Mechanism of tubulin modification enzymes

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

Disruption in tubulin modifications is a hallmark of neurodegeneration, cancers and neurodevelopmental disorders and mutations in tubulin modification enzymes cause several human pathologies, including early onset neurodegeneration in humans. For example, mutations in cytosolic carboxypeptidase 1, an enzyme that deglutamylates tubulin causes early onset neurodgeneration. An essential aspect of understanding the tubulin code and how mutations in tubulin code enzymes cause disease is to understand how the code is written i.e. the mechanism of the enzymes that introduce these modifications and how cooperation and competition between these enzymes gives rise to the complex microtubule modification patterns observed in cells, and how disease states disrupt these patterns. Specifically we aim (1) to determine high-resolution structures of key tubulin modification enzymes in isolation as well as in complex with the microtubule to understand their mechanism as well as design small molecule modulators with potential therapeutic use or to enable monitoring of these modifications in live cells; (2) to map tubulin modification sites on tubulin. For this our model system is the i3Neuron because tubulin modifications are highly abundant in neurons, and because disruption of many tubulin modification enzymes leads to neuronal pathologies; (3) to investigate the biochemical interplay between tubulin modification enzymes and how this gives rise to temporally and spatially regulated modification patterns in cells. This project leverages our ability to make unmodified and recombinant single-isoform engineered human tubulin and coupled with our expertise with an array of structural techniques (X-ray crystallography, cryo-EM and SAXS), high-resolution mass spectrometry, classical kinetics and single molecule fluorescence to answer fundamental questions about the mechanism and regulation of tubulin modification enzymes in health and disease. During this year we focused on TTLL glutamylases and glycylases, the largest family of tubulin modification enzymes. Glutamylation and glycylation involve the post translational ATP-addition of glutamate chains to the tubulin C-terminal tails. Glutamylation is the most abundant tubulin modification in the human brain. During this year we made several important discoveries. (1) We determined the cryo-EM structure of the tubulin glutamylase TTLL6 and elucidated the mechanistic basis for alpha versus beta tubulin recognition within the large TTLL family (3) We showed that TTLL6 reads out the glutamylation status of the microtubule, specifically that on the beta-tubulin tail, establishing cross-talk between the modifications on the alpha and beta-tubulin tails. (3) We have continued our work on identifying mechanism-based inhibitors for TTLL enzymes and characterized them through structural analysis. (4) We characterized the substrate specificity and regulation mechanism of tubulin glycylases TTLL3, 8 and 10 and shed light on a potential mechanism of polyglycine chain length control. (5) We developed methods for the combinatorial modification of microtubules with multiple different modifications (glutamylation, glycylation, acetylation, detyrosination) to be now used to discover how these modifications regulate the activity of microtubule based motors and microtubule associated proteins.(6) We developed a small molecule reporter for glutamylation in live cells and showed its application in diverse cell types. Our work during this year advanced our understanding of the microtubule cytoskeleton by continuing to shed light on the molecular mechanisms of action of the tubulin code, and by developing methodologies and tools for the community that are needed to advance our understanding of tubulin modifications in health and disease.