TANGO1 inhibitors reduce collagen secretion and limit tissue scarring

Excessive scarring and fibrosis, caused by uncontrolled extracellular matrix (ECM) protein secretion (e.g., collagen), pose significant challenges in regenerative medicine. Current therapies are inadequate, highlighting the need for novel approaches to limit collagen release from hyperactive fibroblasts. This study focuses on the TANGO1 family of proteins, essential for collagen export from the endoplasmic reticulum (ER). Specifically, the interaction between TANGO1 and cTAGE5 is critical for this process. Inhibiting this interaction could provide a therapeutic strategy to control pathological collagen hypersecretion. The research explores the use of membrane-permeant peptide inhibitors targeting the TANGO1-cTAGE5 interface to achieve this control. The researchers hypothesize that these peptide inhibitors will effectively reduce collagen secretion, leading to a decrease in scarring and fibrosis.

Significant progress has been made in understanding fibrosis pathophysiology and the role of fibrogenic cytokines. However, effective antifibrotic therapies remain limited. Trials targeting specific pathways like interleukin-4 (IL-4) or TGFβ have yielded disappointing results, likely due to the involvement of multiple concurrent fibrogenic signaling pathways. A consistent hallmark across various fibrotic pathologies is excessive ECM protein secretion, particularly collagen. Therefore, modulating this excess ECM deposition represents a promising therapeutic approach. The discovery of the TANGO1 family of proteins, including TANGO1 and its paralog cTAGE5, has provided insights into the mechanisms governing collagen secretion and ER export. TANGO1's role as a master organizer of ER exit site (ERES) machinery, facilitating the formation of inter-organelle tunnels for increased secretory capacity of bulky cargoes like collagen, has been established. This essential role of TANGO1 in collagen secretion suggests that inhibiting its function could be a viable approach to control pathological collagen hypersecretion. Previous research has shown that TANGO1's function depends on specific interactions, highlighting potential targets for therapeutic intervention.

The researchers designed membrane-permeant peptide inhibitors targeting the coiled-coil region (CC2 domain) responsible for TANGO1-cTAGE5 heterodimerization. AlphaFold2 was used to predict the structure of the TANGO1-cTAGE5 interface. Two peptides, P2 and P5, were synthesized, each corresponding to the coiled-coil regions of TANGO1 and cTAGE5, respectively, and conjugated to a cell-penetrating dodecapeptide for cytoplasmic delivery. Scrambled control peptides (scrP2 and scrP5) were also synthesized. The effects of these peptides were evaluated using various methods: 1. **Cell culture experiments:** U2OS cells (collagen I) and RDEB/FB/C7 fibroblasts (collagen VII) were treated with the peptides, and changes in TANGO1, cTAGE5 protein levels, collagen localization (immunofluorescence microscopy), and collagen secretion (Western blotting) were assessed. 2. **Zebrafish model:** Zebrafish embryos were treated with the peptides from the 2-cell stage until 3 days post-fertilization (dpf). Larval length, phenotypes, and collagen fiber architecture (second harmonic generation (SHG) microscopy) were analyzed. Laser-induced cutaneous wounds in adult zebrafish were also treated with peptides to assess the effect on wound healing. 3. **Human fibroblasts:** Primary human skin fibroblasts and fibroblasts from scleroderma patients were treated with the peptides. Collagen secretion was assessed via Western blotting and mass spectrometry (secretome analysis). The effect of the peptides on TGFβ-activated fibroblasts was also investigated. 4. **Biochemical analyses:** Western blotting was used to quantify TANGO1 and cTAGE5 protein levels. Immunofluorescence microscopy visualized intracellular collagen localization. Mass spectrometry (LC-MS/MS) analyzed the secretome of treated and untreated fibroblasts. Cytotoxicity assays (LDH release) measured the effects of the peptides on cell viability. Statistical analyses, including Student's t-tests, Mann-Whitney U tests, and two-way ANOVAs were employed to compare results between different groups.

The study's key findings include: 1. **Peptide-induced destabilization of TANGO1 and cTAGE5:** Treatment with peptides P2 and P5 reduced TANGO1 and cTAGE5 protein levels in U2OS cells, suggesting that targeting the TANGO1/cTAGE5 heterodimerization interface leads to unstable monomers. 2. **Inhibition of TANGO1/cTAGE5 in zebrafish:** Peptide treatment in zebrafish embryos resulted in shorter body axes and other phenotypes consistent with TANGO1/cTAGE5 inhibition. SHG microscopy revealed altered collagen fiber organization in the larval tails. 3. **Collagen retention in the ER:** Peptide treatment in U2OS and RDEB/FB/C7 cells resulted in increased intracellular collagen co-localized with the ER marker Calnexin, indicating inhibited collagen ER export. 4. **Reduced collagen secretion in primary fibroblasts:** Treatment of primary human skin fibroblasts with P2 and P5, particularly in combination, significantly reduced procollagen I secretion. The effect was reversible, lasting for at least 24 hours after peptide removal. Scrambled control peptides had no effect. 5. **Inhibition of multiple ECM protein secretion:** Mass spectrometry analysis revealed that P2+P5 treatment significantly reduced the secretion of various collagens (types I, III, V, VI, XII), fibrillin-1, fibronectin, and other ECM proteins in primary human fibroblasts. 6. **Reduced scar formation in zebrafish wounds:** Peptide treatment in adult zebrafish with laser-induced wounds led to reduced granulation tissue formation and collagen deposition within the granulation tissue, without causing increased cell death (TUNEL assay). 7. **Inhibition of ECM secretion in activated fibroblasts:** The peptides effectively reduced collagen, fibrillin-1, and fibronectin secretion in TGFβ-activated fibroblasts from both healthy individuals and scleroderma patients.

This study demonstrates that membrane-permeant peptides inhibiting TANGO1 and cTAGE5 function effectively control fibrotic ECM protein secretion across diverse models, from zebrafish to human scleroderma fibroblasts. The success of this approach contrasts with the limited success of previous antifibrotic therapies, likely due to the complexity and redundancy of fibrotic pathways. The peptides' mechanism involves destabilization of the TANGO1-cTAGE5 heterodimer, leading to reduced protein levels and impaired collagen export from the ER. The reversible nature of the inhibition observed in human fibroblasts suggests a potential advantage in terms of therapeutic control and minimal long-term side effects. The effectiveness of the peptides in both healthy and scleroderma fibroblasts underscores their potential as antifibrotic agents, particularly in tackling pathological collagen hypersecretion. The zebrafish model provided valuable in vivo validation, demonstrating the inhibitors' efficacy in controlling collagen deposition during wound healing. The findings support the central role of TANGO1 in secreting various large ECM proteins and suggest a coordinated ER export of these proteins.

This research establishes a novel therapeutic strategy for fibrotic disorders characterized by excessive ECM production. The developed membrane-permeant peptides effectively inhibit TANGO1 and cTAGE5 function, reducing collagen and other ECM protein secretion in multiple models. The reversible nature of the effect and its demonstration in scleroderma fibroblasts suggest significant therapeutic potential. Future research should focus on optimizing peptide delivery, evaluating long-term efficacy and safety, and exploring potential applications in other fibrotic diseases.

The study primarily focuses on in vitro and zebrafish models. While these models offer valuable insights, extrapolation to human disease requires further clinical studies. The long-term effects of peptide treatment, particularly regarding potential compensatory mechanisms or off-target effects, need investigation. The study did not directly measure the disruption of TANGO1-cTAGE5 interaction after peptide treatment due to technical limitations.