Effects of organic acids on tubulin polymerization and associated GTP hydrolysis

Hamel, Ernest; Campo, A A del; Lowe, Michael C.; Waxman, Phyllis G.; Lin, Chii M.

doi:10.1021/bi00532a014

Cited by 50 publications

(47 citation statements)

References 22 publications

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“…BtubA/B assembly was slower with NaCl than with KCl. Organic salts (acetate, glutamate, and Pipes) enhanced polymerization, similar to tubulin (45). In marked contrast with mammalian tubulin (46), BtubA/B polymerizes at low temperatures.…”

Section: Mapping Bacterial Tubulin Similarities and Differences Withmentioning

confidence: 96%

Bacterial Tubulin Distinct Loop Sequences and Primitive Assembly Properties Support Its Origin from a Eukaryotic Tubulin Ancestor

Martín-Galiano

Oliva

Sanz

et al. 2011

Journal of Biological Chemistry

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The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic ␣␤-tubulin but, strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either ␣-or ␤-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide-binding loops are more conserved, and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic cytosolic chaperonin, which is absent from the Prosthecobacter dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than ␣␤-tubulin, forming pairs of protofilaments that coalesce into bundles instead of microtubules, and it lacks the ability to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/ S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like ␤-tubulin. BtubA/B chimera bearing the ␤-tubulin loops M, H1-S2, and S9-S10 in BtubB fold, assemble, and have reduced GTPase activity. However, introduction of the ␣-tubulin loop S9-S10 with its unique eight-residue insertion impaired folding. From the sequence analyses, its primitive assembly features, and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding ␣-and ␤-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution.

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Section: Mapping Bacterial Tubulin Similarities and Differences Withmentioning

confidence: 96%

Bacterial Tubulin Distinct Loop Sequences and Primitive Assembly Properties Support Its Origin from a Eukaryotic Tubulin Ancestor

Martín-Galiano

Oliva

Sanz

et al. 2011

Journal of Biological Chemistry

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“…14,15 In the absence of MAPs, α/β-tubulin heterodimers polymerize only by treatment with high concentrations of glycerol or organic acids such as glutamate. 16 The discovery of numerous compounds from natural sources which display a wide structural diversity and are cytotoxic by perturbation of the dynamic instability of microtubules has attracted much attention within the last two decades. [17][18][19][20] Microtubules have, therefore, become an attractive pharmacological target for anticancer drug discovery.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and structure–activity relationship studies on tryprostatin A, an inhibitor of breast cancer resistance protein

Jain

Zhang

Zhou

et al. 2008

Bioorganic & Medicinal Chemistry

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Tryprostatin A is a potent inhibitor of breast cancer resistance protein, consequently a series of structure-activity studies on the cell cycle inhibitory effects of tryprostatin A analogues as potential antitumor antimitotic agents have been carried out. These analogues were assayed for their growth inhibition properties and their ability to perturb the cell cycle in tsFT210 cells. SAR studies resulted in the identification of the essential structural features required for cytotoxic activity. The absolute configuration L-Tyr-L-pro in the diketopiperazine ring along with the presence of the 6-methoxy substituent on the indole moiety of 1 was shown to be essential for dual inhibition of topoisomerase II and tubulin polymerization. Biological evaluation also indicated the presence of the 2-isoprenyl moiety on the indole scaffold of 1 was essential for potent inhibition of cell proliferation. Substitution of the indole N a -H in 1 with various alkyl or aryl groups, incorporation of various L-amino acids into the diketopiperazine ring in place of L-proline, and substitution of the 6-methoxy group in 1 with other functionality provided active analogues. The nature of the substituents present on the indole N a -H or the indole C-2 position influenced the mechanism of action of these analogues. nalogues 68 (IC 50 = 10 μM) and 67 (IC 50 = 19 μM) were 7-fold and 3.5-fold more potent, respectively than 1 (IC 50 = 68 μM) in the inhibition of the growth of tsFT210 cells. Diastereomer-2 of tryprostatin B 8 was a potent inhibitor of the growth of three human carcinoma cell lines: H520 (IC 50 = 11.9 μM), MCF7 (IC 50 = 17.0 μM) and PC3 (IC 50 = 11.1 μM) and was equipotent with etoposide, a clinically used anticancer agent. Isothiocyanate analogue 71 and 6-azido analogue 72 were as potent as 1 in the tsFT210 cell proliferation and may be useful tools in labeling BCRP.

