A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice.
A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice.
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浦肯野细胞变性 (pcd) 小鼠中 Nna1 的结构和功能分析。
DOI：
10.1096/fj.12-205047
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Morgan,JamesI
中科院分区：
文献类型：
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作者：
Wu,Hui-Yuan;Wang,Taiyu;Li,Leyi;Correia,Kristen;Morgan,JamesI
轴切诱导酶 Nna1 定义了 M14 金属羧肽酶的一个亚家族，其突变是浦肯野细胞变性 (pcd) 小鼠的基础。然而，其催化活性、底物特异性和神经变性/轴突再生的关键过程之间的关系尚不完全清楚。在这里，我们使用了一种转基因救援策略，针对 PCD 小鼠中浦肯野细胞中 Nna1 修饰形式的表达，以确定神经元存活的结构-活性关系，并同时表征了纯化重组 Nna1 的酶学特性。 Nna1 亚家族与天冬氨酸酰化酶独特地共享保守的底物决定残基，当突变时，会导致卡纳万病。同源突变（D1007E 和 R1078E）在体内使 Nna1 失活，其催化谷氨酸（E1094A）的突变也是如此，这意味着酸性底物的代谢对于神经元的生存至关重要。与 Nna1 是微管蛋白谷氨酰酶的报道一致，重组 Nna1（但不是催化突变体）从微管蛋白中去除了谷氨酸。重组 Nna1 代谢具有 2 个或多个 C 端谷氨酸（但不是天冬氨酸）残基的合成底物（3 个谷氨酸的 Vmax 比 2 个谷氨酸高约 7 倍，尽管 KM 相似）。催化作用不依赖于 ATP/GTP，突变 Nna1 的 ATP/GTP 结合位点在体内没有影响。 Nna1 是一种通过水解含聚谷氨酸的底物对神经元存活至关重要的单体酶。—Wu, H.-Y., Wang, T., Li, L., Correia, K., Morgan, J. I. 浦肯野细胞变性 (pcd) 小鼠中 Nna1 的结构和功能分析。
The axotomy-inducible enzyme Nna1 defines a subfamily of M14 metallocarboxypeptidases, and its mutation underlies the Purkinje cell degeneration (pcd) mouse. However, the relationship among its catalytic activity, substrate specificities, and the critical processes of neurodegeneration/axon regeneration is incompletely understood. Here we used a transgenic rescue strategy targeting expression of modified forms of Nna1 to Purkinje cells in pcd mice to determine structure-activity relationships for neuronal survival and in parallel characterized the enzymatic properties of purified recombinant Nna1. The Nna1 subfamily uniquely shares conserved substrate-determining residues with aspartoacylase that, when mutated, cause Canavan disease. Homologous mutations (D1007E and R1078E) inactivate Nna1 in vivo, as does mutation of its catalytic glutamate (E1094A), which implies that metabolism of acidic substrates is essential for neuronal survival. Consistent with reports that Nna1 is a tubulin glutamylase, recombinant Nna1—but not the catalytic mutants—removes glutamate from tubulin. Recombinant Nna1 metabolizes synthetic substrates with 2 or more C-terminal glutamate (but not aspartate) residues (Vmax for 3 glutamates is ∼7-fold higher than 2 glutamates although KM is similar). Catalysis is not ATP/GTP dependent, and mutating the ATP/GTP binding site of Nna1 has no effect in vivo. Nna1 is a monomeric enzyme essential for neuronal survival through hydrolysis of polyglutamate-containing substrates.—Wu, H.-Y., Wang, T., Li, L., Correia, K., Morgan, J. I. A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice.