Tubulin system

A tyrosine ligase present in brain extracts has been shown to add a tyrosine residue specifically to the carboxyl terminal end of a-tubulin (236,249). The function of this enzyme in the assembly of the tubulin system is not clear since both tyrosylated and untyrosylated a-tubulin dimers can polymerize. 

The tubulin system plays a key role during mitosis and disturbing its dynamic equilibrium can prevent cell division and induce apoptosis. Microtubules, dynamic protein polymers composed of a-tubulin and P-tubulin heterodimers, are major components of the cytoskeleton with an important role in variety of cellular functions. They have become well-established cellular targets for... 

Certain proteins endow cells with unique capabilities for movement. Cell division, muscle contraction, and cell motility represent some of the ways in which cells execute motion. The contractile and motile proteins underlying these motions share a common property they are filamentous or polymerize to form filaments. Examples include actin and myosin, the filamentous proteins forming the contractile systems of cells, and tubulin, the major component of microtubules (the filaments involved in the mitotic spindle of cell division as well as in flagella and cilia). Another class of proteins involved in movement includes dynein and kinesin, so-called motor proteins that drive the movement of vesicles, granules, and organelles along microtubules serving as established cytoskeletal tracks. ... 

In addition, the alkaloid colchicine (from Colchicum autumnale) blocks tubulin polymerization by binding to heterodimeric (3-tubulin between amino acids 239 and 254. Since it inhibits the MT-dependent migration of granulocytes into areas of inflammation and their MT-dependent release of proinflammatory agents, it is used to treat attacks of gout. Its antimitotic effect in the gastrointestinal system induces diarrhoea. Nocodazole competes for the binding site of colchicine and has similar effects on heterodimeric (3-tubulin. 

In favorable systems, the coherent movement of neuro-filaments and microtubule proteins provides strong evidence for the structural hypothesis. Striking evidence was provided by pulse-labeling experiments in which NF proteins moved over periods of weeks as a bell-shaped wave with little or no trailing of NF protein. Similarly, coordinated transport of tubulin and MAPs makes sense only if MTs are being moved, since MAPs do not interact with unpolymerized tubulin [31]. 

There has been an intensive examination of the biochemical properties of microtubule proteins over the past 15 years, and most of the work has focused on proteins derived from neural systems. For convenience, we will deal with the molecular properties of tubulin first and then collectively consider the so-called microtubule-associated proteins (MAPs). 

The polymer self-assembly theory of Oosawa and Kasai (1962) provides valuable insights into the nature of the nucleation process. The polymerization nucleus is considered to form by the accretion of protomers, but the process is highly cooperative and unfavorable. Indeed, this is strongly suggested by the observation that thousands of actin or tubulin protomers are found in F-actin and microtubule structures if nucleation of self-assembly were readily accomplished and highly favorable, the consequence would be that many more fibers of shorter polymer length would be observed. The Oosawa kinetic theory for nucleation permits one to obtain information about the size of the polymerization nucleus if two basic assumptions can be satisfied in the experimental system. First, the rate of nuclei formation is assumed to be proportional to the loth power of the protomer concentration with io representing the number of protomers required to create the nucleus. Second, the treat-... 

Bergen and Borisy (1980) used the axoneme-hased approach to explore the commitment to treadmilling, and they also found that the efficiency was quite low. An 5 value of 0.07 0.04 was obtained, corresponding to the net addition of 1.6 0.8 tubulin protomers/second. Interestingly, their estimates of the dissociation constants for the two ends were 2.2 0.6 and 3.2 0.6 iiM for the assembly and disassembly ends, respectively. We calculate that this corresponds to only about 0.2 kcal/mol. In a more recent investigation from the same laboratory (Cote and Borisy, 1981), porcine tubulin was found to exhibit an s value of about 0.26, and the rate of the tubulin flux was about 28 protomers/ second. The authors of the latter study suggested that the discrepancy might be accounted for in terms of the need to use MAP-depleted tubulin with the axoneme system to prevent self-nucleation, or in terms of the temperature differences in the two studies. 

The structure-function relationship of the indole-indoline binary alkaloids was relegated to obscurity until the recent achievement of methodologies for their complete syntheses (see Chapter 2, this volume). Our work with C-20 congeners of VBL has established that the complex interactions between this molecule and tubulin or microtubules can be modified by structural alteration. The various, concentration-dependent reactions of VBL with the microtubule system in vitro are sensitive to subtle modifications at a single molecular locus. In addition, these reactions are distinctive on a mechanistic level as seen from the unique activity profiles of most of our C-20 alkyl congeners. At first light, we can look toward the future with secured optimism. 