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“…Tubulin polymerization was followed in Gilford recording spectrophotometers equipped with electronic temperature controllers. A drug-tubulin preincubation (0.24-ml reaction mixture) without GTP was performed for 15 (12). Reaction mixtures contained 1.0 M monosodium glutamate (pH 6.6), 0.1 M glucose 1-phosphate, albumin (0.5 mg/ml), 1.0 mM MgCl2, 1.0 mM GTP, and tubulin, [3H]colchicine, and inhibitors as indicated.…”

mentioning

confidence: 99%

2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.

D’Amato

Lin

Flynn

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

384

313

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A metabolite of estradiol, 2-methoxyestradiol (2ME), inhibits angiogenesis In the chicken embryo chorioallantoic membrane assay. Since 2ME causes mitotic perturbalions, we ea ined Its interactions with tubulin. In our standard 1.0 M glutamate system (plus 1.0 mM MgCl2 at 3rC), superstoichiometric concentrations (relative to tubulin) of 2ME inhibited the nucleation and propagation phases of tubulin assembly but did not affect the reaction extent. Although polymer formed In the presence of 2ME was more cold-stable than control polymer, morphology was little changed. Under suboptimal reaction conditions (0.8 M glutamate/no MgCl2 at 26WC), substoichiometric 2ME totally inhibited polymerization. No other estrogenic compound was as effective as 2ME as an inhibitor of polymerization or of the binding of colchicine to tubulin. Inhibition ofcolchicine binding was competitive (KM,22 aM). Thus, a m an metabolite of estradiol binds to the colchicine site of tubulin and, depending on reaction conditions, either inhibits assembly or seems to be incorporated into a polymer with altered stability properties.There is growing evidence that estrogenic compounds affect cell division and act directly on microtubules by interacting with tubulin. The most extensively studied of these compounds is the synthetic analog diethylstilbestrol (DES; Fig. 1, compound 1) and related agents (1-3). These drugs cause disturbances in mitosis, inhibit microtubule assembly at relatively high concentrations, and inhibit the binding of colchicine to tubulin. Estradiol (compound 2), the major estrogenic hormone of human beings, also causes disturbances ofmitosis in cultured cells (1,4,5). These perturbations include aneuploidy, multinucleation, and mitotic arrest, and estradiol has been reported to inhibit the polymerization of rat brain tubulin (6). Seegers et al. (5) found that 2-methoxyestradiol (2ME; compound 3), a metabolite of both estradiol (7) and the oral contraceptive agent 17-ethynylestradiol (compound 4), was more potent than estradiol in producing mitotic perturbations and proposed that it was 2ME rather than estradiol that caused the observed disturbances. Although 2-methoxyestrogens are extremely weak in binding to cytosol estrogen receptors (8), 2ME is found in blood and urine after sequential hepatic hydroxylation and methylation ofestradiol (7).We recently found that 2ME inhibited angiogenesis in the chicken embryo chorioallantoic membrane assay of Crum et al. (9). Disks of2ME (100 Mg) produced large avascular zones 48 hr after implantation on a 6-day embryo, similar to in vitro results of others (10). In attempting to define the mechanistic basis, we observed that 2ME inhibited microtubule assembly. This finding led us to study the interactions of 2ME with purified tubulin.MATERIALS AND METHODS Materials. Purified bovine brain tubulin and H2-CSA4 were prepared as described (11,12). Monosodium glutamate (2.0 M) was adjusted to pH 6.6 with HCl. 2ME, 2-fluoroestradiol, 2-methoxy-17-ethynylestradiol, 2-methoxyestradiol 3-0-meth...

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Effects of organic acids on tubulin polymerization and associated GTP hydrolysis

Cited by 50 publications

References 22 publications

Bacterial Tubulin Distinct Loop Sequences and Primitive Assembly Properties Support Its Origin from a Eukaryotic Tubulin Ancestor

Bacterial Tubulin Distinct Loop Sequences and Primitive Assembly Properties Support Its Origin from a Eukaryotic Tubulin Ancestor

Synthesis and structure–activity relationship studies on tryprostatin A, an inhibitor of breast cancer resistance protein

2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.

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