 5 (cTAGE5).

Peptides induce phenotypic changes in zebrafish consistent with TANGO1/cTAGE5 inhibition.

A Zebrafish larvae at 3 dpf. Fish were treated with P2 + P5 (4 μM), or left untreated in E3 (control). Magnified images of the tail and yolk on the right. Scale bars 1 mm. B Quantification of larva length from head to tail (boxplot with median and interquartile range N = 21, 19, 23 in control, 2 μM and 4 μM respectively). Whiskers connect the maximum and minimum. C Quantification of phenotypes observed in fish for n = 21, 19, 23 replicates in control, 2 μM and 4 μM respectively, from three independent experiments. D Maximum projection of the entire Z-stack of SHG images from three different tail fin regions (Areas #1, 2, 3) as indicated in the bright-field images. Images were acquired from three control and treated fish each, with approximately similar regions of the tail chosen as Area #1, 2, and 3. Scale bars 100 μm. E Quantification of collagen fiber direction obtained from the SHG signal in the three segmented areas approximately represented in (D) Top row: Collagen fiber direction histograms from a representative control or treated fish. Bottom row: Collagen fiber dispersion distribution plots from a representative control or treated fish. This measure represents the dispersion of the preferred orientation of collagen fibers in the image. Control measurements are represented in black and P2 + 5 (4 μM) is treated in orange. Kolmogorov–Smirnov test *** p < 0.001.

Uptake of fluorescently labeled peptides into primary human dermal fibroblasts and inhibition of procollagen I secretion.

A Confocal micrographs of primary human dermal fibroblasts showing uptake of P2 + P5 supplied at 40 μM over 20 h in serum-free conditions. Co-staining with an ER marker in red. Bar: 10 μm. B Quantification of uptake of fluorescent peptides (N = 4, mean  +/−  SD of intracellular fluorescent intensity). C, D Representative western blot (C) and quantification of fibroblast lysates treated or not (Ctrl) with P2 + P5 at 40 μM for 20 h. N = 6 (D). E, F Representative western blot from supernatants; Ponceau staining reflects the loading control in the corresponding cell lysates (E). Quantification of supernatants of human fibroblasts treated for 20 h in serum-free conditions with either P2 or P5 at the indicated concentration. N = 6 (F). G, H Representative western blot from supernatants; Ponceau staining reflects the loading control in the corresponding cell lysates (G). Quantification of supernatants from untreated and P2 + P5 treated skin fibroblasts. N = 24; Mann– Whitney U test, two-tailed, **** p < 0.0001 (H). I Protein concentration of untreated and P2 + P5 treated cell lysates. N = 9. J Duration, in hours, of inhibitory effect after removal of P2 + P5. Levels of untreated (Ctrl) were set at 100%. N = 3 per time point, two-way ANOVA with Sidak’s multiple comparisons test. *** p < 0.0001. K Collagen I western blot of supernatants from controls and scrambled (scr) peptide-treated control fibroblasts and β actin western blot from corresponding cell lysates. L Determination of cytotoxicity of control fibroblasts treated with different peptides (measured by lactate dehydrogenase release (L, D, H)). N = 17 (Ctrl)/7 (P2)/7 (P5)/9 (P2 + P5)/8 (P2 scr + P5 scr). N = independent samples. Data shown as mean ± SD.

Effects of peptide treatment on the secretome of human fibroblasts.

A Volcano plot illustrating significantly downregulated (blue symbols) and upregulated (orange symbols) proteins, by treatment of primary human dermal fibroblasts with 40 μM P2 + P5 for 20 h in serum-free conditions in comparison to control samples. Unpaired two-sided Student´s t-test, S₀ = 0.1, N = 3 independent samples. B Gene ontology (GO) terms of proteins affected by peptide treatment. Box plot represents the median, 25^th and 75^th percentiles, max. and min. are connected through whiskers. Outliers are defined as Q₁−1.8 interquartile range (IQR) and Q₃ + 1.8 IQR. An unpaired two-sided Student´s test was performed.

Peptides limit the formation of granulation tissue in zebrafish wounds.

A H&E staining of skin longitudinal sections at 4 dpw from control PBS-treated fish (left), or those treated with P2 + P5 (right). Granulation tissue (gt) area was measured (yellow dashed lines). B Quantification of granulation tissue area in control or peptide-treated animals. * p = 0.0369. C Immunohistochemical analysis of collagen deposition during wound healing in the granulation tissue in control (left) or peptide-treated (right) animals. D Quantification of collagen area in the granulation tissue. *** p = 0.0006. E Higher magnification of immunohistochemical analysis of collagen deposition and nuclei (magenta boxes in C). F Quantification of total nuclei within the granulation tissue. G Quantification of total nuclei divided by the granulation tissue area. * p = 0.0198. H TUNEL staining of 4 dpw wounds in granulation tissue in control (left) or peptide-treated (right) animals. I Quantification of TUNEL-positive cells divided by total DAPI-positive cells in the granulation tissue. Scale bars: A, C = 200 μm; E, H = 50 µm. ep: epidermis. N = 5 fish per treatment, Student’s t-test, two-tailed. Non-significant (NS). Data are shown as mean ± SD.

Peptides inhibit ECM protein secretion by TGFβ-activated healthy and scleroderma fibroblasts.

A Western blot of supernatants from fibroblasts incubated for 20 h in the absence of serum without (Ctrl) or with 40 μM each of P2 + P5 and without or with 10 ng/ml TGFβ. Peptide treatment strongly reduced amounts of secreted (pro)collagen I (top), fibrillin1 (mid) and fibronectin (bottom) in fibroblasts from healthy donors and scleroderma patients before and particularly after stimulation of ECM production by TGFβ. B Quantification of (A). N = 3 independent samples. Data are shown as mean ± SD.