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
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.
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.
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.