A single amino acid residue defines the difference in ovalicin sensitivity between type I and II methionine aminopeptidases
A single amino acid residue defines the difference in ovalicin sensitivity between type I and II methionine aminopeptidases
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DOI：
10.1074/jbc.m307246200
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发表时间：
2004-03-05
影响因子：
4.8
通讯作者：
Crews, CM
中科院分区：
文献类型：
--
作者：
Brdlik, CM;Crews, CM
TNP-470是第一个进入临床试验的抗血管生成小分子，靶向蛋氨酸氨肽酶-2（MetAP-2），一种切割蛋白质N-末端蛋氨酸的金属蛋白酶。以前，生物化学结合，在体内酵母研究，和结合TNP-470和它的类似物烟曲霉素和ovalicin的人甲硫氨酸氨肽酶-2的结构研究表明，这些化合物表现出MetAP-2的特异性超过其家族成员MetAP-1。为了进一步阐明这种特异性的性质，我们开发了一种基于酵母的筛选人类MetAP-2突变的方法，该突变赋予了卵青霉素抗性。在这三个抗性等位基因中，A362 T出现在大多数克隆中，并且被发现对卵蒜素类抑制剂最具抗性。将人MetAP-2与天然抗卵青霉素的人MetAP-1比对，揭示MetAP-1中的类似残基也是苏氨酸。将该残基突变为丙氨酸导致了对卵圆菌素敏感的MetAP-1等位基因，表明该位置处的丙氨酸对于卵圆菌素的抑制是关键的。这些结果为这类抗血管生成剂对MetAP-2表现出的特异性超过MetAP-1提供了分子解释，并且可以证明可用于开发其他MetAP-2特异性治疗剂。
TNP-470, the first anti-angiogenic small molecule to enter clinical trials, targets methionine aminopeptidase-2 (MetAP-2), a metalloprotease that cleaves the N-terminal methionine of proteins. Previously, biochemical binding, in vivo yeast studies, and structural studies of human methionine aminopeptidase-2 bound to TNP-470 and its analogs fumagillin and ovalicin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1. To further elucidate the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer ovalicin resistance. Of the three resistant alleles, A362T appeared in the majority of clones and was found to be the most resistant to the ovalicin class of inhibitors. Alignment of human MetAP-2 with human MetAP-1, which is naturally ovalicin-resistant, revealed that the analogous residue in MetAP-1 is also a threonine. Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that an alanine at this position is critical for inhibition by ovalicin. These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1 and may prove useful in the development of additional MetAP-2-specific therapeutic agents.