Molecular basis for the thiol sensitivity of insulin-degrading enzyme
Molecular basis for the thiol sensitivity of insulin-degrading enzyme
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DOI：
10.1073/pnas.0801261105
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发表时间：
2008-07-15
影响因子：
11.1
通讯作者：
Leissring, Malcolm A.
中科院分区：
文献类型：
--
作者：
Neant-Fery, Marie;Garcia-Ordonez, Ruben D.;Leissring, Malcolm A.
胰岛素降解酶(IDE)是一种普遍存在的锌金属蛋白水解酶，能降解多种病理生理相关的多肽，包括胰岛素和淀粉样β蛋白(Aβ)。IDE被共价修饰半胱氨酸残基的化合物不可逆转地抑制，这一机制可能在2型糖尿病(DM2)或阿尔茨海默病(AD)的病因学中起作用。然而，尽管已有研究，但IDE对硫醇烷基化试剂的敏感性的分子基础尚未阐明。为了解决这个问题，我们对IDE中的13个半胱氨酸残基进行了全面的突变分析。我们的分析表明C178、C812和C819是赋予硫醇敏感性的主要残基。C812和C819是距离催化锌原子很远的残基，参与其中提供了功能证据，表明IDE的活性部位由两个独立的结构域组成，这两个结构域只有在紧密的位置上才能发挥作用。结构分析和其他证据预测，C812和C819的烷基化破坏了底物结合，而C178的烷基化干扰了活性部位结构域的对位，并巧妙地重新定位了锌结合残基。出乎意料的是，C590的烷基化被发现显著激活了Aβ的水解，而对胰岛素没有影响，这表明IDE的化学调控既可以是双向的，也可以是高度底物选择性的。我们的发现解决了一个长期存在的关于IDE基本酶学的谜团，对DM2和AD的病因学具有重要的意义。此外，这项工作揭示了IDE不寻常底物选择性的关键细节，这可能有助于开发具有治疗价值的药物或IDE突变体。
Insulin-degrading enzyme (IDE) is a ubiquitous zinc-metalloprotease that hydrolyzes several pathophysiologically relevant peptides, including insulin and the amyloid beta-protein (A beta). IDE is inhibited irreversibly by compounds that covalently modify cysteine residues, a mechanism that could be operative in the etiology of type 2 diabetes mellitus (DM2) or Alzheimer's disease (AD). However, despite prior investigation, the molecular basis underlying the sensitivity of IDE to thiol-alkylating agents has not been elucidated. To address this topic, we conducted a comprehensive mutational analysis of the 13 cysteine residues within IDE. Our analysis implicates C178, C812, and C819 as the principal residues conferring thiol sensitivity. The involvement of C812 and C819, residues quite distant from the catalytic zinc atom, provides functional evidence that the active site of IDE comprises two separate domains that are operational only in close apposition. Structural analysis and other evidence predict that alkylation of C812 and C819 disrupts substrate binding, whereas alkylation of C178 interferes with the apposition of active-site domains and subtly repositions zinc-binding residues. Unexpectedly, alkylation of C590 was found to activate hydrolysis of A beta significantly, while having no effect on insulin, demonstrating that chemical modulation of IDE can be both bidirectional and highly substrate selective. Our findings resolve a long-standing riddle about the basic enzymology of IDE with important implications for the etiology of DM2 and AD. Moreover, this work uncovers key details about the mechanistic basis of the unusual substrate selectivity of IDE that may aid the development of pharmacological agents or IDE mutants with therapeutic value.