Microtubule-associated polymerization

Microtubule-associated proteins bind to microtubules in vivo and subserve a number of functions including the promotion of microtubule assembly and bundling, chemomechanical force generation, and the attachment of microtubules to transport vesicles and organelles (Olmsted, 1986). Tubulin purified from brain tissue by repeated polymerization-depolymerization contains up to 20% MAPs. The latter can be dissociated from tubulin by ion-exchange chromatography. The MAPs from brain can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 

Several pathological self-polymerizing systems have been biophysi-cally characterized sufficiently to permit identification of protein or peptide species that could serve as molecular targets in a structure-activity relationship. These include transthyretin (TTR) [73-76], serum amyloid A protein (SAA) [77], microtubule-associated protein tau [78-80], amylin or islet amyloid polypeptide (IAPP) [81,82], IgG light chain amyloidosis (AL) [83-85], polyglutamine diseases [9,86], a-synuclein [47,48] and the Alzheimer s (3 peptide [87-96]. A variety of A(3 peptide assay systems have been established at Parke-Davis to search for inhibitors of fibril formation that could be therapeutically useful [97]. 

Paclitaxel is an alkaloid ester derived from the Pacific yew (Taxus brevifolia) and the European yew (Taxus baccata). The drug functions as a mitotic spindle poison through high-affinity binding to microtubules with enhancement of tubulin polymerization. This promotion of microtubule assembly by paclitaxel occurs in the absence of microtubule-associated proteins and guanosine triphosphate and results in inhibition of mitosis and cell division. 

The basic biology and pharmacology of Epo B (as the most potent and most widely studied natural epothilone) have been summarized in several previous review articles.As indicated in Section 1.1, the biological effects of the compound are based on its ability to bind to microtubules and alter the intrinsic stabihty and dynamic properties of these supramolecular structures. In cell-free in vitro systems, this is demonstrated by the prevention of Ca - or cold-induced depolymerization of preformed microtubule polymers as well as by the promotion of tubuhn polymerization (to form microtubule-like polymers) in the absence of either microtubule-associated proteins (MAPs) and/or guanosine triphosphate (GTP), at temperatures significantly below 37 °C, and in the presence of The latter... 

Griseofulvin inhibits fungal mitosis, presumably disrupting the mitotic spindle by interacting with polymerized microtubules. Griseofulvin also may bind to a microtubule-associated protein. 

Ohe conclusions suggested by the kinetic data obtained few years ago were that 1) the microtubule polymerization activity increases during brain development. This evolution seems to be related to the differential expxression of the microtubule-associated proteins, such as Tau proteins, differing in size and in polymerization activity 2) thyroid hormones increase the polymerization activity probably because they accelerate the transition fron the inmature to the more active mature Tau forms. However, little was knewn on the mechanism that generates Tau heterogeneity at different stages of brain development, and on the site of action of thyroid hormones. 

Patients with dialysis dementia respond positively to an antibody against the hyperphosphorylated microtubule-associated protein, tau, that accumulates in AD tangles (Guy et al. 1991). The neurofilament protein tubulin contains an acidic tau binding site to which Al might bind to cause tau accumulation. However, not only do dialysis dementia patients characteristically show no tangle formation, but the tangles seen in animals treated with Al are predominantly made up of neurofilament subunits such as tubulin, rather than microtubule-associated protein. A potential role for Al in G-protein-regulated neurofilament polymerization is discussed in Sect. G. 

Tubulin is a dimer composed of similar but not identical polypeptides, a and )8, one of which (a) is phosphorylated. The molecular weight of tubulin is 100,000, with each of the subunits having a molecular weight of 55,000. Tubulin polymerizes, forming a polar, helical surface lattice. Microtubules are stabilized by Mg + and GTP, which also promote the polymerization. Ca " inhibits polymerization and may promote depolymerization (Weisenberg, 1972 Staprans et al., 1975). Brain tubulin is phosphorylated, possibly by cAMP-stimulated protein kinase, which is one of many microtubule-associated proteins. Colchicine blocks polymerization by binding to the growing end of a micro-... 

Dibutyryl cAMP applied to Chinese hamster ovary cells in culture causes an increase in the number of microtubules per unit volume of cytoplasm (Porter et al., 1974). Microtubules end on microtubule organizing centers. A microtubule-associated protein, tau, will facilitate the initiation of tubulin polymerization and its subsequent elongation (Witman et al., 1976). The a chain of tubulin can be acted on by the enzyme tyrosyltubulin ligase, so as to add tyrosine, phenylalanine, or 3,4-dihydroxyphenylalanine to the C-terminus (Deanin and Gordon, 1976). 

Fig. 4. Transmission electron microscopy of MTP reaction mixtures, (a) An opened area is seen in a microtubule polymerized in the presence of an IC concentration (3 x 10 W) of deoxydesethyl VBL (5). (b) Single spirals are formed from MTP and 10 M 5. (c) Spiral aggregates are formed from MTP and 10 M VBL. (d) MTP incubated with an lCs concentration (2 X 10 Af) of epimethyldeoxydesethyl VBL (4) formed spiral aggregates both free in solution (single arrows) and associated with microtubules (double arrows), (e) Greater magnification of MTP incubated with an 1C , concentration (2 x 10 M) of methyldeoxyde-sethyl VBL (3) displays a free spiral (arrow) and spiralized material on the microtubules. Bar, 0.1 p.m.

The cause of the cell cycle specificity of the bisindole alkaloids may be associated with the ability of these compounds to interact with the protein tubulin and thereby inhibit the polymerization (and depolymerization) of microtubules (16,17). In this respect the cellular pharmacology of vinca alkaloids is similar to that of other cytotoxic natural products such as colchicine or podophyllotoxin. On closer inspection, however, Wilson determined that the specific binding site on tubulin occupied by vinblastine or vincristine is chemically distinct from the site occupied by the other natural products (18). Subsequent experiments have determined that the maytansinoids, a class of ansa-macrocycles structurally distinct from the bisindoles, may bind to tubulin at an adjacent (or overlapping) site (19). A partial correlation of the antimitotic activity of these compounds with their tubulin binding properties has been made, but discrepancies in cellular uptake probably preclude any quantitative relationship of these effects (20). 

The principal cytoskeletal proteins in non-muscle cells are actin, tubulin, and the components of intermediate filaments. Actin can exist either as monomers ( G-actin ) or polymerized into 70 A diameter double filament ( F-actin ). Polymerized actin usually is localized at the margins of the cells, linked by other proteins to the cell membrane. In contrast, tubulin forms hollow filaments, approximately 250 A in diameter, that are distributed within a cell in association, generally, with cell organelles. Stabilized microtubule structures are found in the flagella and cilia of eucaryotic cells however, in other instances - examples being the mitotic apparatus and the cytoskeletal elements arising in directed cell locomotion - the microtubules are temporal entities. Intermediate filaments, which are composed of keratin-like proteins, are approximately 100 A thick and form stable structural elements that impart rigidity, for example, to nerve axons and epithelial cells. 

The pathway of exchangeable-site GTP hydrolysis associated with bovine brain microtubule polymerization was... 

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