RESUMO
Microtubules are organelles that usually occur only in the cytosol. Walss et al. (1999) discovered the ßII isotype of tubulin, complexed with α, in the nuclei of certain cultured cells, in non-microtubule form. When fluorescently labeled tubulins were microinjected into the cells, only αßII appeared in the nucleus, and only after one cycle of nuclear disassembly and reassembly. It appeared as if αßII does not cross the nuclear envelope but is trapped in the nucleus by the re-forming nuclear envelope in whose reassembly ßII may be involved. ßII is present in the cytoplasm and nuclei of many tumor cells. With some exceptions, normal tissues that expressed ßII rarely had ßII in their nuclei. It is possible that ßII is involved in nuclear reassembly and then disappears from the nucleus. Ruksha et al. (2019) observed that patients whose colon cancer cells in the invasive front showed no ßII had a median survival of about 5.5 years, which was more than halved if they had cytosolic ßII and further lessened if they had nuclear ßII, suggesting that the presence and location of ßII in biopsies could be a useful prognostic indicator and also that ßII may be involved in cancer progression. Yeh and Ludueña. (2004) observed that many tumors were surrounded by non-cancerous cells exhibiting cytosolic and nuclear ßII, suggesting a signaling pathway that causes ßII to be synthesized in nearby cells and localized to their nuclei. ßII could be useful in cancer diagnosis, since the presence of ßII in non-cancerous cells could indicate a nearby tumor. Investigation of this pathway might reveal novel targets for chemotherapy. Another possibility would be to combine αßII with CRISPR-Cas9. This complex would likely enter the nucleus of a cancer cell and, if guided to the appropriate gene, might destroy the cancer cell or make it less aggressive; possible targets will be discussed here. The possibilities raised here about the utility of ßII in cancer diagnosis, prognosis, biology and therapy may repay further investigation.
RESUMO
Microtubules, are formed of the protein tubulin, which is a heterodimer of α- and ß-tubulin subunits. Both α- and ß-tubulin exist as numerous isotypes, differing in amino acid sequence and tissue distribution. Among the vertebrate ß isotypes, ßIII has a very narrow distribution, being found primarily in neurons and in advanced cancers. The places in the amino acid sequence where ßIII differs from the other ß isotypes are highly conserved in evolution. ßIII appears to be highly resistant to reactive oxygen species and it forms highly dynamic microtubules. The first property would be very useful in neurons, which have high concentrations of free radicals, and the high dynamicity would aid neurite outgrowth. The same properties make ßIII useful in cancers. Examination of the amino acid sequences indicates a cysteine cluster at positions 124-129 in ßIII (CXXCXC). This occurs in all ßIII isotypes but not in ßI, ßII, or ßIV. ßIII also lacks the easily oxidized C239. Both features could play roles in free radical resistance. Many aggressive tumors over-express ßIII. However, a recent study of breast cancer patients showed that many of them mutated their ßI, ßII, and ßIV at particular places to change the residues to those found at the corresponding sites in ßIII; these are all sites that are highly conserved in vertebrate ßIII. It is possible that these residues are important, not only in the resistance to free radicals, but also in the high dynamicity of ßIII. The cephalopod mollusks are well known to be highly intelligent and can remodel their own brains. Interestingly, several cephalopods contain the cysteine cluster as well as up to 7 of the 17 residues that are highly conserved in vertebrate ßIII, but are not found in ßI, ßII, or ßIV. In short, it is possible that we are looking at a case of convergent evolution, that a ßIII-like isotype may be required for neuronal growth and function and that a structure-function study of the particular residues conserved between vertebrate ßIII and cephalopod tubulin isotypes could greatly increase our understanding of the role of the various tubulin isotypes in neuronal growth and function and could aid in the development of novel anti-tumor drugs.
