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 better understanding of how tubulin, MT, and MT arrays function in the biology of the cell and organism, and how small molecules such as drugs actually work on microtubules and microtubule arrays, we have continued to study MT, tubulin, and tubulin-drug interactions at biochemical, biophysical, and structural levels. These studies include analyzing new compounds with possibly more favorable spectra of actions as well as more facile chemistry. In the report of 2021, we reported on our study of colchicine site ligands with a study of the colchicine site on chicken erythrocyte tubulin. This past year, we extended our study of the colchicine binding site by generating and studying new analogs of colchicine, which were designed to inform the mechanism of binding of this ancient drug. By designing a series of compounds locked in particular configurations, we could show the role of ring rotation in the binding of colchicine. This involved development of a new strategy for the synthesis of alpha-methoxytropones and related compounds. These methods were required to synthesize the simplified 2-ring analogs of the 3-ring colchicine with fixed configuration. One of the new analogs is highly stable to epimerization due to the high rotational barrier energy. Study of this and related analogs demonstrate that the ground state dihedral angles of the new compounds differ significantly from colchicine, yet the compounds still bind to the colchicine site and inhibit microtubule polymerization. In another extension of our earlier work with blood cell tubulin, we performed a structural study of the blood cell tubulin compared to brain tubulin. We did this by adding another microtubule destabilizing ligand, cryptophycin. This compound is one of the most potent mitotic poisons known, which acts by destabilizing microtubules and preventing polymerization through binding to a ligand site different from the colchicine site. When equimolar amounts of tubulin and cryptophycin are combined, the binding results in the production of very stable, highly curved, very uniform rings composed of a single protofilament of tubulin dimers arranged head-to tail in a circle: a-b-a-b-a-b..Crptophycin rings are very uniform and this facilitates determining the structure by cryoelectron microscopy. This was done with both blood cell tubulin and brain tubulin, and the structures compared. In addition to presenting the first experimental structure of blood cell tubulin, we noticed that the rings of blood cell- and brain-tubulin were not identical in size, even though both were composed of 8 tubulin dimers. We showed that this size difference was due to a local compaction in the blood cell-tubulin, identical in size to that observed in compaction / expansion that happens in linear microtubules composed of 13 protofilaments. This slight change is increasingly recognized as a new regulator of microtubule function and was thought to be due to lattice interactions in the microtubule wall. But our results demonstrate that the compaction / expansion can occur in a single protofilament, eliminating a required role for lattice interactions, and also demonstrates that a linear arrangement of dimers is not required. Tubulin contains binding sites other than the colchicine site, and binding to these sites can destabilize MT, like colchicine-site ligands and cryptophycin and related compounds, or stabilize MT, like the widely employed cancer chemotherapeutic Taxol. Though Taxol has been in routine clinical use for decades, and shows clear benefit against numerous cancers, the details of how it works in cells is still under study. It is clear that Taxol stabilizes microtubules in cells, but how this affects broader intracellular physiology is not settled. This is especially so during the interphase period of the cell cycle that accounts for more than 90% of the cells' duration. Since mitochondria and central to many aspects of cell physiology, are distributed throughout the cell, and align and move on microtubules, we investigated the effect of low dose Taxol on mitochondrial function in a number of cancer cell lines. Some of these cancer cell lines are oxidative, relying on mitochondrial oxidative phosphorylation for their production of ATP, while others rely on glycolysis for ATP despite having functional mitochondria and abundant oxygen. This reliance on glycolysis is known as the Warburg effect and has been long observed in many tumors. In addition to these cell lines, we also included a cell line whose mitochondrial oxidative phosphorylation is knocked out by deletion of the mitochondrial DNA, rendering them obligate glycolytic (but whose mitochondria are otherwise functional) and also normal cells. We found a notable difference between oxidative and glycolytic cell lines. We found an increase in mitochondrial ROS and cytochrome c release, suppression of ATP production and oxidative phosphorylation, fragmentation of the mitochondrial network, and disruption of mitochondria-microtubule linkage. We find these changes in oxidative, but not glycolytic, cancer cells. Noncancer cells, which are oxidative, do not show these changes.