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Bio-organic Studies on Inhibition of Tubulin Assembly by Mitosis Inhibitors

有丝分裂抑制剂抑制微管蛋白组装的生物有机研究

基本信息

批准号：
63470127
负责人：
IWASAKI Shigeo
金额：
$ 4.67万
依托单位：
Institute of Applied Microbiology, The University of
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

This research program was aimed to understand the interaction between tubulin and the mitosis inhibitors, and also to clarify structure and function of tubulin. Previously we have demonstrated with porcine brain tubulin that there exists the rhizoxin/maytansine binding site on tubulin which is different from those for colchicine and for vinblastine.In this research program, we could show the followings: EBI cross-linking to cystein residues on bovine brain beta-tubulin to form beta^s was strongly inhibited by rhizoxin as maytansine, whereas it was not inhibited by colchicine and was only partially inhibited by vinblastine. This coincides with the previous finding obtained with porcine brain tubulin. In a variety of fungi, the resistance to rhizoxin and ansamitocin P-3 (a maytansinoid) did not cross to the resistance to benzimidazoles. This also agrees with the above results, since benzimidazoles are known to bind to the colchicine site. In Aspergillus nidulans, amino acid sequences of beta-tubulins were determined for wild type (rhizoxin/maytansine sensitive) and rhizoxin (and maytansine) resistant strains. The difference between their sequences lied only in the 100th amino acid; the sensitive tubulin had Asn^<100> and the resistant tubulin had Ile^<100>. Interestingly, Schizosacchromyces pombe and Saccharomyces cervisiae had Ile^<100> and Val^<100> respectively in their beta-tubulins, and they are insensitive to rhizoxin. Replacement of these amino acids to Asn^<100> by site-directed mutagenesis conferred rhizoxin sensitivity.On the other hand, the side chain structure of rhizoxin was modified to study the structural requirement for binding to tubulin. A variety of derivatives were prepared and among them, a derivative was chosen for photo-affinity labeling to tubulin. Likewise, another derivative for photo-affinity labeling to rhizoxin/maytansin site was prepared also from maytannsinoid.

本研究旨在了解微管蛋白与有丝分裂抑制物之间的相互作用，并阐明微管蛋白的结构和功能。以前我们已经用猪脑微管蛋白证明了微管蛋白上存在不同于秋水仙碱和长春花碱的根毒素/美坦辛结合部位。在本研究中，我们可以证明：EBI与牛脑β-微管蛋白上的半胱氨酸残基发生交联形成β，S被根霉毒素强烈抑制为美坦辛，而秋水仙碱不抑制它，长春新碱只部分抑制它。这与之前用猪脑微管蛋白得到的发现相吻合。在多种真菌中，对根霉毒素和氨曲霉菌素P-3的抗性与对苯并咪唑的抗性没有交叉。这也与上述结果一致，因为苯并咪唑已知与秋水仙碱结合。在Nidulans曲霉中，测定了野生型(根霉毒素/蛋氨酸敏感)和根霉毒素(和梅坦辛)抗性菌株的β-微管蛋白的氨基酸序列。它们的序列差异仅在于第100个氨基酸，敏感的微管蛋白有ASn^&lt；100&gt；，抗性的微管蛋白有Ile^&lt；100&gt；有趣的是，裂殖酵母和酿酒酵母的β-微管蛋白中分别含有Ile^lt；100&gt；和Val^&lt；100&gt；，它们对根霉毒素不敏感。通过定点突变将这些氨基酸替换为ASN^&lt；100&gt；，从而提高了根霉毒素的敏感性。另一方面，对根霉毒素的侧链结构进行了修饰，以研究其与微管蛋白结合的结构要求。制备了多种衍生物，并从中选择了一种衍生物用于微管蛋白的光亲和标记。同样，另一种用于根霉毒素/美坦辛位点的光亲和标记的衍生物也是从美单宁类化合物中制备的。