RESUMO
Microtubules are a major component of the neuronal cytoskeleton. Tubulin, the subunit protein of microtubules, is an α/ß heterodimer. Both α and ß exist as families of isotypes, whose members are encoded by different genes and have different amino acid sequences. The ßII and ßIII isotypes are very prominent in the nervous system. Our previous work has suggested that ßII may play a role in neuronal differentiation, but the role of ßIII in neurons is not well understood. In the work reported here, we examined the roles of the different ß-tubulin isotypes in response to glutamate/glycine treatment, and found that both ßII and ßIII bind to glutathione in the presence of ROS, especially ßIII. In contrast, ßI did not bind to glutathione. Our results suggest that ßII and ßIII, but especially ßIII, may play an important role in the response of neuronal cells to stress. In view of the high levels of ßII and ßIII expressed in the nervous system it is conceivable that these tubulin isotypes may use their sulfhydryl groups to scavenge ROS and protect neuronal cells against oxidative stress.
Assuntos
Glutationa/metabolismo , Células-Tronco Neurais/metabolismo , Neuroblastoma/metabolismo , Tubulina (Proteína)/metabolismo , Diferenciação Celular , Humanos , Estresse OxidativoRESUMO
Tubulin is the target for very widely used anti-tumor drugs, including Vinca alkaloids, taxanes, and epothilones, which are an important component of chemotherapy in breast cancer and other malignancies. Paclitaxel and other tubulin-targeting drugs bind to the ß subunit of tubulin, which is a heterodimer of α and ß subunits. ß-Tubulin exists in the form of multiple isotypes, which are differentially expressed in normal and neoplastic cells and differ in their ability to bind to drugs. Among them, the ßIII isotype is overexpressed in many aggressive and metastatic cancers and may serve as a prognostic marker in certain types of cancer. The underpinning mechanisms accounting for the overexpression of this isotype in cancer cells are unclear. To better understand the role of ß-tubulin isotypes in cancer, we analyzed over 1000 clones from 90 breast cancer patients, sequencing their ß-tubulin isotypes, in search of novel mutations. We have elucidated two putative emerging molecular subgroups of invasive breast cancer, each of which involve mutations in the ßI-, ßIIA-, or ßIVB isotypes of tubulin that increase their structural, and possibly functional, resemblance to the ßIII isotype. A unifying feature of the first of the two subgroups is the mutation of the highly reactive C239 residue of ßI- or ßIVB-tubulin to L239, R239, Y239, or P239, culminating in probable conversion of these isotypes from ROS-sensitive to ROS-resistant species. In the second subgroup, ßI, ßIIA, and ßIVB have up to seven mutations to the corresponding residues in ßIII-tubulin. Given that ßIII-tubulin has emerged as a pro-survival factor, overexpression of this isotype may confer survival advantages to certain cancer cell types. In this mini-review, we bring attention to a novel mechanism by which cancer cells may undergo adaptive mutational changes involving alternate ß-tubulin isotypes to make them acquire some of the pro-survival properties of ßIII-tubulin. These "hybrid" tubulins, combining the sequences and/or properties of two wild-type tubulins (ßIII and either ßI, ßIIA, or ßIVB), are novel isotypes expressed solely in cancer cells and may contribute to the molecular understanding and stratification of invasive breast cancer and provide novel molecular targets for rational drug development.
