Fungal Tubulin Research Articles

Combretastatin A-4 and Derivatives: Potential Fungicides Targeting Fungal Tubulin

Combretastatin A-4, first isolated from the African willow tree Combretum caffrum, is a tubulin polymerization inhibitor in medicine. It was first postulated as a potential fungicide targeting fungal tubulin for plant disease control in this study. Combretastatin A-4 and its derivatives were synthesized and tested against Rhizoctonia solani and Pyricularia oryzae. Several compounds have EC50 values similar to or better than that of isoprothiolane, which is widely used for rice disease control. Structure-activity relationship study indicated the the cis configuration and hydroxyl group in combretastatin A-4 are crucial to the antifungal effect. Molecular modeling indicated the binding sites of combretastatin A-4 and carbendazim on fungal tubulin are totally different. The bioactivity of combretastatin A-4 and its derivatives against carbendazim-resistant strains was demonstrated in this study. The results provide a new approach for fungicide discovery and fungicide resistance management.

Molecular evolution and functional divergence of tubulin superfamily in the fungal tree of life.

Microtubules are essential for various cellular activities and β-tubulins are the target of benzimidazole fungicides. However, the evolution and molecular mechanisms driving functional diversification in fungal tubulins are not clear. In this study, we systematically identified tubulin genes from 59 representative fungi across the fungal kingdom. Phylogenetic analysis showed that α-/β-tubulin genes underwent multiple independent duplications and losses in different fungal lineages and formed distinct paralogous/orthologous clades. The last common ancestor of basidiomycetes and ascomycetes likely possessed two paralogs of α-tubulin (α1/α2) and β-tubulin (β1/β2) genes but α2-tubulin genes were lost in basidiomycetes and β2-tubulin genes were lost in most ascomycetes. Molecular evolutionary analysis indicated that α1, α2, and β2-tubulins have been under strong divergent selection and adaptive positive selection. Many positively selected sites are at or adjacent to important functional sites and likely contribute to functional diversification. We further experimentally confirmed functional divergence of two β-tubulins in Fusarium and identified type II variations in FgTub2 responsible for function shifts. In this study, we also identified δ-/ε-/η-tubulins in Chytridiomycetes. Overall, our results illustrated that different evolutionary mechanisms drive functional diversification of α-/β-tubulin genes in different fungal lineages, and residues under positive selection could provide targets for further experimental study.

Synthesis and fungicidal activity of quinolin-6-yloxyacetamides, a novel class of tubulin polymerization inhibitors

A novel class of experimental fungicides has been discovered, which consists of special quinolin-6-yloxyacetamides. They are highly active against important phytopathogens, such as Phytophthora infestans (potato and tomato late blight), Mycosphaerella graminicola (wheat leaf blotch) and Uncinula necator (grape powdery mildew). Their fungicidal activity is due to their ability to inhibit fungal tubulin polymerization, leading to microtubule destabilization. An efficient synthesis route has been worked out, which allows the diverse substitution of four identified key positions across the molecular scaffold.

Synthesis and fungicidal activity of tubulin polymerisation promoters. Part 3: Imidazoles

A novel class of experimental fungicides has been discovered, which consists of special tetrasubstituted imidazoles. They are highly active against important phytopathogens, such as Botrytis cinerea (grey mould), Uncinula necator (grape powdery mildew), Mycosphaerella graminicola (wheat leaf blotch) and Alternaria solani (potato and tomato early blight). Their fungicidal efficacy is due to their ability to promote fungal tubulin polymerization, which leads to a disruption of microtubule dynamics. These imidazoles are five-membered ring analogs of similar substituted triazolopyrimidines and pyridazines with the same mode of action. A concise four-step synthesis route has been used to prepare them from commercially available starting materials.

Three tubulin genes of Trichoderma harzianum: alpha, beta and gamma

Three tubulin genes of Trichoderma harzianum were cloned followed genomic walking procedure. The tubulins showed high degree of amino acid homology with other fungal tubulins and were homologous with each other with 32 to 38% amino acid identity. Three measures for the degree of codon usage bias indicated the presence of bias in all the sequences, suggesting high expression levels in all the genes. Protein structures were modeled to provide the basis for understanding the tubulin's properties and its interactions with microtubule-associated proteins. Potential motifs were also postulated.

