A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin.
A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin.
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发表时间：
1999
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通讯作者：
P. Steinert;Y. Chou;V. Prahlad;D. Parry;L. Marekov;K. C. Wu;S. Jang;R. Goldman
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作者：
P. Steinert;Y. Chou;V. Prahlad;D. Parry;L. Marekov;K. C. Wu;S. Jang;R. Goldman
BHK-21成纤维细胞含有III型波形蛋白/结蛋白中间丝(IF)蛋白，在体外实验中，这些蛋白通常与某些高分子量蛋白共分离和共循环。在这里，我们报道了其中一个的纯化，并证明了它实际上是第六型IF蛋白Nestin。巢蛋白在几种成纤维细胞系中表达，但在上皮样细胞系中不表达。我们发现，巢蛋白可以形成同源二聚体和同源四聚体，但如果在体外单独形成则不会形成。在混合物中，巢蛋白优先与纯化的波形蛋白或IV型IF蛋白α-Interexin共组装形成异二聚体卷曲分子。如果巢蛋白的总量不超过约25%，这些分子可以共组装成10 nm。然而，巢蛋白不与I/II型角蛋白IF链二聚化。Nestin蛋白的大部分由一个长的羧基末端的尾巴组成，该尾巴由各种高电荷的多肽重复组成。通过与更大的神经纤维链的类比，我们假设这些序列在IF和/或其他细胞骨架成分之间起到交叉桥或间隔物的作用。通过这种方式，我们认为将少量的巢蛋白直接掺入细胞质III型和IV型IFs的骨架中，为调节它们在细胞中的动态超分子组织和功能提供了一种简单而灵活的方法。
BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein alpha-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.