Assuntos
Neoplasias da Mama/genética , Microtúbulos/metabolismo , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Antineoplásicos , Sequência de Bases , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Galinhas , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Camundongos , Paclitaxel/farmacologia , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/genética , Salmão , Análise de Sequência de DNA/veterinária , Homologia de Sequência de Aminoácidos , Tubulina (Proteína)/metabolismo , Xenopus laevisRESUMO
The subunit protein of microtubules is tubulin, which has been the target for some of the most successful and widely used anti-tumor drugs. Most of the drugs that target tubulin bind to the ß subunit. There are many isotypes of ß-tubulin and their distributions differ among different tissues. The ßIII isotype is over-expressed in many tumors, particularly those that are aggressive, metastatic, and drug resistant. We have previously reported the design and synthesis of a series of compounds to fit the colchicine site on ßIII but not on the other isotypes. In the current study, we tested the toxicity and the anti-tumor activity of one of these compounds, CH-35, on the human breast tumor MDA-MB-231 over-expressing ßIII in a xenogeneic mouse model. We found that CH-35 was as toxic as Taxol® in vivo. Although the ßIII-over-expressing cells developed into very fast-growing tumors, CH-35 was more effective against this tumor than was Taxol. Our results suggest that CH-35 is a promising candidate for future drug development.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Colchicina/análogos & derivados , Tubulina (Proteína)/genética , Animais , Antineoplásicos/toxicidade , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Colchicina/química , Colchicina/farmacologia , Colchicina/toxicidade , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Paclitaxel/farmacologia , Paclitaxel/toxicidade , Testes de ToxicidadeRESUMO
Our previous work identified an intermediate binding site for taxanes in the microtubule nanopore. The goal of this study was to test derivatives of paclitaxel designed to bind to this intermediate site differentially depending on the isotype of ß-tubulin. Since ß-tubulin isotypes have tissue-dependent expression--specifically, the ßIII isotype is very abundant in aggressive tumors and much less common in normal tissues--this is expected to lead to tubulin targeted drugs that are more efficacious and have less side effects. Seven derivatives of paclitaxel were designed and four of these were amenable for synthesis in sufficient purity and yield for further testing in breast cancer model cell lines. None of the derivatives studied were superior to currently used taxanes, however computer simulations provided insights into the activity of the derivatives. Our results suggest that neither binding to the intermediate binding site nor the final binding site is sufficient to explain the activities of the derivative taxanes studied. These findings highlight the need to iteratively improve on the design of taxanes based on their activity in model systems. Knowledge gained on the ability of the engineered drugs to bind to targets and bring about activity in a predictable manner is a step towards personalizing therapies.
Assuntos
Desenho de Fármacos , Microtúbulos/metabolismo , Taxoides/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação , Western Blotting , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Docetaxel , Humanos , Concentração Inibidora 50 , Microtúbulos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Paclitaxel/química , Paclitaxel/farmacologia , Permeabilidade/efeitos dos fármacos , Polimerização/efeitos dos fármacos , Taxoides/química , Termodinâmica , Tubulina (Proteína)/metabolismoRESUMO
Tubulin, the protein subunit of microtubules (MTs), is an α/ß heterodimer. In this chapter, a hypothesis on the evolution of the tubulin molecule is proposed, based in part on recent reports on the structures and functions of different forms of tubulin and its relatives. The concentration is on three main areas. 1) Evolution of the vertebrate ß-tubulin isotypes. In addition to providing a clear idea about the relationships among these isotypes, recent data suggest that tubulin may have functions that do not involve being in a MT, namely, that it can function as an isolated α/ß dimer or as a non-MT polymer. 2) Examination of the entire tubulin superfamily, which includes not only tubulins α, ß, γ, δ, ε, η, and others but also a variety of prokaryotic proteins. The hypothesis is presented that the common ancestor of all these proteins formed a filamentous curving polymer that used the energy of GTP hydrolysis to apply force to nucleic acids and/or membranes and that this common ancestor may have been coeval with the first cells. A variety of chaperones, motors and MT-associated proteins may have coevolved with tubulin and their histories illuminate that of tubulin. The branched, highly negatively charged C-terminal domain present on α- and ß-tubulin appears to be a relatively recent addition to tubulin. 3) The hypothesis is presented that the C-terminal domain may have been of prebiotic origin and that it gradually developed into a protein serving particular metabolic functions whose gene eventually became fused with those of α- and ß-tubulin. Finally, some experiments are proposed that could illuminate the probability of these hypotheses.