Vinblastine, vincristine, and structurally related analogs inhibit microtubule polymerization by 50% at concentrations in the range 0.1-1 xM, and the process of tubulin addition to preformed microtubules, at steady state, is comparably sensitive to inhibition by these agents (5). As shown in Table I, the differences in values for inhibition of steady-state tubulin addition by vinblastine, vincristine, vindesine, and vinepidine were relatively small, but the pattern of activity in the tubulin addition system did not parallel that observed when the compounds were evaluated for effects on the proliferation of B16 melanoma cells in vitro. Vinepidine was more than twice as potent as vinblastine as an inhibitor of steady-state tubulin addition but nearly 10-fold less potent than vinblastine as an inhibitor of cell growth (i). 

A wide variety of other biochemical effects has been reported to be associated with treatment of cells with vinblastine, vincristine, and related compounds (S). These effects include inhibition of the biosynthesis of proteins and nucleic acids and of aspects of lipid metabolism it is not clear whether such effects contribute to the therapeutic or toxic actions of vincristine and vinblastine. Vinblastine and vincristine inhibit protein kinase C, an enzyme system that modulates cell growth and differentiation (9). The pharmacological significance of such inhibition has not been established, however, and it must be emphasized that the concentrations of the drugs required to inhibit protein kinase C are several orders of magnitude higher than those required to alter tubulin polymerization phenomena (10). 

The threshold concentration of monomer that must be exceeded for any observable polymer formation in a self-assembling system. In the context of Oosawa s condensation-equilibrium model for protein polymerization, the cooperativity of nucleation and the intrinsic thermodynamic instability of nuclei contribute to the sudden onset of polymer formation as the monomer concentration reaches and exceeds the critical concentration. Condensation-equilibrium processes that exhibit critical concentration behavior in vitro include F-actin formation from G-actin, microtubule self-assembly from tubulin, and fibril formation from amyloid P protein. Critical concentration behavior will also occur in indefinite isodesmic polymerization reactions that involve a stable template. One example is the elongation of microtubules from centrosomes, basal bodies, or axonemes. 

Boekelheide K. 1987. 2,5-Hexanedione alters microtubule assembly. I. Testicular atrophy, not nervous system toxicity, correlates with enhanced tubulin polymerization. Toxicol AppI Pharmacol 88 370-382. 

Uncouples anaerobic oxidative phosphorylation in tapeworm mitochondria, causing decreased ATP synthesis Binds to tubulin and inhibits polymerization Sensitizes microfilaria, entraps them in reticuloendothelial system Increases membrane permeability, unmasks surface proteins Inhibits fumarate reductase, ATP synthesis binds to tubulin Uncouples oxidative phosphorylation... 

Yet another cytotoxic agent that lacks the glutamate side chain illustrates the breadth of the SAR in this series. It is of note that the three nitrogen atoms in the fused six-membered ring system are disposed so as to form a pyridopyrazine rather than a pyrimidopyrimidine, as in piritrexim. The observation that this agent also inhibits tubulin may play an additional role in its activity. Nucleophylic aromatic... 

Vinblastine is an alkaloid derived from the periwinkle plant Vinca rosea. Its mechanism of action involves inhibition of tubulin polymerization, which disrupts assembly of microtubules, an important part of the cytoskeleton and the mitotic spindle. This inhibitory effect results in mitotic arrest in metaphase, bringing cell division to a halt, which then leads to cell death. Vinblastine and other vinca alkaloids are metabolized by the liver P450 system, and the majority of the drug is excreted in feces via the biliary system. As such, dose modification is required in the setting of liver dysfunction. The main adverse effects are outlined in Table 54-4, and they include nausea and vomiting, bone marrow suppression, and alopecia. This agent is also a potent vesicant, and care must be taken in its administration. It has clinical activity in the treatment of Hodgkin s... 

The mode of action of benzimidazole anthelmintics has proved the subject of much debate. Although they were first thought to inhibit the fumarate-reductase enzyme system of helminths, it now seems likely that their mode of action is the inhibition of tubulin polymerization which leads to a number of degenerative processes. This is consistent with their somewhat slower mode of action than most other anthelmintics which generally cause rapid paralysis of the helminths followed by expulsion. 

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