Chemical interrogation of microtubule eraser enzymes

Descripción del proyecto

Conocer mejor las enzimas que borran las modificaciones postraduccionales

Las modificaciones postraduccionales (MPT) regulan la actividad, la estabilidad y las interacciones de las proteínas mediante la adición de grupos químicos. Estas modificaciones son eliminadas dinámicamente por enzimas conocidas como borradores, que se han convertido en dianas farmacológicas prometedoras. Sin embargo, estudiar los borradores es todo un reto debido a su capacidad para reconocer múltiples sustratos. El equipo del proyecto CHEMTUBIO, financiado por el CEI, introduce un método innovador para detectar «in situ» la actividad de borrado directamente en complejos proteicos. Este método se aplicará a las enzimas que eliminan las MPT de la tubulina, un componente estructural clave del citoesqueleto. Entender cómo funcionan estas enzimas revelaría nuevas vías terapéuticas para los trastornos cardiacos, neuronales y musculares relacionados con la regulación alterada de la tubulina.

Objetivo

Erasers of protein post-translational modifications are ubiquitous regulatory enzymes that control cellular function and are targeted for the treatment of disease. One key example are erasers of histone acetylation, where inhibitors have reached the clinic as cancer chemotherapy. Currently, however, multiple other erasers lack tools of study to determine their relevance and generate clinical candidate molecules. One main reason for this limitation is that erasers display multivalent substrate recognition mechanisms that are difficult to replicate with standard assays in vitro and can jeopardise data translation. I have recently developed a solution to detect histone deacetylase activity in situ not on peptides, as it commonly done, but on semi-synthetic protein substrates and complexes, and still applicable to in vitro screening. My preliminary data supports that this methodology can be applied as a more general strategy, enabling studies and drug development on erasers currently inaccessible. Moreover, it holds great potential to localise eraser activity in living systems and generate maps of enzyme action on endogenous biological structures. CHEMTUBIO aims to generalise in situ eraser activity detection through application to the erasers of tubulin tail detyrosination. These enzymes generate gradients of detyrosination across the cytoskeleton lattice through mechanisms unknown and, even though inhibitors are attractive to treat cardiac and neuronal disorders, no probes are available for further investigation. Here, we will address these knowledge gaps and provide in situ sensors and chemical probes to advance the therapeutic application of detyrosinase inhibition. Furthermore, CHEMTUBIO will develop semi-synthesis methods to allow the study of microtubule lumen modifications. Erasers of luminal acetylation are attractive targets against neuromuscular disease and cancer, and we will provide key mechanistic knowledge to boost future therapeutic developments.