Letai A, Coulombe P A, Fuchs E
Howard Hughes Medical Institute, Department of Molecular Genetics, University of Chicago, Illinois 60637.
J Cell Biol. 1992 Mar;116(5):1181-95. doi: 10.1083/jcb.116.5.1181.
Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.
中间丝（IF）组装过程十分显著，因为它似乎是由中间丝蛋白的一级序列自我驱动的。中间丝蛋白家族分子量在40 - 220kd之间，序列多样，但二级结构相似。每个中间丝多肽都有一个由310个氨基酸残基组成的中央α - 螺旋杆状结构域，参与卷曲螺旋二聚体的形成。该杆状结构两端有两段短序列（约10个氨基酸残基），其保守程度高于其余部分，尽管其分子基础尚不清楚。此外，杆状结构被三个位于保守位置但序列不同的短非螺旋连接区隔开。为了研究不同保守的螺旋和非螺旋杆状序列在二聚体、四聚体及更高阶相互作用中所起作用的程度，我们在I型角蛋白的整个杆状结构的残基中引入了脯氨酸突变，并去除了连接区现有的脯氨酸残基。为了进一步探究杆状结构末端的作用，我们在COOH末端附近引入了更细微的突变。我们研究了这些突变对（a）体内中间丝网络形成以及（b）体外10纳米细丝组装的影响。令人惊讶的是，所有位于卷曲螺旋杆状结构域深处的脯氨酸突变对细丝网络形成和10纳米细丝组装的影响都相当小。此外，去除现有的脯氨酸残基在体内没有明显影响，在体外，这些突变体组装成10纳米细丝，有聚集倾向，但外观正常。在杆状结构末端的突变对细丝网络形成和中间丝组装产生了最显著的影响。这些数据表明，杆状结构域中不同序列在细丝网络形成和10纳米细丝组装中的作用并不相同。具体而言，虽然杆状结构内部片段似乎能够耐受α - 螺旋构象的相当大变化，但保守的末端对于形成一种非常特殊的结构似乎至关重要，在这种结构中，即使是很小的扰动也可能导致中间丝稳定性丧失和正常细胞相互作用的破坏。这些发现对角蛋白K5或K14的点突变引起的单纯性大疱性表皮松解症具有重要意义。
