Microtubules, composed of αβ-tubulin heterodimers, are crucial targets for chemotherapeutic agents and possess eight binding sites. Our previous study identified cevipabulin as the only one agent capable of simultaneously binding to two different sites (Vinblastine site and The Seventh site). Binding to The Seventh site by cevipabulin induces tubulin degradation. This study aimed to investigate whether it is binding to the Vinblastine site and The Seventh site exhibited an interactive cellular effect. Surprisingly, we discovered that cevipabulin induced abnormal tubulin protofilaments polymerization, a previously undefined tubulin morphology, and we proved it was an interactive effect of Cevipabulin's binding to both Vinblastine site and The Seventh site. Immunofluorescence and transmission electron microscopy confirmed cevipabulin induced the formation of linear tubulin protofilaments and their subsequent aggregation into irregular tubulin aggregates. Competition binding assays and the αY224G mutation revealed that binding of cevipabulin to both sites was necessary for the tubulin protofilaments polymerization effect. Moreover, we found that co-treatment with a microtubule stabilization agent binding the Vinblastine site and a microtubule destabilization agent binding at the intra-dimer interface of tubulin could also induce similar tubulin protofilaments polymerization. We proposed a mechanism where a microtubule stabilization agent on the Vinblastine site enhances longitudinal interactions between tubulin dimers, while, a microtubule destabilization agent binding at the intra-dimer interface prevents the adoption of a straight conformation of the tubulin dimer and disrupts lateral interactions between tubulins, consequently leading to tubulin protofilaments polymerization. This study reported a new inhibitor-induced-tubulin-morphology-change and would provide insight into tubulin dynamic instability and also guide further study of cevipabulin.

中文翻译：

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西维帕布林通过与长春花碱位点和第七位点结合诱导异常微管蛋白原丝聚合

微管由 αβ-微管蛋白异二聚体组成，是化疗药物的重要靶点，具有八个结合位点。我们之前的研究确定西维巴布林是唯一一种能够同时结合两个不同位点（长春碱位点和第七位点）的药物。西维巴布林与第七位点的结合诱导微管蛋白降解。本研究旨在调查其是否与长春花碱位点结合，并且第七位点表现出相互作用的细胞效应。令人惊讶的是，我们发现西维帕布林诱导异常的微管蛋白原丝聚合，这是一种先前未定义的微管蛋白形态，并且我们证明这是西维帕布林与长春花碱位点和第七位点结合的相互作用效应。免疫荧光和透射电子显微镜证实西维巴布林诱导线性微管蛋白原丝的形成及其随后聚集成不规则的微管蛋白聚集体。竞争结合测定和αY224G突变表明，西维巴布林与两个位点的结合对于微管蛋白原丝聚合作用是必需的。此外，我们发现，与结合长春花碱位点的微管稳定剂和结合在微管蛋白二聚体内界面的微管去稳定剂共同处理也可以诱导类似的微管蛋白原丝聚合。我们提出了一种机制，其中长春花碱位点上的微管稳定剂增强微管蛋白二聚体之间的纵向相互作用，而结合在二聚体内界面的微管去稳定剂阻止微管蛋白二聚体采用直构象并破坏微管蛋白之间的横向相互作用，从而导致微管蛋白原丝聚合。这项研究报告了一种新的抑制剂诱导的微管蛋白形态变化，将提供对微管蛋白动态不稳定性的深入了解，并指导西维巴布林的进一步研究。