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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

在天然存在的1型和2A型蛋白磷酸酶（PP1和PP2A）抑制剂中，塔图霉素（tatutomycin， TM）的独特之处是，它首先从螺旋体链霉菌（Streptomyces spiroverticillatus）中分离出来，与PP2A相比，它能以更高的亲和力结合并抑制PP1。PP1- tm相互作用的解离常数与PP2A- tm相互作用的解离常数之比（PP1/PP2A比值）在0.01-0.03之间。到目前为止，还没有确定其对酶的特性亲和力的具体结构因素。在本实验中，我们评估了TM的C1-C16片段对其与PP1和PP2A结合的贡献。根据目前的结合模型，这个相对疏水的片段，包含一个带有两个侧链的螺旋基序，应该被容纳在一个稍微远离酶的催化中心的位置。因此，我们预计对该片段进行化学修饰可能会产生一些衍生物，以保持可测量的抑制活性。我们还特别感兴趣的是，螺旋酮的对映体形式存在于OA分子中，与TM相反，OA对PP2A的亲和力比PP1高得多。由于有许多大分子与相对较小的配体的立体特异性相互作用的例子，因此似乎很自然地推测螺旋体的立体化学可能是决定毒素亲和力特征的关键因素。由于这些原因，我们化学合成了两种TM类似物：TM1中去除了螺旋状结构的侧链，但保留了其立体化学结构；TM2中TM1的螺旋状基元被其对映体形式所取代。我们观察到这些衍生物确实对PP1和PP2A具有相当大的抑制活性。通过使用PP1的天然催化亚基（PP1C）和PP2A以及PP1的重组γ亚型（PP1γ）进行剂量抑制分析，PP1/PP2A的比值在TM1的0.2-0.5和TM2的5-10范围内。TM与TM1和TM2衍生化导致的比例显著增加表明，螺旋酮的立体化学及其侧链的存在是TM与PP1和PP2A特征亲和的重要因素。少