Mary Carmen Roach

Preparation of a monoclonal antibody specific for the class I isotype of β-tubulin: The β isotypes of tubulin differ in their cellular distributions within human tissues

Tubulin, the subunit protein of microtubules, is an alpha/beta heterodimer. In many organisms, both alpha and beta consist of various isotypes. Although the isotypes differ in their tissue distributions, the question of whether the isotypes perform different functions in vivo is unanswered. In mammals, the betaI and betaIV isotypes are quite widespread, and betaII is less so, while betaIII and betaVI have narrow distributions and betaV distribution is unknown. As a tool for localizing the isotypes, we report the preparation of a monoclonal antibody specific for betaI, to add to our previously described monoclonal antibodies specific for betaII, betaIII, and betaIV [Banerjee et al., J. Biol. Chem. 263:3029-3034, 1988; 265:1794-1799, 1990; 267:5625-5630, 1992]. In order to prepare this antibody, we have purified betaI-rich rat thymus tubulin. We have used our battery of antibodies to localize the beta isotypes in four human tissues: oviduct, skin, colon, and pancreas. We have found striking differences in their tissue distributions. There is little or no betaIII in these tissues, except for the columnar epithelial cells of the colon. BetaII is restricted to very few cells, except in the skin, where it is concentrated in the stratum granulosum. BetaI is widespread in all the epithelia. In the skin it is found in the entire stratum malpighii. In the oviduct, betaI is found largely in the nonciliated epithelial cells. In the exocrine pancreas, betaI occurs only in the centroacinar cells and not in the acinar cells; the latter do not stain with any of these antibodies. BetaIV is present at very low levels in skin and pancreas. By contrast, it is prominent in the colon and also in the oviduct, where it occurs in all the epithelial cells, especially in the ciliated cells, with the highest concentrations in the cilia themselves. These results suggest that the regulation of the expression and localization of isotypes in tissues is very complex.

Interaction of ustiloxin a with bovine brain tubulin

Ustiloxin A is a modified peptide derived from false smut balls on rice panicles, caused by the fungus Ustilaginoidea virens; structurally, it resembles phomopsin A. Ustiloxin A is cytotoxic and is an inhibitor of microtubule assembly in vitro. Because of its resemblance to phomopsin A, we examined its interaction with tubulin and compared the results with those obtained with phomopsin A and dolastatin 10, both of which were found previously to have very similar effects. We determined that ustiloxin A inhibited the formation of a particular intra-chain cross-link in beta-tubulin, as do vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, halichondrin B and homohalichondrin B; this is in contrast to colchicine and podophyllotoxin which do not inhibit formation of this cross-link. Ustiloxin A also inhibited the alkylation of tubulin by iodo[14C]acetamide, as do phomopsin A and dolastatin 10; vinblastine was almost as potent as inhibitor of alkylation as ustiloxin A, whereas maytansine, halichondrin B and homohalichondrin B have little or no effect. In addition, ustiloxin A inhibited exposure of hydrophobic areas on the surface of the tubulin molecule. In this respect, ustiloxin A was indistinguishable from phomopsin A but slightly more effective than dolastatin 10 and considerably more effective than vinblastine; this provides a strong contrast to maytansine, rhizoxin, and homohalichondrin B which have no effect on exposure of hydrophobic areas and to halichondrin B which enhances exposure. Lastly, ustiloxin A strongly stabilized the binding of [3H]colchicine to tubulin. The combination of ustiloxin A with cholchicine stabilized tubulin with a half-life of over 8 days, comparable with results obtained with phomopsin A and colchicine. A comparison of the structures of ustiloxin A, phomopsin A and dolastatin 10 raised the possibility that the strong stabilization of the tubulin structure may require a short segment of hydrophobic amino acids such as the modified valine-isoleucine sequence present in all three compounds. The rest of the structure, specifically the large ring of ustiloxin A and phomopsin A, may serve to place this sequence in an appropriate conformation to interact with tubulin.

Interaction of halichondrin B and homohalichondrin B with bovine brain tubulin.

