Differences in paclitaxel and docetaxel interactions with tubulin detected by mutagenesis of yeast tubulin.
Differences in paclitaxel and docetaxel interactions with tubulin detected by mutagenesis of yeast tubulin.
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DOI：
10.1002/cmdc.200800288
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发表时间：
2008-12
期刊：
影响因子：
3.4
通讯作者：
Himes, Richard H.
中科院分区：
文献类型：
--
作者：
Winefield, Robert D.;Entwistle, Ruth A.;Foland, Travis B.;Lushington, Gerald H.;Himes, Richard H.
紫杉醇和半合成类似物多西紫杉醇（方案）是两种紫杉醇类抗肿瘤药物，用于治疗多种癌症紫杉烷与微管中二聚体蛋白α的β-亚基、β-微管蛋白以1:1的摩尔比结合，导致微管的动态特性降低，导致有丝分裂停止和细胞凋亡死亡紫杉烷还促进微管蛋白组装成微管多西紫杉醇在体外促进哺乳动物脑微管蛋白组装的效果是紫杉醇的2 ~ 3倍，其结合常数比紫杉醇大相同因子我们利用酿酒酵母β-微管蛋白的定点诱变技术研究了微管蛋白中紫杉烷的结合位点。虽然野生型酿酒葡萄球菌微管蛋白不结合紫杉烷，但我们能够通过在β-微管蛋白[5]中制造5个突变来植入紫杉烷结合选择这五个位点进行突变的基本原理是基于哺乳动物脑小管蛋白-紫杉醇复合物的电子晶体结构该结构表明，氨基酸侧链K19、V23、D26、H227和F270在紫杉烷结合中起重要作用。在酿酒酵母β-微管蛋白中，这些位点被不同的氨基酸A19、T23、G26、N227和Y270占据。当我们将这5个残基替换为脑β-微管蛋白中的残基时，酵母微管蛋白能够结合紫杉醇我们目前正在通过系统地逆转原始突变来确定每个残基对紫杉烷结合的相对重要性。作为测量变化影响的筛选，我们正在使用一种基于细胞的测定方法，其中我们检查突变对细胞增殖的影响。为了能够使用基于细胞的试验，我们将突变的β-微管蛋白基因引入到减少多药运输活性的酵母菌株中，以产生对紫杉醇（AD1-8-tax）敏感的菌株在这些研究过程中，我们发现不同菌株对紫杉醇和多西紫杉醇的敏感性存在有趣的差异。两种紫杉烷对菌株生长的影响见表1。字母B和Y分别指在脑和酿酒酵母微管蛋白中发现的五种残基。结果表明，25µM的任一紫杉醇均能完全抑制含有脑β-微管蛋白残基（BBBBB）的菌株的生长，但对含有酵母残基（YYYYY）的菌株没有影响。当酵母β-微管蛋白的23、26、270位改变为残基时，菌株（BYBBB、BBYBB、BBBBY）对这两种紫杉烷不敏感，表明这些残基对紫杉烷结合的重要性。然而，在第19位（YBBBB）或227位（BBBYB）替换酵母残留物并不影响对紫杉醇的敏感性。
Paclitaxel and a semi-synthetic analogue docetaxel (Scheme) are two taxane anti-tumor agents that are used against a number of cancers.[1] The taxanes bind to the β-subunit of the dimeric protein α, β-tubulin in microtubules in a 1: 1 molar ratio, resulting in a decrease in the dynamic nature of microtubules leading to mitotic arrest and apoptotic cell death.[2] The taxanes also promote the assembly of tubulin into microtubules.[3] Docetaxel is two to three times as effective as paclitaxel in promoting the assembly of mammalian brain tubulin in vitro and has a binding constant that is greater than that of paclitaxel by the same factor.[4] We have been using site-directed mutagenesis of Saccharomyces cerevisiae β-tubulin to examine the taxane binding site in tubulin. Although wild-type S. cerevisiae tubulin does not bind taxanes, we were able to instill taxane binding by making five mutations in β-tubulin.[5] The rationale for choosing the five sites to mutate was based on the electron crystal structure of the mammalian brain tubulin-paclitaxel complex.[6] This structure indicated that the amino acid side chains, K19, V23, D26, H227 and F270, are important in taxane binding. In S. cerevisiae β-tubulin these sites are occupied by different amino acids, A19, T23, G26, N227, and Y270. When we exchanged the five residues for those that occur in brain β-tubulin, yeast tubulin was able to bind paclitaxel.[5] We are currently in the process of determining the relative importance of each residue to taxane binding by systematically reversing our original mutations. As a screen to measure the effects of the changes, we are using a cell-based assay in which we examine effects of the mutations on cell proliferation. To be able to use a cell-based assay we introduced the mutated β-tubulin gene into a yeast strain that has diminished multidrug transport activity [7] to produce a strain that is sensitive to paclitaxel (AD1-8-tax).[8] In the course of these studies we found interesting differences in the sensitivity of the various strains to paclitaxel and docetaxel.The effect of the two taxanes on the growth of the strains is presented in Table 1. The letters B and Y refer to the five residues found in brain and S. cerevisiae tubulin, respectively. The data show that 25 µM of either taxane completely inhibited growth of the strain with brain β-tubulin residues at the five positions (BBBBB), but had no effect on the strain with yeast residues (YYYYY). When positions 23, 26, and 270 were changed back to the residues in yeast β-tubulin, the strains (BYBBB, BBYBB, BBBBY) were insensitive to both taxanes, indicating the importance of these residues to taxane binding. However, substituting a yeast residue at position 19 (YBBBB) or 227 (BBBYB) did not affect the sensitivity to paclitaxel,
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影响因子：
64.8
作者：
SCHIFF, PB;FANT, J;HORWITZ, SB
通讯作者：
HORWITZ, SB
DOI：
10.1073/pnas.0403459101
发表时间：
2004-07-06
影响因子：
11.1
作者：
Ganesh, T;Guza, RC;Kingston, DGI
通讯作者：
Kingston, DGI
DOI：
10.1073/pnas.051309398
发表时间：
2001-04-24
影响因子：
11.1
作者：
Snyder, JP;Nettles, JH;Nogales, E
通讯作者：
Nogales, E
影响因子：
2.1
作者：
GASTEIGER, J;MARSILI, M
通讯作者：
MARSILI, M
影响因子：
15
作者：
Paik, Younkee;Yang, Chao;Kingston, David G. I.
通讯作者：
Kingston, David G. I.