Assuntos
Evolução Molecular , Multimerização Proteica/fisiologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Células Eucarióticas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Células Procarióticas/metabolismoRESUMO
The G protein-coupled oestrogen receptor GPER1, also known as GPR30, has been implicated in oestrogen signalling, but the physiological importance of GPER1 is not fully understood. The GPER1 agonist G-1 has become an important tool to assess GPER1-mediated cellular effects. Here, we report that this substance, besides acting via GPER1, affects the microtubule network in endothelial cells. Treatment with G-1 (3 µM) for 24 h reduced DNA synthesis by about 60 % in mouse microvascular endothelial bEnd.3 cells. Treatment with 3 µM G-1 prevented outgrowth of primary endothelial cells from mouse aortic explants embedded in Matrigel. Treatment with G-1 (0.3-3 µM) for 24 h disrupted bEnd.3 cell and HUVEC microtubule structure in a concentration-dependent manner as assessed by laser-scanning confocal immunofluorescence microscopy. G-1-induced (3 µM) disruption of microtubule was observed also after acute (3 and 6 h) treatment and in the presence of the protein synthesis inhibitor cycloheximide. Disruption of microtubules by 3 µM G-1 was observed in aortic smooth muscle cells obtained from both GPER1 knockout and wild-type mice, suggesting that G-1 influences microtubules through a mechanism independent of GPER1. G-1 dose dependently (10-50 µM) stimulated microtubule assembly in vitro. On the other hand, microtubules appeared normal in the presence of 10-50 µM G-1 as determined by electron microscopy. We suggest that G-1-promoted endothelial cell anti-proliferation is due in part to alteration of microtubule organization through a mechanism independent of GPER1. This G-1-promoted mechanism may be used to block unwanted endothelial cell proliferation and angiogenesis such as that observed in, e.g. cancer.
Assuntos
Ciclopentanos/farmacologia , Células Endoteliais/metabolismo , Microtúbulos/efeitos dos fármacos , Quinolinas/farmacologia , Receptores de Estrogênio/agonistas , Receptores Acoplados a Proteínas G/agonistas , Moduladores de Tubulina/farmacologia , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Multimerização Proteica/efeitos dos fármacos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Técnicas de Cultura de Tecidos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
A growing body of evidence suggests that abnormal elements of the cytoskeleton may be associated with the pathophysiology of schizophrenia. Isoforms of a major cytoskeleton protein, ß-tubulin, were recently demonstrated to have distinct roles in neuronal differentiation and cell viability. For these reasons, we tested the hypothesis that there are differences in the expression of ß-tubulin isoforms (ßI-ßIV) in the brain in schizophrenia, using western blot analysis in an elderly group of subjects with this illness and a control group. We found that ßI-tubulin protein expression was decreased in the anterior cingulate cortex and increased in the dorsolateral prefrontal cortex, but not changed in superior temporal gyrus or hippocampus in schizophrenia. Our data supports the growing body of evidence suggesting abnormalities of the cytoskeleton in schizophrenia.
Assuntos
Encéfalo/metabolismo , Esquizofrenia/patologia , Tubulina (Proteína)/metabolismo , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Encéfalo/patologia , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Isoformas de Proteínas/metabolismo , Lobo Temporal/metabolismo , Lobo Temporal/patologiaRESUMO
Tubulin, the subunit protein of microtubules, is an α/ß heterodimer. Both α- and ß-tubulin exist as numerous isotypes, differing in their amino acid sequences and encoded by different genes. The differences are highly conserved in evolution, suggesting that they are functionally significant. Neurons are a potentially very useful system for elucidating this significance, because they are highly differentiated cells and rich in tubulin isotypes. We have examined the distribution of ß-tubulin isotypes in mouse primary cultured cortical neurons from embryonic fetus, newborn pups and adults. Neurons from both embryonic and adult mouse brains express the ßI, ßII, and ßIII isotypes, but apparently not ßIV or ßV. ßI, ßII, and ßIII are found in both cell bodies and neurites. However, the situation is different in newborn mice. Although ßI and ßIII are present in these neurons in both cell bodies and neurites and ßIV is absent, just like in embryonic and adult mice, two striking differences were noted in the neurons from newborn mice. First, ßV is apparently present evanescently in the neurons in both cell bodies and neurites. Interestingly, the ßV was expressed strongly in newborn neurons after one day of culture; expression became much weaker after 3days, and almost disappeared after 5days. Second, the distribution of ßII is different from other isotypes. After newborn mouse neurons were cultured for 3days, ßII started to disappear partly from the cell bodies; this was much more pronounced after five days in culture. Our findings suggest that ßII's major function may involve the neurites and not the cell body. They also raise the possibility that ßV has a unique role in the neurons of newborn mice.
