Functional analysis of tubulin detyrosination. – TYRTUB

The breakthrough, which allowed for the identification of tubulin carboxypeptidase was a result of the development of covalent inhibitor, which was compatible with “click chemistry”. This allowed for a post-inhibition attachment of azide beads and affinity purification of the tubulin carboxypeptidase enzyme followed by its identification.

The main achievements until now include discovery of the identity of tubulin carboxypeptidase and development of original method to design the inhibitors for this enzyme. In addition, we have determined that tubulin detyrosination plays an essential role in germline stem cell differentiation in Drosophila.

The results obtained in the course of this project are the basis of an application for standard maturation project with Satt Axlr Montpellier entitled: “Development of drug candidates for treating neurodegenerative diseases”. The main objective of this project is the creation of a start-up company.

International patent application entitled: «Development of a novel method to design TCP inhibitors«. CNRS number DI n° 09672-01.

Microtubules (MTs) are hollow tubes formed by the assembly of alpha- and beta-tubulin heterodimers. As major constituents of the cellular cytoskeleton, they are involved in a number of functions including cell division, intracellular transport, cell motility and cell morphology. The main question we are trying to answer in our lab is how can MTs adapt to all these diverse functions? One way to regulate protein functions is by changing their properties with posttranslational modifications. The first tubulin related modification to be discovered was detyrosination. This modification is specific to alpha-tubulin and it consists of the removal of the tyrosine residue found at the very C-terminus of most alpha-tubulins. Elimination of the hydrophobic tyrosine residue results in unmasking of negatively charged glutamates and leads to drastic changes in the biochemical properties of the alpha-tubulin tail. Since tubulin tails are exposed on the MT surface where they provide the binding sites for MT associated proteins (MAPs) and molecular motors, changing their nature could regulate these interactions and adapt MT to different functions. However, at the moment very little is known about the function of detyrosination due to the fact that the identity of the enzyme involved in its generation, termed tubulin carboxypeptidase (TCP), remains unknown. To overcome this problem, we have developed an alternative approach to study the role of detyrosination, which is based on the in vivo mutagenesis of alpha-tubulin. Using this approach we have already demonstrated that tubulin detyrosination plays an essential role in germ line stem cell differentiation in Drosophila. Blocking detyrosination leads to “tumor like” stem cell overproliferation during both spermatogenesis and oogenesis. Here we propose to carry out an extensive identification of the MAPs that are regulated by the status of the C-terminus of alpha-tubulin and thus serve as detyrosination effectors. Knowledge about the identity of these effectors will greatly facilitate the understanding of the molecular mechanisms underlying the stem cell defects caused by the lack of detyrosination. In addition, to identify the TCP we have elaborated a novel biochemical approach, which is based on the development of a highly specific and covalent inhibitor of this enzyme compatible with affinity purification methods. Using this inhibitor we will identify TCP and address the role of this enzyme in ciliary functions. Taken together our project will greatly advance the knowledge about tubulin detyrosination and open up a new field in MT research.