Characterization of the active site and a unique uncompetitive inhibitor of the PPM1-type protein phosphatase PPM1D
Characterization of the active site and a unique uncompetitive inhibitor of the PPM1-type protein phosphatase PPM1D
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DOI：
10.2174/092986608785849236
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发表时间：
2008-09-01
影响因子：
1.6
通讯作者：
Sakaguchi, Kazuyasu
中科院分区：
文献类型：
--
作者：
Chuman, Yoshiro;Yagi, Hiroaki;Sakaguchi, Kazuyasu
蛋白磷酸酶镁依赖性1 - δ (PPM1D)是PPM1（以前称为PP2C）蛋白磷酸酶家族的一员，在DNA损伤时被诱导。PPM1D的过表达被认为通过抑制肿瘤抑制蛋白发挥致癌作用。与其他磷酸酶相比，PPM1D对底物的一级序列具有较高的选择性，但该酶识别底物的机制尚不清楚。在我们目前的研究中，我们希望确定活性中心并进一步阐明PPM1D的底物偏好，并为此在人类PPM1型磷酸酶之间进行序列比对。这一分析结果清楚地表明PPM1D的推定活性位点残基在PPM1家族成员中是高度保守的。利用PPM1D突变体进行的磷酸酶分析进一步确定了金属螯合残基和磷酸结合残基。在使用一系列磷酸化p53肽类似物的动力学分析中，将酸性残基引入去磷酸化位点两侧的区域增强了它们与PPM1D的亲和力。对PPM1D的同源性建模还发现，PPM1D含有两个特征环，一个富pro -残基环位于活性位点的对面，一个富碱性残基环位于活性位点附近。有趣的是，从酸性p53肽类似物中提取的不可水解的AP4-3E肽以非竞争性的方式非常有效地阻断PPM1D的活性，这表明AP4-3E肽可能是开发新型PPM1D抑制剂的有用先导化合物。
Protein phosphatase magnesium-dependent 1, delta (PPM1D) is a member of the PPM1 (formerly PP2C) protein phosphatase family, and is induced in response to DNA damage. The overexpression of PPM1D is thought to exert oncogenic effects through the inhibition of tumor suppressor proteins. PPM1D shows high selectivity for the primary sequence in its substrates when compared with other phosphatases, but the mechanisms underlying substrate recognition by this enzyme is not clearly known. In our present study we wished to identify the active center and further elucidate the substrate preference of PPM1D, and to this end performed sequence alignments among the human PPM1 type phosphatases. The results of this analysis clearly showed that the putative active site residues of PPM1D are highly conserved among the PPM1 family members. Phosphatase analyses using PPM1D mutants further identified the metal-chelating residues and a phosphate binding residue. In kinetic analyses using a series of phosphorylated p53 peptide analogs, the introduction of acidic residues into the region flanking the sites of dephosphorylation enhanced their affinity with PPM1D. Homology modeling of PPM1D also revealed that PPM1D contains two characteristic loops, a Pro-residue rich loop on the opposite side of the active site and a basic-residue rich loop in the vicinity of the active site in the catalytic domain. Interestingly, nonhydrolyzable AP4-3E peptides derived from the acidic p53 peptide analogs very effectively blocked PPM1D activity in an uncompetitive manner, suggesting that AP4-3E peptides may be useful lead compounds in the development of novel inhibitors of PPM1D.