Assuntos
Encéfalo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Contagem de Células , Células Cultivadas , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo , Tubulina (Proteína)/químicaRESUMO
Tubulin, an α/ß heterodimer, has had most of its 3D structure analyzed; however, the carboxy (C)-termini remain elusive. Importantly, the C-termini play critical roles in regulating microtubule structure and function. They are sites of most of the post-translational modifications of tubulin and interaction sites with molecular motors and microtubule-associated proteins. Simulated annealing was used in our molecular dynamics modeling to predict the interactions of the C-terminal tails with the tubulin dimer. We examined differences in their flexibility, interactions with the body of tubulin, and the existence of structural motifs. We found that the α-tubulin tail interacts with the H11 helix of ß-tubulin, and the ß-tubulin tail interacts with the H11 helix of α-tubulin. Tail domains and H10/B9 loops interact with each other and compete for interactions with positively-charged residues of the H11 helix on the neighboring monomer. In a simulation in which α-tubulin's H10/B9 loop switches on sub-nanosecond intervals between interactions with the C-terminal tail of α-tubulin and the H11 helix of ß-tubulin, the intermediate domain of α-tubulin showed more fluctuations compared to those in the other simulations, indicating that tail domains may cause shifts in the position of this domain. This suggests that C-termini may affect the conformation of the tubulin dimer which may explain their essential function in microtubule formation and effects on ligand binding to microtubules. Our modeling also provides evidence for a disordered-helical/helical double-state system of the T3/H3 region of the microtubule, which could be linked to depolymerization following GTP hydrolysis.
Assuntos
Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Simulação por Computador , Humanos , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: A maximum entropy approach is proposed to predict the cytotoxic effects of a panel of colchicine derivatives in several human cancer cell lines. Data was obtained from cytotoxicity assays performed with 21 drug molecules from the same family of colchicine compounds and correlate these results with independent tubulin isoform expression measurements for several cancer cell lines. The maximum entropy method is then used in conjunction with computed relative binding energy values for each of the drug molecules against tubulin isotypes to which these compounds bind with different affinities. RESULTS: We have found by using our analysis that alphabetaI and alphabetaIII tubulin isoforms are the most important isoforms in establishing predictive response of cancer cell sensitivity to colchicine derivatives. However, since alphabetaI tubulin is widely distributed in the human body, targeting it would lead to severe adverse side effects. Consequently, we have identified tubulin isotype alphabetaIII as the most important molecular target for inhibition of microtubule polymerization and hence cancer cell cytotoxicity. Tubulin isotypes alphabetaI and alphabetaII are concluded to be secondary targets. CONCLUSIONS: The benefit of being able to correlate expression levels of specific tubulin isotypes and the resultant cell death effect is that it will enable us to better understand the origin of drug resistance and hence design optimal structures for the elimination of cancer cells. The conclusion of the study described herein identifies tubulin isotype alphabetaIII as a target for optimized chemotherapy drug design.
Assuntos
Antineoplásicos/farmacologia , Colchicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias/tratamento farmacológico , Tubulina (Proteína)/biossíntese , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Moduladores de Tubulina/farmacologiaRESUMO
The differences among the vertebrate beta isotypes of tubulin are highly conserved in evolution, suggesting that they have functional significance. To address this, we have used differentiating neuroblastoma cells as a model system. These cells express the betaI, betaII, and betaIII isotypes. Although there is no difference prior to differentiation, a striking difference is seen after differentiation. Both betaI and betaIII occur in cell bodies and neurites, while betaII occurs mostly in neurites. Knocking down betaI causes a large decrease in cell viability while silencing betaII and betaIII does not. Knocking down betaII causes a large decrease in neurite outgrowth without affecting viability. Knocking down betaIII has little effect on neurite outgrowth and only decreases viability if cells are treated with glutamate and glycine, a combination known to generate free radicals and reactive oxygen species. It appears, therefore, that betaI is required for cell viability, betaII for neurite outgrowth and betaIII for protection against free radicals and reactive oxygen species.