Halichondrin B is a polyether macrolide of marine origin which binds to tubulin and inhibits microtubule assembly in vitro and in vivo. As is the case with phomopsin A and dolastatin 10, halichondrin B is a non-competitive inhibitor of vinblastine binding to tubulin. Analogous to maytansine, which by contrast is a competitive inhibitor of vinblastine binding, halichondrin B has no effect on colchicine binding, which is greatly stabilized by phomopsin A and dolastatin 10, but not by maytansine. We have previously developed assays which allow sensitive discrimination among the interactions of various ligands with tubulin, and examined the effects of ligands on the reactivity of tubulin sulfhydryl groups and the exposure of hydrophobic areas on the surface of the tubulin molecule. To classify the nature of the interaction between halichondrin B and tubulin, in this study we examined the effects of halichondrin B and its closely related analogue, homohalichondrin B, by these assays. We found that: (1) halichondrin B and homohalichondrin B both inhibited formation of an intra-chain cross-link between two sulfhydryl groups in beta-tubulin, as do phomopsin A, dolastatin 10, maytansine, and vinblastine; (2) halichondrin B resembles maytansine in that it had no effect on alkylation of tubulin sulfhydryl groups by iodoacetamide, unlike phomopsin A, dolastatin 10 and vinblastine, all of which inhibit alkylation; (3) halichondrin B differs from other anti-mitotic drugs in that it enhanced exposure of hydrophobic areas on tubulin; (4) homohalichondrin B, like maytansine and in contrast to phomopsin A, dolastatin 10 and vinblastine, had no effect on exposure of hydrophobic areas; and (5) homohalichondrin B, contrary to maytansine, inhibited alkylation of tubulin sulfhydryl groups in the presence of GTP and MgCl2. In their interactions with the tubulin molecule, halichondrin B and homohalichondrin B appear to have unique conformational effects which differ from those of other drugs and also from the effects of each other as well.

Contrasting effects of maytansine and vinblastine on the alkylation of tubulin sulfhydryls

Abstract The ansa macrolide maytansine is a competitive inhibitor of vinblastine for binding to tubulin. Both drugs are potent inhibitors of microtubule assembly in vitro but maytansine, unlike vinblastine, is unable to induce tubulin aggregation or to stabilize colchicine binding. In this study, the effects of maytansine and vinblastine on the accessibility of tubulins sulfhydryl groups were compared. It was found that 10 μ m vinblastine inhibited the reaction of bovine brain tubulin with [14C]iodoacetamide by 45%. In contrast, maytansine, even up to 100 μ m , had no effect on the reaction. However, when the two drugs were tested in combination, maytansine was a potent inhibitor of vinblastines effect, consistent with the two drugs competing for the same or overlapping sites, but suggesting that the nature of the binding was different. In contrast, maytansine did not affect the suppression of alkylation induced by colchicine and podophylotoxin, consistent with these drugs binding to different sites. Maytansine and vinblastine were each able to increase the formation of β ∗ by the bifunctional reagent, N,N′-ethylenebis-(iodoacetamide); β ∗ is the designation for an electrophoretically faster migrating form of β-tubulin which apparently contains an intrachain crosslink. Thus, in at least the portion of the tubulin molecule involved in β ∗ formation, the two drugs have similar effects. Since maytansine does not appear to suppress any competing alkylation reactions, it is possible that the enhancement of β ∗ formation represents a genuine conformational effect. Since the sulfhydryl groups of tubulin may be involved in regulating microtubule assembly, it is likely that maytansine and vinblastine differ in the manner in which they inhibit microtubule assembly.

The interaction of phomopsin A with bovine brain tubulin

Phomopsin A is an anti-mitotic compound from the fungus Phomopsis leptostroniformis which is a potent inhibitor of microtubule assembly in vitro; like maytansine, it is known to compete with vinblastine for binding to tubulin (E. Lacey, J. A. Edgar, and C. C. J. Culvenor (1987) Biochem. Pharmacol. 36, 2133-2138). A major difference between the effects of maytansine and vinblastine is that vinblastine is a potent inhibitor of tubulin decay, whereas maytansine has little or no effect on decay. Since phomopsin A is structurally distinct from either maytansine or vinblastine, tubulin decay may be measured by either the time-dependent loss of the ability to bind to [3H]colchicine or the time-dependent increase in the binding of bis(8-anilinonaphthalene 1-sulfonate) (BisANS) to tubulin. By either method, phomopsin A was found to be a much stronger inhibitor of tubulin decay than is vinblastine or any other drug yet tested, and in fact, when decay is measured by the increase of BisANS binding, phomopsin A appears to stop the process entirely. This may prove to be useful in the determination of the higher-order structure of the tubulin molecule.

