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Intracellular distribution and regulatory function of protein phosphatases

蛋白磷酸酶的细胞内分布和调节功能

基本信息

批准号：
11480161
负责人：
TAKAI Akira
金额：
$ 8.38万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480161/
关键词：
Protein phosphatases Protein phosphorylation Signal transduction Okadaic acid Microcystins Tautomycin NMR Natural toxins 酵素阻害剤 NMR

项目摘要

Among the naturally occurring inhibitors of type 1 and type 2A protein phosphatases (PP1 and PP2A), tatutomycin (TM), first isolated from the bacterium Streptomyces spiroverticillatus, is unique in that it binds to and inhibits PP1 with higher affinity than it does PP2A . The ratio of the dissociation constant for the PP1-TM interaction to that for the PP2A-TM interaction (the PP1/PP2A ratio) is in the range 0.01-0.03. No specific structural factors responsible for its characteristic affinity to the enzymes have been identified until now.In the present experiments we have evaluated the contributions of the C1-C16 segment of TM to its binding to PP1 and PP2A.According to the current binding model, this relatively hydrophobic segment, which contains a spiroketal motif with two side chains, are supposed to be accommodated in a site which is slightly apart from the catalytic center of the enzymes. We therefore expected that chemical modifications of this segment might yield some derivative … More s retaining a measurable inhibitory activity. We were also particularly interested in the fact that the enantiomeric form of the spiroketal is present in the molecule of OA, which, in contrast to TM, exhibits exceedingly higher affinity to PP2A than to PP1. Since there are numerous examples of stereospecific interaction of a macromolecule with a relatively small ligand, it seemed natural to speculate that the stereochemistry of the spiroketal might be a key factor determining the affinity characteristics of the toxins.For these reasons we have chemically synthesized two TM analogues : TM1 in which the side chains of the spiroketal is removed but its stereochemistry is retained, and TM2 in which the spiroketal motif of TM1 is replaced with its enantiomeric form. We have observed that these derivatives indeed retain considerable inhibitory activities against PP1 and PP2A.The PP1/PP2A ratio, determined by dose-inhibition analyses using the native catalytic subunits of PP1 (PP1C) and PP2A and a recombinant γ isoform of PP1(PP1γ), is in the range 0.2-0.5 with TM1 and 5-10 with TM2. The marked increases in the ratio caused by the derivatization of TM to TM1 and TM2 indicate that the stereochemistry of the spiroketal as well as the presence of its side chains is an important factor for the characteristic affinities of TM to PP1 and PP2A. Less

在1型和2A型蛋白磷酸酶（PP 1和PP 2A）的天然存在的抑制剂中，首先从细菌螺旋轮枝链霉菌（Streptomycesspiroverticillatus）中分离的达图霉素（TM）是独特的，因为它以比PP 2A更高的亲和力结合并抑制PP 1。PP 1-TM相互作用的解离常数与PP 2A-TM相互作用的解离常数之比（PP 1/PP 2A比）在0.01-0.03的范围内。本实验研究了TM的C1-C16片段对TM与PP 1和PP 2A结合的影响，根据目前的结合模型，TM的C1-C16片段是一个相对疏水的片段，含有一个螺缩酮基序和两个侧链，被认为被容纳在稍微远离酶的催化中心的位置。因此，我们预计，对这一片段进行化学修饰可能会产生一些衍生物， ...更多信息保留可测量的抑制活性。我们还特别感兴趣的是，螺缩酮的对映体形式存在于OA分子中，与TM相反，OA对PP 2A的亲和力比对PP 1的亲和力高得多。由于存在大量的大分子与相对小的配体的立体特异性相互作用的实例，因此推测螺缩酮的立体化学可能是决定毒素的亲和特性的关键因素似乎是自然的。出于这些原因，我们化学合成了两种TM类似物：其中螺缩酮的侧链被除去但其立体化学被保留的TM 1，和其中TM 1的螺缩酮基序被其对映体形式取代的TM 2。我们已经观察到这些衍生物确实保留了对PP 1和PP 2A的相当大的抑制活性。通过使用PP 1的天然催化亚基（PP 1C）和PP 2A以及PP 1的重组γ同种型（PP 1 γ）的剂量抑制分析确定的PP 1/PP 2A比率对于TM 1在0.2-0.5范围内，对于TM 2在5-10范围内。由TM衍生为TM 1和TM 2引起的比率的显著增加表明螺缩酮的立体化学以及其侧链的存在是TM对PP 1和PP 2A的特征亲和力的重要因素。少