Assuntos
Diferenciação Celular , Neurônios/citologia , Neurônios/metabolismo , Tubulina (Proteína)/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Imunofluorescência , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Glicina/farmacologia , Humanos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Tretinoína/farmacologiaRESUMO
Microtubules are the primary target for many anti-cancer drugs, the majority of which bind specifically to beta-tubulin. The existence of several beta-tubulin isotypes, coupled with their varied expression in normal and cancerous cells provides a platform upon which to construct selective chemotherapeutic agents. We have examined five prevalent human beta-tubulin isotypes and identified the colchicine-binding site as the most promising for drug design based on specificity. Using this binding site as a template, we have designed several colchicine derivatives and computationally probed them for affinity to the beta-tubulin isotypes. These compounds were synthesized and subjected to cytotoxicity assays to determine their effectiveness against several cancerous cell lines. We observed a correlation between computational-binding predictions and experimentally determined IC(50) values, demonstrating the utility of computational screening in the design of more effective colchicine derivatives. The most promising derivative exhibited an IC(50) approximately threefold lower than values previously reported for either colchicine or paclitaxel, demonstrating the utility of computational design and assessment of binding to tubulin.
Assuntos
Antineoplásicos/química , Colchicina/química , Moduladores de Tubulina/química , Sequência de Aminoácidos , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Colchicina/síntese química , Colchicina/toxicidade , Simulação por Computador , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cinética , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Termodinâmica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/toxicidadeRESUMO
Tubulin, the primary subunit of microtubules, is remarkable for the variety of small molecules to which it binds. Many of these are very useful or promising agents in cancer chemotherapy. One of the most useful of these is paclitaxel. The tubulin molecule is itself an alpha/beta heterodimer, both alpha- and beta-tubulin monomers existing as multiple isotypes. Despite the success of paclitaxel as an anticancer drug, resistance often occurs in cancer cells and has been associated with variations in tubulin isotype expression, most notably with the increased expression of betaIII-tubulin. Paclitaxel is thought to reach its binding site on beta-tubulin by diffusion through nanopores in the microtubule wall. It has been suggested that a transitional step in this process may be the binding of paclitaxel to an intermediate site within a nanopore, from which it moves directly to its binding site in the microtubule interior facing the lumen. To test this hypothesis, we have computationally docked paclitaxel within a microtubule nanopore and simulated its passage to the intermediate binding site. Targeted molecular dynamics was then used to test the hypothesis that paclitaxel utilizes the H6/H7 loop as a hinge to move directly from this intermediate binding site to its final position in the luminal binding site. We observed that this motion appears to be stabilized by the formation of a hydrogen bond involving serine 275 in beta-tubulin isotypes I, IIa, IIb, IVa, IVb, V, VII, and VIII. Interestingly, this residue is replaced by alanine in the betaIII and VI isotypes. This observation raises the possibility that the observed isotype difference in paclitaxel binding may be a kinetic effect arising from the isotype difference at this residue. We are now able to suggest derivatives of paclitaxel that may reverse the isotype-specificity or lead to an alternate stabilizing hydrogen-bond interaction with tubulin, thus increasing the rate of passage to the luminal binding site and hopefully offering a therapeutic advantage in paclitaxel resistant cases.
Assuntos
Antineoplásicos/metabolismo , Microtúbulos/metabolismo , Paclitaxel/metabolismo , Antineoplásicos/química , Sítios de Ligação , Modelos Moleculares , Paclitaxel/químicaRESUMO
The C termini of beta-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human betaI and betaIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from betaIII and 16 from betaI can assemble normally. Interestingly, removal of the next residue (Ala(428)) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human betaI and betaIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1-429 of human beta-tubulins make no contribution to microtubule assembly. Ala(428), in the C-terminal sequence motif N-QQYQDA(428), lies at the end of helix H12 of beta-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala(428) may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr(429) plays a more complex role. In the betaI isotype of tubulin, Thr(429) is not at all necessary for assembly; however, in the betaIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of betaIII as well as with the observation that evolutionary conservation is total for Ala(428) and frequent for Thr(429).
