Neurofilament (NF) assembly; Divergent characteristics of human and rodent NF-L subunits
Neurofilament (NF) assembly; Divergent characteristics of human and rodent NF-L subunits
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DOI：
10.1074/jbc.273.9.5101
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发表时间：
1998-02-27
影响因子：
4.8
通讯作者：
Lazzarini, RA
中科院分区：
文献类型：
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作者：
Carter, J;Gragerov, A;Lazzarini, RA
先前的研究表明，啮齿动物的神经丝（NF）是专门的异聚物，需要NF- l加上NF- m或NF- h才能形成细丝。我们评估了人类NF- l和NF- m的组装能力，发现与大鼠的NF- l不同，人类NF- l能够自组装，然而，人类NF- m不能形成均聚物，需要NF- l的存在才能并入细丝。在酵母“相互作用陷阱”系统中分析了人类NF-L、人类NF-RI和啮齿动物NF-L的棒状结构域或全长亚基。这些研究表明，在那些不形成均聚物的神经丝中，纤维形成的基本障碍是在二聚体形成的水平上。我们预测了哪些氨基酸差异可能是造成大鼠和人类NF-L棒状结构域不同二聚化电位的原因，我们使用位点特异性突变对这些预测进行了测试。有趣的是，设计用于恢复或消除卷曲卷曲倾向的棒状结构域的单个氨基酸变化分别被发现将大鼠NF-L转化为能够均聚的亚基，而将人类NF-L转化为不再能够自组装的蛋白质。我们的研究结果还表明，人类NF-L的L12连接区域（通常被认为具有延长的β -sheet构象）的功能特性与α -螺旋一致，α -螺旋将其前后的七聚体重复序列定位在一个允许盘绕二聚化的方向上。这些研究揭示了人类和啮齿动物NF-L亚基组装特性之间的重要差异，可能表明两种物种神经丝组装的启动事件可能不同。
Previous studies have shown that rodent neurofilaments (NF) are obligate heteropolymers requiring NF-L plus either NF-M or NF-H for filament formation, We have assessed the competence of human NF-L and NF-M to assemble and find that unlike rat NF-L, human NF-L is capable of self-assembly, However, human NF-M cannot form homopolymers and requires the presence of NF-L for incorporation into filaments, To investigate the stage at which filament formation is blocked, the rod domains or the full-length subunits of human NF-L, human NF-RI, and rodent NF-L were analyzed in the yeast "interaction trap" system, These studies demonstrated that the fundamental block to filament formation in those neurofilaments that do not form homopolymers is at the level of dimer formation, Based on theoretical biophysical considerations of the requirements for the formation of coiled-coil structures, we predicted which amino acid differences were likely to be responsible for the differing dimerization potentials of the rat and human NF-L rod domains, We tested these predictions using site-specific mutagenesis, Interestingly, single amino acid changes in the rod domains designed to restore or eliminate the coiled-coil propensity were found respectively to convert rat NF-L into a subunit capable of homopolymerization and human NF-L into a protein that is no longer able to self-assemble, Our results additionally suggest that the functional properties of the L12 linker region of human NF-L, generally thought to assume an extended beta-sheet conformation, are consonant with an alpha-helix that positions the heptad repeats before and after it in an orientation that allows coiled-coil dimerization, These studies reveal an important difference between the assembly properties of the human and rodent NF-L subunits possibly suggesting that the initiating events in neurofilament assembly may differ in the two species.