Microtubule regulation by isotype expression, post translational modification, and by small molecules.

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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. . A major drug binding site on tubulin, the subunit protein of microtubules, is named for its first known ligand, colchicine. This site, and this ligand, have had significant presence in this project for many years. The number of compounds now known to bind to this site is quite large, and many have been investigated for use in various diseases conditions. Rigosertib is a compound that binds to tubulin at the colchicine site. We demonstrated that rigosertib has promise as a therapeutic for pediatric rhabdomyosarcoma, a disease with poor prognosis whose current treatment includes use of vinca alkaloids, drugs that bind to tubulin at a different binding site, and which can show severe toxicity as a side-effect. We show that, while rigosertib can inhibit oncogenic RAS signaling, its cytotoxic activity is due to inhibition of microtubule function, such as causing mitotic arrest in rhabdomyosarcoma cell lines, leading to cell death. 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 the previous reporting period, we extended 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. In previous reports, we have also reported development of probes which are sensitive to oxidative damage to cells via producing fluorescence upon reacting covalently with (mostly) protein carbonyls. These are covalent products of reaction of oxidizing species (Reactive Oxygen Species, or ROS) with cellular proteins. Here we demonstrate the utility of a new fluorogenic carbonyl probe for detection of oxidative damage in living cells and in kidney disease. . In the previous reporting year, we additionally focused on the small molecule post translational modifications (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. We have continued to study this interesting PTM of tubulin by developing an antibody to this modification, which allows us to trace its presence in different cells and in different tissues, as well as in disease states compared to normals.