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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/216043
Targeting the TGF-ß pathway, SMAD proteins and cofactors
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[eng] TGF-ꞵ signaling is key for many biological processes as embryo development, tissue homeostasis and immune system regulation. When altered, this pathway can lead to diseases such as cancer, fibrosis and rare syndromes. Key elements of the pathway are the SMAD family of transcription factors, which translate the extracellular signal received by the TGF-ꞵ receptor to the nucleus for regulation of gene expression. SMAD proteins have a characteristic structure which is shaped by an MH1 domain, for specific DNA recognition, a flexible linker region, and their MH2 domain, which can form complexes with other SMAD proteins and co-factors. This last domain is often mutated in disease, especially in the case of SMAD4 for which single point mutations and deletions have been identified in the literature. In this work, I focused on the study of SMAD4 variants associated with diseases, such as cancer, Juvenile Polyposis Syndrome, Hemorrhagic Hereditary Telangiectasia and Myhre Syndrome. With this aim, we produced different recombinant protein constructs to study the effect of these variants in their fold and binding properties. Firstly, I started with the characterization of the variants R496C- and I500V/M/T- SMAD4, associated with Myhre Syndrome. This is a gain-of-function disease that begins during embryonic development, and the alterations observed lead to the dysfunction of multiple organs. We could confirm that these specific SMAD4 variants had increased levels of SMAD4 protein in cells, possibly related to decreased ubiquitination and degradation of the protein, among other possible causes are loss-of-function variants, as in gastrointestinal cancers and Juvenile Polyposis. In this case, our work showed that the complexes with R-SMADs and the variants lead to several different stoichiometries compared to those of the wild type (WT) protein.
The second section of this thesis is focused on the search for small-molecules as SMAD4 binders. We used single molecule biophysics and structural biology to identify pharmacological strategies based on targeting SMAD4 to modulate TGF-ꞵ signaling. This search was conducted through a target-based in vitro approach using purified SMAD4 MH2 domain and large libraries of compounds. Among these compounds, we included FDA-approved drugs in case we could identify hits that could be repurposed to treat individuals suffering from very rare syndromes. Validated hits have affinities of interaction ranging between low and high micromolar and will be further developed and tested. Some interesting. approved drugs were identified as SMAD4 binders.
In the last chapter of this project, I focused on the DNA recognition ability of Ras Responsive Element Binder 1 (RREB1). RREB1 plays a key role in communication between RAS and TGF-ꞵ signaling to regulate epithelial-to-mesenchymal transition
(EMT) during embryonic development and maintenance of healthy tissue, but also during cancer progression. RREB1 is a zinc finger (ZF) protein with multiple isoforms. In particular, I studied a well-conserved evolutionary ZF pair located at the C-terminus of the protein.
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TORNER BLANCAFORT, Carles. Targeting the TGF-ß pathway, SMAD proteins and cofactors. [consulta: 26 de novembre de 2025]. [Disponible a: https://hdl.handle.net/2445/216043]