Binding to microtubules

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). 

Certain drugs bind to microtubules and thus interfere with their assembly or disassembly. These include colchicine (used for treatment of acute gouty arthritis), vinblastine (a vinca alkaloid used for treating certain types of cancer), paclitaxel (Taxol) (effective against ovarian cancer), and griseoflilvin (an antifungal agent). 

In erythrocytes and most other cells, the major structural link of plasma membranes to the cytoskeleton is mediated by interactions between ankyrin and various integral membrane proteins, including Cf/HCOj antiporters, sodium ion pumps and voltage-dependent sodium ion channels. Ankyrin also binds to the =100 nm, rod-shaped, antiparallel a(3 heterodimers of spectrin and thus secures the cytoskeleton to the plasma membrane. Spectrin dimers self-associate to form tetramers and further to form a polygonal network parallel to the plasma membrane (Fig. 2-9D). Neurons contain both spectrin I, also termed erythroid spectrin, and spectrin II, also termed fodrin. Spectrin II is found throughout neurons, including axons, and binds to microtubules, whereas spectrin I occurs only in the soma and dendrites. 

Etoposide (VePesid) is a semisynthetic derivative of podophyllotoxin that is produced in the roots of the American mandrake, or May apple. Unlike podophyllotoxin and vinca alkaloids, etoposide does not bind to microtubules. It forms a complex with the enzyme topoiso-merase II, which results in a single-strand breakage of DNA. It is most lethal to cells in the S- and Gj-phases of the cell cycle. Drug resistance to etoposide is thought to be caused by decreased cellular drug accumulation. 

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. 

Adociasulfates 1-6 (380-385) were isolated from a Haliclona (aka Adocia) sp. from Palau and were all inhibitors of kinesin motor proteins [331]. Adociasulfate 2 (381) had earlier been shown to inhibit the activity of the motor protein kinesin by interference with its binding to microtubules [332], An Adocia sp. from the Great Barrier Reef contained adociasulfates 1 (380), 7 (386) and 8 (387), which inhibit vacuolar H -ATPase [333]. Adociasulfates 5 (384) and 9 (388) were obtained from Adocia aculeata from the Great Barrier Reef [334], The structure of adociasulfate 1 (380) was confirmed by an enantioselective total synthesis [335]. Adociasulfate 10 (389) from Haliclona sp. from Palau also inhibits the kinesin motor proteins [336]. 

Another group of drugs that bind to microtubules are benzimidazole and related compounds. These have been used widely to treat infection by parasitic nematodes in both humans and animals. Unfortunately resistance has developed rapidly. 

Herrmann, H., and Wiche, G. (1987). Plectin and IFAP-300K are homologous proteins binding to microtubule-associated proteins 1 and 2 and to the 240 kilodalton subunit of spectrin./. Biol. Chem. 262, 1320-1325. 

Fig. 5. 3D EM shows how kinesin and tau bind to microtubules. (A) Reconstruction of a microtubule decorated with kinesin heads (ochre). One kinesin head binds per afi-tubulin heterodimer (grey) and, due to its asymmetric form, can be used to distinguish between the subunits. (B) Inside view of a microtubule that was coassembled with gold-labeled tau and decorated with kinesin heads. The kinesin heads can be seen on the outside through the holes between the protofilaments. The labeled repeat motif of tau binds to the inside face of microtubule. The averaged nanogold density (yellow), which is attached to a repeat motif of tau through a linker, can only be seen near the Taxol binding site of -tubulin, but not on the a subunit (Kar et al, 2003a). The ribbon diagram of the refined zinc-sheet structure is also shown for reference (see Figure 3).

Orbit/MAST proteins, also known as CLIP-associated proteins (CLASPs), are involved in the regulation of microtubule dynamics and bind to microtubule plus ends via CLIP115 or CLIP170. Active CLASP suppresses microtubule assembly and axon outgrowth (Lee et al., 2004), whereas activated adenomatous polyposis coli protein (APC see below) promotes microtubule assembly and axon outgrowth. 

The natural products eleutherobin (1), epothilone (23), paditaxel (24), nonataxel (25), and discodermolide (26) (Figure 6) show a similar mode of action. Furthermore, competitive inhibition of paditaxel binding to microtubules by epothilone, eleutherobin, and discodermolide is observed, and so a common pharmacophore and the existence of a common tubulin binding site are therefore strongly suggested [15]. The identification of comparable structural characteristics is complicated, since conformations established by NMR spectroscopy or X-ray structure analysis do not necessarily correspond to the binding conformations [16]-... 

Kinetics of MSA Binding to Microtubules. How Do They Reach Their Site in the Microtubules ... 

Fig. 6 Competition between 3H-paclitaxel and 14C-docetaxel for binding to microtubules. 11.3 pM tubulin was assembled at 37°C in PEDTA, ImM GDP, ImM GTP, 8mM MgCl2, pH6.7 by the addition of paclitaxel and docetaxel at a total concentration of 20 pM, at different molar ratios of paclitaxel to docetaxel. The total concentration of microtubules was 11.0 0.10 pM the concentration of tubulin in supernatants (not polymerized tubulin) varied between ca. 0.4 (in paclitaxel excess) and 0.2 pM (in docetaxel excess). Open circles, 3H-paclitaxel bound per polymerized tubulin dimer solid circles, 14C-docetaxel bound squares, total ligand (paclitaxel plus docetaxel) bound per polymerized tubulin dimer. The solid lines are the best fit to the data, employing a simple competition model of the two ligands for the same site, taken from [35]...

The kinetics of taxane binding to microtubule were subsequently determined taxanes and epothilones [10, 22, 23, 44] bind to microtubules extremely fast (Fig. 8, Table 1). 

Table 1 Kinetic rates of taxane site ligands binding to microtubules (37°C)...

From the kinetics of ligand binding to microtubules we can obtain valuable information about the way paclitaxel microtubule stabilizing agents reach their binding site. They bind to an external binding site located in the pores of the microtubule wall from which they are totally or partially relocated to an inner luminal site. 

In brain tissues, specific isoforms of glycolytic enzymes are also expressed there are specific brain isoforms for PFK (PFK-C), fructose-1,6-bisphosphate aldolase (aldolase C), enolase (enolase y), but not for GAPDH. The isoforms bear the same catalytic functions however, they could be specialized to form different ultrastructural entities. For example, muscle PFK (a dissociable tetrameric form) binds to microtubules and bundle them [94, 95], however, the brain isoenzyme (stable tetramer) does not [96]. 

Li G, Yin H, Kuret J. 2004a. Casein kinase 18 phosphorylates tau and disrupts its binding to microtubules. J Biol Chem 279 15938-15945. 

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