Interactions of Vinblastine and Maytansine with Tubulin

The Vinca alkaloids, vinblastine and vincristine (FIGURE l), are 9-ringed compounds purified from the Madagascar periwinkle Vinca rosea.’ They bind to tubulin with high affinity and prevent microtubule assembly.2 Clinically, vinblastine is the drug of choice to treat Hodgkin’s disease and vincristine to induce remission of acute lymphocytic Maytansine (FIGURE 1) is a macrocyclic ansa macrolide isolated from African plants of the genera Maytenus and P ~ t t e r l i c k i a . ~ ~ ~ It also binds tightly to tubulin and blocks microtubule assembly.2 Although it has been found to be active against a variety of cancers, maytansine’s toxicity is too high for it to be a useful therapeutic tool. The reason we are considering maytansine and the Vinca alkaloids together in the same article is that, despite their structural dissimilarity, they appear to bind to the same site or sites on the tubulin molecule. Interestingly, other than the fact that they both inhibit microtubule assembly, maytansine’s effects on the tubulin molecule are profoundly different from those of vinblastine.

N,N-Bis(α-iodoacetyl)-2,2′-dithiobis(ethylamine), a reversible crosslinking reagent for protein sulfhydryl groups

Abstract This paper describes the synthesis and analysis of N,N′-bis(α-iodoacetyl)-2-2′-dithiobis(ethylamine) (BIDBE), a bifunctional reagent with the structure ICH2CONH-(CH2)2SS(CH2)2NHCOCH2I. BIDBE is shown to react with the sulfhydryl groups of tubulin and to generate a cleavable crosslink between aldolase subunits. BIDBE may be the only reagent hitherto described which can generate a cleavable crosslink between nonadjacent sulfhydryl groups in proteins.

Interaction of the cyanobacterial thiazoline-containing lipid curacin A with bovine brain tubulin

Curacin A is a thiazoline‐containing lipid from the marine cyanobacterium Lyngbya majuscula. Despite being a potent inhibitor of microtubule assembly and of colchicine binding to tubulin, curacin A bears little or no structural resemblance to colchicine or to any other tubulin ligand. We investigated the interaction of curacin A with bovine brain tubulin using three different approaches. We first examined its effect on the intra‐chain formation of a cross‐link in β‐tubulin by N,N′‐ ethylenebis(iodoacetamide). Formation of this cross‐link, between cys239 and cys354, is blocked by colchicine and its A‐ring analogues as well as by various other inhibitors of colchicine binding; C‐ring analogues do not inhibit its formation. Curacin A strongly inhibited formation of this cross‐link. Second, we examined the effect of curacin A on the time‐dependent exposure of sulfhydryl groups on tubulin as measured by alkylation with iodo[14C]acetamide. Curacin A inhibited this very strongly, more so than either colchicine or podophyllotoxin. Last, we investigated the effect of curacin A on the time‐dependent exposure of hydrophobic areas on the tubulin molecule. We found that curacin A had only a small effect on this process, comparable in magnitude to that of podophyllotoxin. Curacin A thus appears to have an unusual interaction with tubulin. Its binding site on tubulin is likely to overlap with that of the A‐ring of colchicine. Drug Dev. Res. 40:223–229, 1997.

Formation of aminoacyl-tRNA-guanylyl-5′-methylene diphosphonate-elongation factor complex

Abstract A procedure for the preparation of a ternary complex, guanylyl-5′-methylene diphosphonate-EF-Tu-Phe-tRNA is described. This complex is sufficiently stable to persist purification by gel filtration chromatography. Such a complex can bind to ribosomes in the presence of poly U.