Structural studies of recombinant TGIF1 and FBP28 WW domains using NMR and peptide ligation strategies
The present thesis is divided in three different but related projects. In the first project, the interaction between TGIF1 and SMAD proteins is investigated. TGIF1 is a transcriptional suppressor that prevents the gene transcription due to its interaction with DNA and protein suppressor complexes. T...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/400299 |
| Acceso en línea: | http://hdl.handle.net/10803/400299 |
| Access Level: | acceso abierto |
| Palabra clave: | Cèl·lules animals Celulas animales Animal cells Pèptids Péptidos Peptides Ressonància magnètica nuclear Resonancia magnètica nuclear (Física) Nuclear magnetic resonance Ciències de la Salut 577 |
| Sumario: | The present thesis is divided in three different but related projects. In the first project, the interaction between TGIF1 and SMAD proteins is investigated. TGIF1 is a transcriptional suppressor that prevents the gene transcription due to its interaction with DNA and protein suppressor complexes. TGIF1 has a role in different signalling pathways such as retinoid, Wnt or TGF-β. Focusing on TGF-β, this is one of the main signalling pathways that regulates a plethora of functions in metazoans, including cell differentiation, proliferation and tissue homeostasis, among many others. SMAD proteins are the centrals mediators of the pathways, carrying the signal from the TGF-β receptors at the plasma membrane to the DNA. The aim of this project was to describe, from a structurally point of view, the interaction between TGIF1 and SMAD proteins, already detected biochemically. Using NMR spectroscopy and EMSA assays we could observe the interaction between TGIF1 and the MH1 domain of SMAD2 and SMAD4. Furthermore, the addition of SMAD2/4-MH1 disrupts the TGIF1-DNA interaction. On the other hand, the binding between SMAD2-MH2 and TGIF1 (256-347) was not detected in our experimental conditions. Moreover, we found that p38α and CK1 kinases phosphorylate serines 286, 291 and 294 of TGIF1 (256-347) in vitro. While these serines are located in a region where TGIF1 interacts with many proteins - including SMAD2, HDAC, or Axin-2 - , the phosphorylation could indicate a regulation mechanism. However, the phosphorylation does not change the overall structure of the TGIF1 fragment. Finally, we have detected an interaction between TGIF1 fragments 150-248 and 256-347, suggesting the presence of open and closed conformations of full-length TGIF1. The second project is related to the ligation reaction between two peptides. Our study demonstrates that the addition of HOBt to the cysteine-free direct aminolysis ligation reaction between one thioester peptide and one N-terminal free peptide increase the conversion but not the rate of the ligation. This effect is especially increased when sterically hindered amino acid (such as valine or leucine) are present in the ligation junction. The reaction is also compatible with phosphorylated peptides but intramolecular cyclisation side-reactions appear when the peptide thioester is not protected at the N-terminus. Lastly, six selected mutants structures of FBP28-WW2 were determined de novo by NMR spectroscopy. The six mutant structures maintain the main characteristics of WW domain structure, even for the mutations introducing deletions at the N and C termini. The structures were the experimental confirmation of the effect of this set of mutations in the structure. The existence of the WW fold in all these mutations was key to provide the grounds for the simulated folding curves generated using the UNRES force field. |
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