Desarrollo de péptidos fotoconmutables para el control de la actividad celular

Abstract

Las interacciones proteína-proteína son determinantes para las funciones biológicas y constituyen dianas terapéuticas de gran interés. Muchas de ellas son mediadas por péptidos cortos donde una secuencia lineal adopta una estructura helicoidal. Moléculas que imitan esos péptidos se han usado con éxito como inhibidores de las interacciones en que participan. La posibilidad de usar versiones de esos péptidos diseñadas para poder controlar con luz su estructura y actividad inhibidora abre nuevas vías para la investigación de los procesos biológicos a escala molecular y la intervención terapéutica en los mismos.

En esta tesis se desarrolla un panel de péptidos de estructura fotoconmutable mediante acoplamiento de azobenceno a su estructura. Estos péptidos derivan de la parte de la proteína β-arrestina que interacciona con β-adaptina 2 y tienen como objetivo la inhibición fotocontrolada de esa interacción. Se estudian los factores de diseño molecular que arrojan mejores resultados de fotontrol de la estructura y la actividad inhibidora, incluyendo diferentes posiciones y distancias de acoplamiento de azobenceno y uso de aminoácidos no naturales en la secuencia peptídica. Asimismo, se aplican algunos de los péptidos desarrollados al fotocontrol en células vivas del proceso de endocitosis mediada por clatrina, un proceso de importancia cardinal en la biología celular de los organimos eucariotas, logrando una alteración del mismo dependiente de la iluminación. En conjunto, estos trabajos suponen una demostración de aplicabilidad de la imitación directa de estructuras biológicas en el diseño de moléculas fotocontroladas para el control de las interacciones y actividades biológicas de las estructuras imitadas. Los principios de diseño empleados son potencialmente aplicables a otras interacciones proteicas intracelulares de gran relevancia biológica y clínica.

Protein-protein interactions are crucial for biological functions and constitute therapeutic targets of great interest. Many protein-protein interactions are mediated by short peptides in which a linear sequence adopts a helical structure. Molecules that mimic these peptides have been successfully used as inhibitors of the interactions in which they are involved. The possibility of using versions of such peptides modified by design in order to control their structure and inhibitory activity by optical means opens new avenues for the investigation of biological processes at the molecular level and therapeutic intervention in their disorders.

In this thesis a panel of peptides with photoswitchable secondary structure is developed by cross-coupling azobenzene to selected pairs of cysteine lateral chains introduced in their sequences. These peptides are derived from the C-terminal region of the protein β-arrestin, known to interact with β-adaptin 2. Furthermore, a peptide termed BAP-long corresponding to that region is known to bind β-adaptin 2 as well as to adopt a helical conformation when bound to it while remaining unstructured when free. 20-mer BAP-long is an appealing candidate for photocontrol of secondary structure with potential for the photocontrol of binding affinity and of biological activity related to the interaction of β-arrestin with β-adaptin 2 in living cells.

The first part of the thesis introduces the work and reviews literature on chemical photoswitches as well as their application to biomolecules and to molecular mechanisms of biological functions. Different photoswitches are presented and different approaches to the control of biomolecules are discussed separating pharmacological applications from applications requiring genetic modification (optogenetics). The focus is on the photocontrol of secondary structure of biomolecules, particularly peptides and proteins, as a strategy to achieve photocontrol of their functions. A final sub-section introduces the system in which the assays presented have taken place: clathrin-mediated endocytosis of membrane receptors.

The second part of the thesis investigates molecular design factors that yield superior results in terms of fotocontrol of structure and inhibitory activity of versions of BAP-long. These features include different coupling positions and distances of the azobenzene photoswitch along the peptide structure as well as use of unnatural amino acids in the peptide sequence. In this part of the thesis the hypothesis that photocontrol of peptide structure results in photocontrol of affinity and ability to competitively inhibit the interaction of β-arrestin with β-adaptin is verified in vitro. Furthermore, the observation is made that for this particular system and potentially for others of similar characteristics the better photoswitchable inhibitors of the interaction are the least structured peptides when free in solution.

In the third part of the thesis some of the peptides are applied in living cells to photocontrol clathrin-mediated endocytosis, a process of cardinal importance in eukaryotic organisms and in which the interaction of β-arrestin with β-adaptin plays an important role. Photocontrolled alteration of endocytosis of the transferrin receptor is achieved with the same light dependence observed in the previous section for binding. This is in agreement with the second hypothesis that photocontrol of affinity results in photocontrol of the biological activity associated to the interaction, probably by competition of the photoswitchable peptides against the natural proteins for the available binding sites. The results also indicate an ability of the peptides to cross cell membranes confirmed by tracking of fluorescent peptides. Photocontrolled alteration of clathrin-containing structures is also observed.

Together, these studies represent a demonstration of the applicability of the direct imitation of biomolecules in designing photoswitchable inhibitors to control the interaction and biological activities of the mimicked structures. The design principles used are potentially applicable to other intracellular protein-protein interactions of biological and clinical relevance.