Structural and biological characterization of the tubulin interaction with dinitroanilines

Characteristics of the interaction of dinitroaniline compounds with tubulin molecules have an extremely high selectivity: these substances efficiently bind to the tubulins of both plant and protozoan origins and practically do not interact with any animal and fungal tubulins despite a very high similarity between the corresponding sequences. This work summarizes and comprehensively analyzes the specific structural features and mechanisms of these interactions, in particular, the patterns of the structure and arrangement of dinitroaniline binding sites on the surface of different tubulin subunits and tubulins of various origins. Dinitroaniline binding sites are localized to the surface of longitudinal contacts between tubulin subunits and contain diamine amino acid residues (lysine or arginine), which bind the nitrile group of dinitroanilines. The localizations of these sites on the surface of identical subunits of different origins (for example, α-tubulins of plants and protozoans) coincide; however, the location of these binding sites on the surfaces of tubulin α- and β-subunits is different. The characterized sites can also be potential binding sites for other antimicrotubule compounds, in particular, cyanoacrylates.

Carbendazim Inhibits Cancer Cell Proliferation by Suppressing Microtubule Dynamics

Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells, including drug- and multidrug-resistant and p53-deficient cell lines. Because of its promising preclinical anti-tumor activity, it has undergone phase I clinical trials and is under further clinical development. Although it weakly inhibits polymerization of brain microtubules and induces G(2)/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC(50), 10 microM) and half-maximally arrests mitosis at a similar concentration (8 microM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 microM, with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (K(d), 42.8 +/- 4.0 microM). Unlike some benzimidazoles that bind to the colchicine site in tubulin, carbendazim neither competes with colchicine nor competes with vinblastine for binding to brain tubulin. Thus, carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest.

Molecular basis for fungal selectivity of novel antimitotic compounds.

Compounds that selectively disrupt fungal mitosis have proven to be effective in controlling agricultural pests, but no specific mitotic inhibitor is available for the treatment of systemic mycoses in mammalian hosts. In an effort to identify novel mitotic inhibitors, we used a cell-based screening strategy that exploited the hypersensitivity of a yeast alpha-tubulin mutant strain to growth inhibition by antimitotic agents. The compounds identified inhibited yeast nuclear division and included one structural class of compounds shown to be fungus specific. MC-305904 and structural analogs inhibited fungal cell mitosis and inhibited the in vitro polymerization of fungal tubulin but did not block mammalian cell microtubule function or mammalian tubulin polymerization. Extensive analysis of yeast mutations that specifically alter sensitivity to MC-305904 structural analogs suggested that compounds in the series bind to a site on fungal beta-tubulin near amino acid 198. Features of the proposed binding site explain the observed fungal tubulin specificity of the series and are consistent with structure-activity relationships among a library of related compounds.

A lineage-restricted and divergent beta-tubulin isoform is essential for the biogenesis, structure and function of blood platelets.

Mammalian megakaryocytes release blood platelets through a remarkable process of cytoplasmic fragmentation and de novo assembly of a marginal microtubule band. Cell-specific components of this process include the divergent beta-tubulin isoform beta1 that is expressed exclusively, and is the predominant isoform, in platelets and megakaryocytes. The functional significance of this restricted expression, and indeed of the surprisingly large repertoire of metazoan tubulin genes, is unclear. Fungal tubulin isoforms appear to be functionally redundant, and all mammalian beta-tubulins can assemble in a variety of microtubules, whereas selected fly and worm beta-tubulins are essential in spermatogenesis and neurogenesis. To address the essential role of beta1-tubulin in its natural context, we generated mice with targeted gene disruption. beta1-tubulin(-/-) mice have thrombocytopenia resulting from a defect in generating proplatelets, the immediate precursors of blood platelets. Circulating platelets lack the characteristic discoid shape and have defective marginal bands with reduced microtubule coilings. beta1-tubulin(-/-) mice also have a prolonged bleeding time, and their platelets show an attenuated response to thrombin. Two alternative tubulin isoforms, beta2 and beta5, are overexpressed, and the total beta-tubulin content of beta1-tubulin(-/-) megakaryocytes is normal. However, these isoforms assemble much less efficiently into platelet microtubules and are thus unable to compensate completely for the absence of beta1-tubulin. This is the first genetic study to address the essential functions of a mammalian tubulin isoform in vivo. The results establish a specialized role for beta1-tubulin in platelet synthesis, structure, and function.

