Microtubule regulation by isotype expression and by small molecules.

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In pursuit of newer microtubule-targeting agents with more favorable spectra of actions as well as more facile chemistry, we have previously developed new methods for synthesizing variants of MT targeting agents. In the report of 2018, we applied our methods to the microtubule-targeting natural product drug dictyostatin, demonstrating that this method allowed synthetic extension of particular sites on precursor molecules to produce new variants of dictyostatin that demonstrated significantly different biological activity. In the report for 2017, we described the development of new analogs of epothilone, a microtubule-stabilizing drug that is already in clinical use. In the current reporting year, we additionally focused on the small molecule PTMs of tubulin, having uncovered a PTM that is new to protein science. The group of PTMs occur at the carboxyl terminal end of both alpha and beta tubulin monomers. One of the earliest discovered PTMs happens on the carboxyl terminus of alpha tubulin. This normally ends with a Tyr residue, but this can be removed enzymatically when the tubulin is polymerized to MT, uncovering the penultimate Glu residue. The Tyr is restored when the MT disassembles. This PTM alters the binding properties of the MT, acting to regulate binding of other proteins to the MT surface. We discovered that there is a previously unrecognized additional regulation step involving addition of a new residue to the Glu instead of the Tyr residue. Surprisingly this residue is taurine. Taurine is a beta-amino acid, known to science for more than 200 years and known to be the most abundant free amino acid in the human body, accounting for about 100 g of a 100 kg human. It has never previous been found covalently incorporated into a protein, even as a PTM. It has been found as a modification of a mitochondrial tRNA, and its function in that role is sufficient to cause clinical disease if absent. The role of the PTM of alpha tubulin with taurine is not known yet, but is under study. Also the tissue and developmental distribution of the taurine-tubulin PTM has not been completely uncovered, though we have preliminary evidence showing that this is not at all uniform through different cell lines and tissue types. Further research will reveal the distribution and function of this unique form of taurine and the significance to MT function of this singular PTM. Often study of the regulation of microtubules and microtubule arrays and the effects of small molecules on those arrays depends on tools of microscopy, both for in vitro assays with purified proteins and especially for assays that address microtubule arrays inside cells, and the effects of small molecules on the biology of cells mediated by effects on the MT arrays. For this reason we have continued our work on new methods that use fluorescence methods and advanced microscope applications to pursue these aims. We have previously published methods to use genetically encoded, FRET-based oxygen sensors based on a myoglobin-mCherry construct to map the intracellular oxygen levels and show how they are affected by varying subatmospheric extracellular oxygen levels, as occur in body tissues. In this reporting period, we extend this approach, by combining mapping of intracellular oxygen levels via the Myoglobin-mCherry sensor with measurement of the cytoplasmic redox level. The redox level is measured noninvasively by using two-photon excitation fluorescence lifetime imaging (FLIM) of free and enzyme-bound NAD(P)H and FAD. This allows a contemporaneous reading of metabolic activity through real-time, non-invasive, cell-by-cell intracellular oxygen level and coenzyme status redox monitoring in living cells. Our next application will be to perturb these cells using the small molecules that are known to alter MT arrays and also known to be therapeutically significant by inducing variation in reactive oxygen generation within the cytoplasm of the exposed cells.