Skip to main content
European Commission logo
English English
CORDIS - EU research results
CORDIS
CORDIS Web 30th anniversary CORDIS Web 30th anniversary

Transforming Growth Factor – Bench To Bedside. Towards a better understanding of TGF-β isoform specific signalling in health and disease

Periodic Reporting for period 2 - TGF-BTB (Transforming Growth Factor – Bench To Bedside. Towards a better understanding of TGF-β isoform specific signalling in health and disease)

Reporting period: 2022-06-01 to 2023-05-31

The overarching goal of the TGF-BTB (Transforming Growth Factor – Bench To Bedside) project is to unravel the distinct roles played by TGF-β isoforms, a group of growth factor molecules that, much like hormones, circulate within our body and engage specific cell surface receptors, thereby initiating signalling pathways that regulate a multitude of critical physiological processes. TGF-βs exert profound influence on cell proliferation, wound healing, cancer progression, and immune responses. Elevated TGF-β signalling often manifests in conditions such as cancer, fibrotic diseases, and chronic inflammation. Following cardiac infarction or other forms of cardiac injury, TGF-β overexpression can lead to life-altering complications.
A central focus of TGF-BTB is the exploration of the enigmatic co-receptor, Betaglycan. Betaglycan is referred to as a "co-receptor" because TGF-β can bind to it without triggering signal transmission. However, it has been demonstrated that Betaglycan enhances TGF-β activity by presenting it to the signaling receptors. This enhancement is particularly crucial for one isoform, TGF-β2, as its natural binding affinity to the signaling receptors is insufficient for signaling without Betaglycan's presence. To gain deeper insights into the mechanisms underpinning Betaglycan's signaling potentiation and to appreciate the significance of preserving three distinct TGF-β isoforms and their selective inhibition, TGF-BTB has established the following objectives:
• To attain a comprehensive molecular understanding of the TGF-β:Betaglycan complex.
• To observe TGF-β:Betaglycan complex formation and the transfer of the ligand to the signalling receptor, both in vitro and in vivo.
• To design a peptide-based inhibitor capable of effectively blocking TGF-β signalling, suitable for functional studies and with potential clinical applications.
Throughout the course of this project, we have gained a comprehensive understanding of the interaction between Betaglycan and TGF-β Growth Factors. This knowledge has allowed us to elucidate how Betaglycan can bind to TGF-β while still providing space for partial binding of the signalling receptor, enabling the seamless transfer of TGF-β. The structural insights obtained have clarified the mechanisms underlying ligand specificity, shedding light on which isoforms are most susceptible to the loss of Betaglycan subdomains.
Furthermore, during the project's duration, we embarked on the endeavor to design peptide-based inhibitors tailored for the TGF-β signaling pathway. These inhibitors were intended to complement checkpoint anticancer therapy, enhancing its efficacy, and addressing persistent inflammatory conditions associated with lung remodeling in COPD or asthma. After multiple iterations, our designed peptide-based inhibitors have proven to be effective and highly specific for the TGF-β pathway.
The molecular picture revealing TGF-β as bound to betaglycan, the main objective of this phase of the project, was successfully obtained. Due to the complexity of this system, we initially recorded many failed attempts, however applying the “divide and conquer” strategy allowed us to advance the project. Thanks to this decision only selected parts of the picture began to emerge, but they were of excellent quality. Subsequently, the full picture could be assembled by assimilating many independent experimental results obtained using different techniques. High-resolution structures demonstrated how betaglycan embraces the TGF-β ligand with 1:1 stoichiometry, but in a manner that still leaves capacity for one additional molecule of signalling receptor to bind. Also the new CryoEM structure of O-domain in complex with TGF-b ligand and extracellular domain of TbRII obtained during incoming phase reveald how the TbRII affinity is enhanced by the presence of O-domain
This mode of binding is consistent with the previously proposed potentiation mechanism. With the employment of artificial membrane with attached fluorescently labelled extracellular domains of the receptors and time resolved FRET signal detection, we could demonstrate that in the presence of betaglycan the singlaling triggered by TGF-B2 is significantly more efficient and close to the TGF-b1.
In parallel the TGF-BTB project was focused on the development of a peptide-based inhibitor of the TGF-β signalling pathway for functional studies as well as for clinical applications. This aim was divided into three separate tasks: In silico inhibitor design, followed by production and biophysical/biochemical evaluation of the inhibitor potency and finally activity assessment in tissue culture.
After a series of experiments, it became apparent that the theory of deriving small peptides from the highly structured – disulphide constrained donors would not work efficiently. Learning from this, efforts were subsequently focused on the improvement and adoption of minimal negative TGF-ß that blocks TGF-ß signalling. Due to the presence of four disulphides, it adopts a very specific, structurally defined conformation. Nonetheless, it also poses challenges, one of which is poor folding upon expression in mammalian cells. That was first point to address. Performing experiments that aimed to reengineer the peptide core, it was possible to simplify it without impairing the inhibitory activity, whilst significantly improving production in mammalian cells.
Partial results of this project were presented during the Molecular Biophysics & Structural Biology Symposium hosted annually by the University of Pittsburgh Graduate Programme as well as during the FASEB TGF-β meetings organized in 2020 (online) and 2022 (in Dublin) for which the TGF-BTB project was awarded the best poster presentation. During the incoming phase results of this project were presented during the “Single Molecule Network Meeting” 2023 as well as during the seminars, presentations and chalk talks to the public, students, and other researchers.
During the outgoing phase of the TGF-BTB project, a fruitful collaboration was established with research groups led by Dr Theresa Whiteside and Dr Greg Delgoffe at the UPMC Hillman Cancer Centre in Pittsburgh, who expressed their interest in the novel TGF-ß signalling pathway inhibitor for use as an adjuvant in two-component anticancer therapy. This collaboration allowed the project to expand the research towards new applications. Immunotherapies for cancer that aim to block checkpoint molecules have dramatically changed cancer treatment. The inhibitor developed in the TGF-BTB project increased the efficiency of anticancer checkpoint blockade in mice, particularly improving the tumor infiltration of immune cells and thus promoting the antitumor response. Results of this collaboration were reported in the Molecular Cancer Therapeutics journal in 2021.

Data obtained by the TGF-BTB project will greatly help scientists to understand the mechanism behind TGF-β signalling potentiation by betaglycan and will be important for the TGF-β biology field and beyond. Also, the experience gained will further advance the field in future projects that rely upon Cryo-EM analysis of ligand:receptor complexes as well as use of NMR in protein-protein complex analysis. Last but not least, this new strategy for a TGF-β signalling inhibitor may lead to development of improved checkpoint immunotherapy that can benefit cancer patients worldwide.
Project