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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剂之间的相互作用，并阐明微管蛋白的结构和功能。之前我们已经用猪脑微管蛋白证明了微管蛋白上存在与秋水仙碱和长春碱不同的Rhizoxin/maytansine结合位点。在本研究计划中，我们可以证明：EBI与牛脑β-微管蛋白上的半胱氨酸残基交联形成β-微管蛋白被根霉素如美登素强烈抑制，而秋水仙碱不抑制，长春碱仅部分抑制。这与先前用猪脑微管蛋白获得的发现一致。在各种真菌中，对根霉素和安丝菌素P-3（美登木素生物碱）的抗性与对苯并咪唑类的抗性没有交叉。这也与上述结果一致，因为已知苯并咪唑与秋水仙碱位点结合。在构巢曲霉中，确定野生型（根霉素/美登素敏感性）和根霉素（和美登素）抗性菌株的β-微管蛋白的氨基酸序列。它们之间的序列差异仅在于第100个氨基酸;敏感微管蛋白具有Asn^<100>，而抗性微管蛋白具有Ile^<100>。有趣的是，粟酒裂殖酵母和酿酒酵母<100><100>在其β-微管蛋白中分别具有Ile^和瓦尔^，并且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^<100>，赋予根霉素敏感性。另一方面，对根霉素的侧链结构进行修饰，以研究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。同样地，也从美登木素生物碱制备用于光亲和标记至根霉素/美登木素位点的另一衍生物。