Regulation of Insulin-Regulated Membrane Aminopeptidase Activity by Its C-Terminal Domain
Regulation of Insulin-Regulated Membrane Aminopeptidase Activity by Its C-Terminal Domain
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DOI：
10.1021/bi101893w
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发表时间：
2011-04-05
期刊：
影响因子：
2.9
通讯作者：
Parker, Michael W.
中科院分区：
文献类型：
--
作者：
Ascher, David B.;Cromer, Brett A.;Parker, Michael W.
胰岛素调节的氨肽酶（TRAP）（一种膜结合的锌金属肽酶）的抑制剂的开发是发现用于治疗记忆丧失（例如与阿尔茨海默病相关的记忆丧失）的药物的有希望的方法。然而，在文献中没有关于抑制发生的机制的共识。序列比对，二级结构预测和同源性模型的基础上最近确定的相关金属肽酶的结构表明，胞外区由四个结构域。部分蛋白水解和质谱报告确认了一些域的边界。我们已经产生了纯化的重组片段的人IRAP的基础上，这些数据，并检查其动力学和生化特性。全长细胞外结构组装为二聚体，不同的非重叠片段也二聚化，这表明扩展的二聚体界面。只有含有结构域1和2的重组片段具有氨肽酶活性，并结合放射性标记的六肽抑制剂血管紧张素IV（Ang IV）。然而，缺乏结构域3和4的片段具有降低的活性，尽管它们仍然以与较长片段相同的亲和力结合一系列抑制剂。在血管紧张素IV的存在下，IRAP是抗蛋白水解，这表明显着的构象变化后发生的抑制剂的结合。我们发现，TRAP有第二个锌+结合位点，不与催化区，这是失去后，结合血管紧张素IV。由结构域3和4引起的活性调节与调节进入IRAP活性位点的构象变化一致。
The development of inhibitors of insulin-regulated aminopeptidase (TRAP), a membrane-bound zinc metallopeptidase, is a promising approach for the discovery of drugs for the treatment of memory loss such as that associated with Alzheimer's disease. There is, however, no consensus in the literature about the mechanism by which inhibition occurs. Sequence alignments, secondary structure predictions, and homology models based on the structures of recently determined related metallopeptidases suggest that the extracellular region consists of four domains. Partial proteolysis and mass spectrometry reported here confirm some of the domain boundaries. We have produced purified recombinant fragments of human IRAP on the basis of these data and examined their kinetic and biochemical properties. Full-length extracellular constructs assemble as dimers with different nonoverlapping fragments dimerizing as well, suggesting an extended dimer interface. Only recombinant fragments containing domains 1 and 2 possess aminopeptidase activity and bind the radiolabeled hexapeptide inhibitor, angiotensin IV (Ang IV). However, fragments lacking domains 3 and 4 possess reduced activity, although they still bind a range of inhibitors with the same affinity as longer fragments. In the presence of Ang IV, IRAP is resistant to proteolysis, suggesting significant conformational changes occur upon binding of the inhibitor. We show that TRAP has a second Zn2+ binding site, not associated with the catalytic region, which is lost upon binding Ang IV. Modulation of activity caused by domains 3 and 4 is consistent with a conformational change regulating access to the active site of IRAP.