Mechanism of tubulin modification enzymes

National Institute Of Neurological Disorders And Stroke

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Abstract

Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. They are subject to highly diverse, abundant and evolutionarily conserved posttranslational modifications. Disruption of tubulin modification levels and patterns leads to cancers, neuropathologies and defective axonal regeneration. An essential aspect of deciphering the tubulin code 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. 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 substrate specificity and catalytic mechanism; (2) to map tubulin modification sites for all modification enzymes; (3) to investigate the biochemical interplay between tubulin modification enzymes and how this gives rise to temporally and spatially regulated modification patterns. 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 will answer fundamental questions about the mechanism and regulation of tubulin modification enzymes. We have continued to make progress towards these goals. Specifically, 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. It is the most abundant tubulin modification in the human brain. We have continued our work on identifying mechanism-based inhibitors for TTLL enzymes and characterizing them through structural analysis. Our inhibitor studies will serve as a springboard for the design of inhibitors for therapeutic intervention in neurodegenerative disorders characterized by tubulin hyper-glutamylation. We also have developed methods for the combinatorial modification of microtubules to be now used to discover how these modifications regulate the activity of microtubule based motors and microtubule associated proteins.