Microtubules of the mycorrhizal fungus Glomus intraradices in symbiosis with tomato roots

In this study the presence and orientation of fungal microtubules were recorded in arbuscular mycorrhizal symbiosis for the first time. Visualization of the fungal microtubules was achieved by using a protocol specifically labelling only fungal tubulins. Microtubules of external mycelium, intraradical hyphae, arbuscules, and vesicles of the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith were examined when in symbiosis with tomato (Lycopersicon esculentum Mill.). Microtubules were organized as bundles in both external and intraradical hyphae. The bundles of microtubules extended directly from intraradical hyphae into the arbuscules, where the microtubules remained as bundles in the larger hyphae. In the fine fungal branches of the arbuscules, microtubules were seen as thinner filaments. Fungal microtubules were seen to connect the intraradical hyphae and arbuscules. In addition, microtubules of adjacent arbuscules could continue directly from one arbuscule to another. Microtubules reached to the basal cone of each vesicle, but the live vesicles, containing many nuclei, seemed devoid of any microtubular labelling.Key words: cytoskeleton, endomycorrhiza, filamentous fungi, tomato, tubulin, Zygomycota.

Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi.

Microsporidia are obligate intracellular parasites that were thought to be an ancient eukaryotic lineage based on molecular phylogenies using ribosomal RNA and translation elongation factors. However, this ancient origin of microsporidia has been contested recently, as several other molecular phylogenies suggest that microsporidia are closely related to fungi. Most of the protein trees that place microsporidia with fungi are not well sampled, however, and it is impossible to resolve whether microsporidia evolved from a fungus or from a protistan relative of fungi. We have sequenced beta-tubulins from 3 microsporidia, 4 chytrid fungi, and 12 zygomycete fungi, expanding the representation of beta-tubulin to include all four fungal divisions and a wide diversity of microsporidia. In phylogenetic trees including these new sequences, the overall topology of the fungal beta-tubulins generally matched the expected relationships among the four fungal divisions, although the zygomycetes were polyphyletic in some analyses. The microsporidia consistently fell within this fungal diversification, and not as a sister group to fungi. Overall, beta-tubulin phylogeny suggests that microsporidia evolved from a fungus sometime after the divergence of chytrids. We also found that chytrid alpha- and beta-tubulins are much less divergent than are tubulins from other fungi or microsporidia. In trees in which the only fungal representatives were the chytrids, microsporidia still branched with fungi (i.e., with chytrids), suggesting that the affiliation between microsporidian and fungal tubulins is not an artifact of long-branch attraction.

Tubulin and actin protein patterns in Scots pine (Pinus sylvestris) roots and developing ectomycorrhiza with Suillus bovinus

The role of tubulin and actin in the development of Scots pine (Pinus sylvestris) roots and in the formation of the ectomycorrhiza with the basidiomycete Suillus bovinus was studied by immunoblotting of 2D-gels with anti-tubulin and anti-actin antibodies. In the short roots the α-tubulin pattern was different from that in the other root types due to the more acidic pI of the two α-tubulins. During the formation of the ectomycorrhiza, two new α-tubulins were detected in the acidic α-tubulin cluster. No such variation occurred in the plant β-tubulin patterns. The fungal tubulins dominated in the ectomycorrhiza, but no changes in tubulin polypeptide patterns from those in the S. bovinus mycelium were observed. Contrary to the tubulins, plant actin dominated in the mycorrhiza. The specific α-tubulin patterns of uninfected and infected short roots indicate that α-tubulin is involved in the morphogenesis of Pinus sylvestris short roots. The high level of plant actin at early stage of the mycorrhiza formation suggests a significant role of this protein in the interaction between plant cells and fungal hyphae.

