Detyrosination is an important process for the function of microtubules, which form the skeleton of all our cells. The team of Oncode Investigator Thijn Brummelkamp (NKI) previously identified Vasohibins as detyrosinating enzymes, but most cells contain detyrosinated microtubules even when the two Vasohibin genes are deleted. With a combination of genome-wide mutagenesis screens, the team now identified MATCAP as the a new enzyme involved in this process. Together with the team of Oncode Investigator Anastassis Perrakis (NKI) they then used structural biology methods (X-ray crystallography, computational docking, and single-particle cryo-EM in the NKI/Oncode facility), to show how MATCAP works (as an atypical metalloprotease) and how it engages with microtubules. Besides this, generating mice lacking the detyrosination activity of both enzymatic systems – Vasohibins and MATCAP – they showed that this mainly affects brain development. The results of this study are published today in Science and may shed light on side effects of commonly used anticancer drugs.

“Our results address a long-standing question and pave the way to study the roles of tubulin detyrosination” says Lisa Landskron, postdoc in Brummelkamp’s lab and first author of this newly published research. “Knowing the identity of the enzymatic machinery, allows us now to explore how detyrosination is coordinated with other posttranslational modifications of microtubules. This is important to understand how different functional fates are assigned to microtubule populations” she adds.

The evolving history of microtubule detyrosination

Our cells have their own skeleton, which is largely composed by microtubules. Microtubules help a cell keep and adjust its shape, transfer vesicles, and to divide. The microtubule cytoskeleton is constantly modified to generate microtubules necessary to achieve these specific functions. The first discovered important posttranslational modification of microtubules is detyrosination – the removal of the genetically encoded terminal tyrosine of α-tubulin - but the enzymes involved remained unknown for decades.

"This project has been incredible fun in many ways. The genetic search for the elusive enzyme, pointed to an unstudied gene that wasn’t even categorized as protease before”, says Thijn Brummelkamp. “The highly collaborative aspect was enjoyable on itself but also yielded a view on this chemical reaction from many perspectives. Last, this work will make it possible to study the role of tubulin detyrosination in the many different biomedical processes in which it has been implicated”.

Tubulin detyrosination, was first observed more than 40 years ago. It has been implicated in the fidelity of mitosis, cardiac function, brain development, and in neuropathy, a frequent unwanted side effect of cancer chemotherapy. Because the enzymes carrying out this modification were unknown, Brummelkamp’s team used mutagenesis in haploid human cells to identify the corresponding enzymes. In 2017 this led to the identification of Vasohibins in complex with their co-factor SVBP.

This was unexpected because Vasohibins were until then known as secreted factors influencing angiogenesis. Inactivation of Vasohibins diminished - but did not ablate - tubulin detyrosination, indicating that additional enzymes must exist. Using genetics in Vasohibin-knockout cells, Landskron identified KIAA0895L, an unstudied protein that was not annotated as a protease. In collaboration with the Perrakis’s team, she then established that KIAA0895L was an atypical metalloprotease and renamed it MATCAP. Tassos Perrakis explains: “Having previously collaborated with Thijn’s team to determine the structure of Vasohibins, finding out that the crystal structure of MATCAP was so different, was amazing. This was very helpful to understand how MATCAP recognizes and cleaves the tyrosine in the tubulin tail”. Later, PhD student Jitske Bak determined the cryo-EM structure of MATCAP bound to fully tyrosinated microtubules, someting that would not had been possible without the cryo-electron micorscope at the NKI, which Oncode helped to fund and establish.

The next steps

For the future, the two Oncode teams will continue their work with an NWO EW “M2” grant, which allows groups from different backgrounds to collaborate and synergistically answer important research questions. They would like to understand why two different types of enzymes are needed – do they have a specialisation for microtubules with other modifications or do they act in specfiic contexts? They will also work to better understand the role of detyrosination in health - for example the fidelity of chromosome segregation - and disease such as in chemotherapy-induced neuropathy.