Design of peptides able to modulate Protein-protein interactions (PPIs) in mutated p53

Abstract

[eng] Protein-protein interactions (PPIs) play a crucial role in the homeostasis of the cells; therefore, they are important therapeutic targets. Typically, the goal in targeting PPIs is to impede them. On the contrary, in this project, we aim to stabilise the interactions between the homo-tetrameric p53 tetramerisation domain (TD). Tumour suppressor p53 is a transcription factor protein, also called 'genome guardian,' due to its ability to bind the DNA, check its integrity, and decide the cell fate depending on the seriousness of DNA damage. It is universally recognised that the protein is biologically active as a homo-tetramer, and only in this oligomeric state can it fully fulfil its functions. Nevertheless, it has been shown that also dimeric p53 has biological activities. In spite of these evidences, the tetramerisation event is still not fully understood, and it has been suggested that the oligomerisation is sophisticatedly regulated to control protein activity. When the protein is mutated, the populations of the different p53 species change, having an impact on the protein activity. For these reasons, understanding the mechanism of tetramerisation is extremely important to face cancer and to understand cancer-related biological functions better. In this project, we focused on the protein's tetramerization domain. We used a short version of it to study the equilibrium between different species, including the wild type (WT) and six cancer-related mutations. Moreover, we aimed to design mutants able to stabilise either the dimer or the tetramer by cross-linking two monomers. Finally, we designed and synthesised peptides as ligands able to interact with p53TD to stabilise the mutated form R337H. Concerning the study of p53TD oligomerisation, a variety of biophysical techniques, such as native MS, CD, NMR, and thermophoresis were applied. We found that the WT is tetrameric in all the conditions studied, it is the most stable among our peptides. The WT tetramer dissociates only with the increase of the voltage in the gas phase, giving a mixture of mainly monomers and tetramers. All the mutations studied destabilise the tetramer, although to different extents. The mutation T329I in the β-strain has a weakly destabilising effect on the TD, whereas the others on the α-helix strongly destabilise the tetramer. To simplify our system and reduce the number of equilibria between p53 oligomeric forms, we designed mutants to stabilise the dimer in one case (L330C) and the tetramer in the other (L344C) by cross-linking two monomers. L330C mutant is mainly dimeric and it is able to tetramerise. Most probably, the introduction of the Cys residue modifies the peptide structure making the β-strand shorter and causing the α-helix to recede. These structural changes may destabilise the domain, giving lower thermal stability and resulting in a more fluctuating structure in the MD

simulations. In comparison, the L344C mutant has a more similar structure to the WT, and apparently, the introduction of the Cys does not affect the structure of the peptide. It is a tetramer in the gas phase, and also more stable than the WT when increasing the voltage. Nevertheless, NMR and MST experiments suggested that it is mainly dimeric in solution. Finally, we designed two generations of peptides to bind to the hydrophobic pocket of p53TD with the aim of stabilising the mutated form R337H. The binding was studied by several biophysics techniques, such as chemical shift perturbation NMR, CD, fluorescence, thermophoresis, native MS, and HSQC NMR. Concerning the first generation, thermal stability CD showed a shift of the Tm of ca. 3 degrees. Native MS showed the binding with the second-generation ligands, and the stability of the complexes was investigated by increasing the voltage in the gas phase. From these experiments, two ligands were selected, Rab4 and cRys2R2, to be further studied by NMR experiments, and preliminary biological parameters, such as their stability in human serum and their cytotoxicity, were determined. Both peptides are stable in serum, and none of them is cytotoxic over 24 h in HeLa cells, having an IC50 value in the range of 50 – 150 µM.

[spa] Las interacciones proteína-proteína (IPPs) constituyen importantes dianas terapéuticas. En la mayoría de los casos en los que se utiliza una IPP como diana, lo que se pretende es inhibir la interacción. Por el contrario, en este proyecto, nuestro objetivo es reforzar las IPPs que estabilizan el dominio de tetramerización (TD) homo-tetramérico de p53. La proteína p53 es un importante factor de transcripción implicado en la reparación del material genético. La proteína es biológicamente activa en forma de homo-tetrámero aunque, recientemente, se ha observado que también la p53 dimérica tiene actividades biológicas. En este proyecto nos hemos centrado, en primer lugar, en un estudio detallado del mecanismo de tetramerzación de p53 utilizando una variedad de técnicas biofísicas. Para ello, hemos utilizado una versión minimizada del TD que nos ha permitido estudiar con más detalle el equilibrio entre diferentes especies, incluyendo el tipo salvaje (WT) y seis mutaciones relacionadas con cáncer. Hemos abordado también el diseño de mutantes capaces de estabilizar el dímero o el tetrámero mediante la reticulación de dos monómeros. Finalmente, hemos diseñado y sintetizado péptidos de pequeño tamaño capaces de interactuar con p53TD y estabilizar la forma mutada R337H. La presente tesis ha permitido, por un lado, establecer el estado de oligomerización de distintos mutantes de p53 relacionados con el cáncer. Esta información será importante de cara al futuro para abordar el bloqueo de posibles actividades biológicas de dichos mutantes. En segundo lugar, se han obtenido variantes covalentes del dominio de tetramerización nativo de p53, variantes que son capaces de modular la posición de los equilibrios monómero/dímero y dímero/tetrámero de la proteína. Finalmente, se ha sintetizado una nueva familia de péptidos capaces de interaccionar con las formas mutadas del dominio de tetramerización de p53 y modificar su estabilidad. Se trata de péptidos policatiónicos, tanto lineales como cíclicos, que han resultado ser excelentes ligandos del mutante R337H. Los dos compuestos más prometedores han resultado ser estables en suero, y ninguno de ellos se ha mostrado citotóxico frente a células HeLa.