Characterization of early assembly intermediates of recombinant human keratins
Characterization of early assembly intermediates of recombinant human keratins
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DOI：
10.1006/jsbi.2002.4466
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发表时间：
2002-01-01
影响因子：
3
通讯作者：
Aebi, U
中科院分区：
文献类型：
--
作者：
Herrmann, H;Wedig, T;Aebi, U
中间丝（IF）形成细胞骨架的主要结构元件。在体外分析这些纤维蛋白揭示了非常不同的装配性能的核和细胞质IF蛋白。然而，角蛋白，特别是最大的和最异质性组的细胞质IF蛋白，一直难以分析，由于其快速组装动力学下的近生理条件下用于其他IF蛋白。我们在这里展示了角蛋白，像其他细胞质IF蛋白一样，经历了组装成全宽可溶性复合物的阶段，即，"单位长度长丝"（ULF）。然而，与其他IF蛋白相反，角蛋白ULFs纵向退火成长丝准与它们的形成一致。IF蛋白体外组装成细丝可通过增加组装缓冲液的离子强度和/或降低其pH值来启动。我们现在的文件，23-mer肽从头部结构域的各种IF蛋白可以诱导丝的形成，即使在低盐和高pH值的条件下。这表明，“头”参与的形成和纵向协会的ULF。使用在pH 9下引起可溶性低聚物形成的Tris缓冲方案，表皮角蛋白K5/14形成不太规则的细丝，并且比简单的上皮角蛋白KS/18效率低。然而，在磷酸钠缓冲液（pH 7.5）中，K5/14能够形成长的部分解开的细丝，其在加入20 mM KCl后压缩成延伸的规则细丝。将相同的组装方案应用于突变体K14 R125 H表明，引起严重疾病表型和形态学细丝异常的突变可以在体外以惊人的效率形成长的规则细丝。(C)2002 Elsevier Science（美国）。
The intermediate filaments (IFs) form major structural elements of the cytoskeleton. In vitro analyses of these fibrous proteins reveal very different assembly properties for the nuclear and cytoplasmic IF proteins. However, keratins in particular; the largest and most heterogenous group of cytoplasmic IF proteins, have been difficult to analyze due to their rapid assembly dynamics under the near-physiological conditions used for other IF proteins. We show here that keratins, like other cytoplasmic IF proteins, go through a stage of assembling into full-width soluble complexes, i.e., "unit-length filaments" (ULFs). In contrast to other IF proteins, however, longitudinal annealing of keratin ULFs into long filaments quasi-coincides with their formation. In vitro assembly of IF proteins into filaments can be initiated by an increase of the ionic strength and/or lowering of the pH of the assembly buffer. We now document that 23-mer peptides from the head domains of various IF proteins can induce filament formation even under conditions of low salt and high pH. This suggests that the "heads" are involved in the formation and longitudinal association of the ULFs. Using a Tris-buffering protocol that causes formation of soluble oligomers at pH 9, the epidermal keratins K5/14 form less regular filaments and less efficiently than the simple epithelial keratins KS/18. In sodium phosphate buffers (pH 7.5), however, K5/14 were able to form long partially unraveled filaments which compacted into extended, regular filaments upon addition of 20 mM KCl. Applying the same assembly regimen to mutant K14 R125H demonstrated that mutations causing a severe disease phenotype and morphological filament abnormalities can form long; regular filaments with surprising efficiency an vitro. (C) 2002 Elsevier Science (USA).