Caracterización estructural y funcional de proteínas quinasas C y su interacción con proteínas moduladoras y sustratos

Laburpena

In the present Doctoral Thesis, the biophysical and structural analysis of important proteins belonging to the PKC kinase family has been carried out. In addition, the study has also been related to proteins with which they interact, Fascin1, an important substrate of PKCα with a fundamental role in the spatiotemporal dynamics within the F-actin cytoskeleton and, on the other hand, a protein with a paramount role in the cellular localization in the membrane of PKCs, RACK1. All of this in conjunction with various combinations of additives (CaCl2, the phorbol ester P13A and ADP-MgCl2), which are important in modulating the activity of these kinases. By means of X-ray crystallography and cryo-electron microscopy techniques, an extensive study of crystallization screenings and preparation of CryoEM grids for PKCs kinases together with the modulatory proteins and substrates was performed. Unfortunately, no crystals were obtained with the presence of any of the PKCs under study neither information by CryoEM that would allow atomic or near-atomic structural determination of the PKCs proteins. Nevertheless, through CryoEM we managed to determine preliminary conditions, resulting in 2D classifications in which we could interpret a possible interaction model for the PKCα-Fascin1 complex. Thus, with a great potential to be able to structurally determine the protein complex by improving the initial data acquisition. We also performed several biophysical techniques such as Thermal Shift Assay that allowed us to infer different binding modes for PKCα and Fascin1 depending on the combination of additives added to the medium. By SPR, it was also determined that the highest affinity between Fascin1 and PKCα occurred with the wild-type version of Fascin1 together with all the additives tested (CaCl2, the phorbol ester P13A and ADP-MgCl2). Cryo-electron microscopy tomography studies allowed us to determine the binding of Fascin1 to F-actin filaments in a discrete manner by using a Fascin1 mutant affecting one of the two main sites of interaction with F-actin. The project is currently at an advanced stage prior to the atomic structural determination of this interaction. On the other hand, drug rescue assays were also carried out for pharmacological substances that could be able to interact with the PIP2-binding pocket of the C2 domain of PKCα and that could influence the ability of the kinase to recognize the membrane. However, after performing biophysical assays such as FRET and lipid sedimentation assays, as well as cellular assays coupled to confocal microscopy, none of the drugs under study could be assigned with a role in disrupting the ability of the protein recognizing the membrane. However, by X-ray crystallography it was determined that one of the compounds under study was able to interact with the PIP2 binding pocket. Despite the enormous structural challenges presented by these PKC proteins (asymmetry, small size and the ability to adopt multiple conformations making them very flexible proteins) and the fact that to date there is no complete structural information for any of their isoforms, it has been possible to obtain highly valuable information with the potential to advance in the structural determination at an atomic resolution for the protein complex PKCα-Fascin1. This could help to overcome the big issue that exists today in the development of specific drugs for this family of kinases and thus be able to fight cancer, one of the main diseases in which PKC proteins are involved.