Uncontrolled secretion of extracellular matrix (ECM) proteins such as collagen can lead to excessive scarring and fibrosis, and compromise tissue function. Researchers at the Center for Genomic Regulation and at the University of Cologne have now developed an experimental strategy to tackle scarring and fibrosis that uses peptide inhibitors to limit the amount of collagen released from hyperactive fibroblasts. The team’s experiments with patient-derived human cells and in animal models showed the strategy to be effective and non-toxic and found that its effects were reversible.

ICREA research professor Vivek Malhotra, PhD, a researcher at the Center for Genomic Regulation (CRG), and colleagues reported on their findings in Nature Communications, in a paper titled “TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.”

Scarring occurs from the secretion and accumulation of various components—primarily collagens—into the space between individual cells, and usually occurs as a response to injury or damage. “After tissue injury, platelets, endothelial cells, and inflammatory cells induce fibroblasts to initiate extracellular matrix (ECM) deposition,” the authors wrote. Excessive collagen secretion can also cause the buildup of fibrotic tissue, a more serious condition where excess connective tissue is formed to the extent that it compromises the function of tissues and sometimes entire organs. Around 45 percent of deaths in the industrialized world are attributed to some form of tissue fibrosis. However, the authors further noted, “Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking.”

Treatment options for both scarring and fibrosis are usually limited to surgery. Outside the body, scar tissue is often beneath the outer layer of the skin. Since most topical creams are not able to penetrate deeply enough to reach the affected areas effectively, their ability to remodel or heal the tissue is limited. Inside the body, scarring and fibrosis can affect many different tissues and organs, each with its unique environment and challenges and with no one-size-fits-all treatment option. “In spite of significant progress in understanding the pathophysiology of the underlying processes of fibrosis and identifying the role of several fibrogenic cytokines, few specific therapeutic strategies are available to reduce or control connective tissue formation during wound healing and fibrosis,” the team stated. Co-corresponding author, Malhotra, added, “Existing treatment options are usually ineffective because they try and fail to mop the excessive collagen up.”

The investigators explained that while targeting excess ECM deposition could represent a promising disease modifying therapeutic approach to fibrosis, achieving targeted control of collagen deposition would require a detailed understanding of the mechanisms governing the secretion of these large proteins. “Of particular importance is their export from the endoplasmic reticulum (ER), a process that has only recently become amenable to molecular analysis after the discovery of the TANGO1 family of proteins, including TANGO1 and its paralog cTAGE5.” The new strategy involves using peptides to block the export of collagen from inside cells, by disrupting key interaction between TANGO1 and cTAGE5.

Both proteins bind to each other and are essential for the export of collagens from their site of synthesis inside the cell to the exterior. The two proteins “sit” at the endoplasmic reticulum exit site, a place in the cell where materials like proteins are packaged and transported out the cell.

The researchers further explained that targeted control of collagen deposition requires a detailed understanding of the mechanisms governing the secretion of these large proteins. “Of particular importance is their export from the endoplasmic reticulum (ER), a process that has only recently become amenable to molecular analysis after the discovery of the TANGO1 family of proteins, including TANGO1 and its paralog cTAGE5 … TANGO1 family proteins have emerged as master organizers of ER exit site (ERES) machinery.”

Malhotra stated, “In this work, we tried a completely different idea: to block the floodgates at the cellular level. The strategy works at the cellular level, releasing enough collagen so that tissues don’t fall apart while protecting them from excessive amounts that impairs their function.”

Proteins are like puzzle pieces. To know how two pieces fit together, you need to see their shapes clearly. Both TANGO1 and cTAGE5 are large, complex proteins which constantly shapeshift. To date, their exact structure remains unknown, which in turn means we don’t understand how they connect at the molecular level, hindering efforts to design drugs that can block the interaction. “Biologists have traditionally considered ER export un-druggable because its machinery is essential for cell viability and acts constitutively, such that targeting any component could produce pleiotropic, off-target effects on secretion,” the researchers noted.

They overcame this challenge by using AlphaFold2, an artificial intelligence program that can guess the shapes of the two proteins without needing structural data about their 3D shape. Commented Ishier Raote, PhD, study first author, who carried out the work at the Center for Genomic Regulation. “Targeting the endoplasmic reticulum exit site has been historically considered impossible because a third of all human proteins go through it, so inhibiting its activity would likely have many off-target, toxic effects. In other words, it’s been ‘undruggable’. Only recently have there been indications that there is some specificity for the secretory materials. In this study we aimed to achieve this specificity by inhibiting the interface between TANGO1 and cTAGE5 with targeted precision.”

The predictions made by AI allowed the authors of the study to design peptides which can pass through a cell membrane and disrupt the interaction between TANGO1 and cTAGE5. They then tested the peptides using normal human fibroblasts, the most common type of cell found in connective tissue. The peptides successfully inhibited collagen export, causing it to accumulate inside the cells. The effect was also reversible, with collagen levels increasing again after the peptides were removed within a 48-hour period.

They observed similar effects in experiments with fibroblasts from patients with scleroderma, a complex autoimmune disease characterized by fibrosis of the skin and internal organs. “Inhibiting TANGO1/cTAGE5 resulted in a severe reduction of collagen secretion also in these cells suggesting that myofibroblasts in patients with scleroderma can be targeted by the inhibitory peptides.”

The peptides were then tested using zebrafish, a common animal model to study tissue development and wound healing. The strategy visibly reduced collagen deposition in wound areas. “…  our precise targeting of the interface of ERES proteins, TANGO1 and cTAGE5, controls their activity specifically, showing that it is possible to control ER export of specific ECM proteins, without major off-target toxicity,” they stated. “Our results suggest a promising avenue for therapeutic intervention via targeted inhibition of ER export, for currently intractable fibrotic disorders characterized by excessive ECM production.”

The researchers next plan to evaluate the efficacy of the peptides in pig skin because it closely resembles human skin. They will also finetune the properties of the peptides to increase their potency.

“We believe this represents a new strategy to control the effects of collagen hypersecretion. This could range from alleviating the cosmetic effects of skin scarring to the treatment of autoimmune diseases like scleroderma, as well as to manipulate post-surgery related events associated with wound healing to prevent fibrosis,” Malhotra concluded. The authors further noted, “Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes … In addition to the potential implications for anti-fibrotic therapeutic development, it is exciting that these peptide inhibitors were active in zebrafish, as they provide an  experimental handle to control ECM secretion selectively, acutely, and reversibly, in almost any metazoan system without the confounds of cells or tissues adapting to the genetic loss of TANGO1.”

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