Regulation and evolution of the single alpha‐tubulin gene of the ciliate Tetrahymena thermophila

The single alpha-tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha-tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha-tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha-tubulins. The single alpha-tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha-tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha-tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha-tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha-tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha-tubulin message amounts via message decay. However the complex control of alpha-tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates.

Binding selectivity of rhizoxin, phomopsin A, vinblastine, and ansamitocin P-3 to fungal tubulins: Differential interactions of these antimitotic agents with brain and fungal tubulins

The binding of four potent antimitotic agents, rhizoxin (RZX), phomopsin A (PMS-A), ansamitocin P-3 (ASMP-3), and vinblastine (VLB), to tubulins from RZX-sensitive and -resistant strains of Aspergillus nidulans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae was investigated. Mycelial extracts to which RZX could bind contained β-tubulin with Asn as the 100th amino acid residue (Asn-100) in all cases, and those without affinity for RZX contained β-tubulins with either Ile-100 or Val-100. Though PMS-A shares the same binding site as RZX and ASMP-3 on porcine brain tubulin (Asn-100), only ASMP-3 bound Asn-100 fungal tubulins in a competitive manner with respect to RZX. PMS-A and VLB, which strongly bind to porcine brain tubulin, did not bind to any of the fungal mycelial extracts examined. The results indicate differential interactions of these antimitotic agents with brain and fungal tubulins.

Interaction of thiabendazole with fungal tubulin

Thiabendazole, 2-(4′-thiazolyl)benzimidazole, at 80 μM completely inhibits mitosis in hyphae of Aspergillus nidulans, growing in liquid culture. DNA and RNA synthesis and mycelial growth are only partially inhibited at this concentration. Binding studies with cell-free mycelial extracts from Penicillium expansum showed that thiabendazole competitively inhibits [ 14C]carbendazim binding to tubulin, which suggests that the antimitotic activity of thiabendazole is based on interference with microtubule assembly. Tubulin from a thiabendazole-resistant and carbendazim-highly sensitive mutant of P. expansum has a lower affinity to thiabendazole and a higher affinity to carbendazim than tubulin from a wild-type strain. This indicates that in this mutant the structure of the binding site is affected. The data presented suggest that several sites of both the tubulin and ligand molecule are involved in the binding of benzimidazole compounds to fungal tubulin.

Differential binding of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans.

The antimitotic compound methyl benzimidazol-2-yl carbamate (MBC) formed a complex in vitro with a protein present in mycelial extracts of fungi. The binding protein of Aspergillus nidulans showed a set of properties which is unique for tubulin. Binding occurred rapidly at 4 degrees C and was competitively inhibited by oncodazole and colchicine. Other inhibitors of microtubule function such as podophyllotoxin, vinblastine sulfate, melatonin, and griseofulvin did not interfere with binding of MBC. Electrophoretic analysis of partially purified preparations of the binding protein revealed the presence of proteins with similar mobilities as mammalian tubulin monomers. Hence it is concluded that the binding protein is identical with fungal tubulin. The effect of MBC on mycelial growth of mutant strains of A. nidulans was positively correlated with the affinity of the binding sites for this compound. The apparent binding constant for MBC and tubulin from a wild type was estimated at 4.5 X 10(5), from a resistant strain at 3.7 X 10(4), and from a strain with increased sensitivity to MBC at 1.6 X 10(6) liters/mol. Mutants showing resistance and increased sensitivity to MBC are candidates to have alterations in tubulin structure. Affinity of tubulin for MBC is probably a common mechanism of resistance to this compound in fungi. Low affinity of tubulin for MBC is probably a common mechanism of resistance binding constant of 2.5 X 10(3) liters/mol.