Chemical interrogation of microtubule eraser enzymes

Projektbeschreibung

Einblicke in Enzyme, die posttranslationale Modifikationen rückgängig machen

Posttranslationale Modifikationen (PTM) regulieren die Aktivität, Stabilität und Interaktionen von Proteinen durch die Anlagerung chemischer Gruppen. Diese Modifikationen werden dynamisch von Enzymen rückgängig gemacht, die als „Radiergummis“ bekannt sind und vielversprechende Ziele für Medikamente darstellen. Die Untersuchung dieser Radiergummis ist jedoch nicht einfach, da sie mehrere Substrate erkennen können. Das ERC-finanzierte Projekt CHEMTUBIO führt eine innovative Methode zum Nachweis von Radiergummi-Aktivitäten in situ direkt an Proteinkomplexen ein. Dieser Ansatz soll bei Enzymen angewendet werden, die PTMs von Tubulin, einer wichtigen Strukturkomponente des Zytoskeletts, entfernen. Das Verständnis der Funktionsweise dieser Enzyme könnte neue therapeutische Möglichkeiten eröffnen, darunter Herz-, Nerven- und Muskelerkrankungen, die mit einer gestörten Tubulinregulierung einhergehen.

Ziel

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.