Assuntos
Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Células HeLa , Humanos , Microtúbulos/genética , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Tubulina (Proteína)/genéticaRESUMO
Tasidotin, an oncolytic drug in phase II clinical trials, is a peptide analog of the antimitotic depsipeptide dolastatin 15. In tasidotin, the carboxyl-terminal ester group of dolastatin 15 has been replaced by a carboxy-terminal tert-butyl amide. As expected from studies with cemadotin, [(3)H]tasidotin, with the radiolabel in the second proline residue, was hydrolyzed intracellularly, with formation of N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5), a pentapeptide also present in dolastatin 15 and cemadotin. P5 was more active as an inhibitor of tubulin polymerization and less active as a cytotoxic agent than tasidotin, cemadotin, and dolastatin 15. [(3)H]P5 was not the end product of tasidotin metabolism. Large amounts of [(3)H]proline were formed in every cell line studied, with proline ultimately becoming the major radiolabeled product. The putative second product of the hydrolysis of P5, N,N-dimethylvalyl-valyl-N-methylvalyl-proline (P4), had little activity as either an antitubulin or cytotoxic agent. In seven suspension cell lines, the cytotoxicity of tasidotin correlated with total cell uptake of the compound and was probably affected negatively by the extent of degradation of P5 to proline and, presumably, P4. The intracellular enzyme prolyl oligopeptidase probably degrades tasidotin to P5. When CCRF-CEM human leukemia cells were treated with N-benzyloxycarbonylprolylprolinal (BCPP), an inhibitor of prolyl oligopeptidase, there was a 30-fold increase in the IC(50) of tasidotin and a marked increase in intracellular [(3)H]tasidotin. BCPP also caused a 4-fold increase in the IC(50) of P5, so the enzyme probably does not convert P5 to P4. Inhibiting degradation of P5 should have led to a decrease in the IC(50) obtained for P5 in the presence of BCPP.
Assuntos
Antineoplásicos/toxicidade , Depsipeptídeos/toxicidade , Oligopeptídeos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/patologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Depsipeptídeos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Concentração Inibidora 50 , Oligopeptídeos/metabolismo , Oligopeptídeos/toxicidade , Fatores de TempoRESUMO
The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the betaIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin- and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the betaIII isotype of tubulin was generated to evaluate the effect of betaIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the betaIII-tubulin-overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the betaIII-tubulin-overexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms. The ability of the taccalonolides to overcome clinically relevant mechanisms of drug resistance in vitro and in vivo confirms that the taccalonolides represent a valuable addition to the family of microtubule-stabilizing compounds with clinical potential.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Microtúbulos/efeitos dos fármacos , Esteroides/farmacologia , Taxoides/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Camundongos Endogâmicos C3HRESUMO
Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the guanosine-5'-triphosphate composition of the growing and shortening microtubule tips. It is the main objective of this article to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed.
Assuntos
Microtúbulos/química , Microtúbulos/ultraestrutura , Modelos Químicos , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestrutura , Misturas Complexas/química , Simulação por Computador , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Conformação ProteicaRESUMO
Tubulin is the target for some very powerful anti-mitotic and anti-tumor drugs. The betaIII tubulin isotype is found in very few normal tissues, but is often found in tumors, where it has been implicated in resistance to anti-tumor drugs. The betaIII isotype occurs in fish, amphibians, birds and mammals and its unique features are highly conserved in evolution. One of these features is the replacement of cys239 by ser239. Cys239 is unusual in being highly sensitive to oxidation; in fact, oxidation of this residue inhibits microtubule assembly. The betaIII isotype also has a very unusual cys124, where other beta isotypes have ser/ala124. The striking conservation in betaIII of vertebrates strongly suggests that cys124 and ser239 play functional roles. We have prepared the C124S and S239C mutants of betaIII and tested their effects on the functional properties of tubulin. We have found that both the betaIII C124S and betaIII S239C mutants bind colchicine less well than does wild-type alphabetaIII, and also make transfected HeLa cells more resistant to colchicine. However, the double mutant, betaIII C124S/S239C, binds colchicine still less well than do either of the single mutants, but in contrast to the former, the double mutant increases the cells' sensitivity to colchicine. Our results indicate that the roles that these residues play in colchicine binding and microtubule integrity are far more complex than previously imagined and that the specific residues at which betaIII differs from the other isotypes act collectively to keep betaIII in a functional conformation.