Structural Features Of Keratin And Related IF

角蛋白及相关IF的结构特征

• 批准号: 6967751
• 负责人: ALASDAIR C. STEVEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6967751
• 关键词: RNA interference; X ray crystallography; cell component structure /function; conformation; crosslink; cytoskeletal proteins; disulfide bond; fibrous protein; gene mutation; intercellular connection; intermediate filaments; keratin; keratinization; keratinocyte; molecular assembly /self assembly; protein protein interaction; protein purification; protein sequence; protein structure function; skin; structural biology; synthetic peptide; tissue /cell culture; vimentin

Intermediate filaments (IF) are the ubiquitous constituents of the cytoskeletons of eukaryotic cells. They consist of five different types, of which the most numerous and complex are the type I and type II keratins that are widely expressed in epithelia. We are interested also in the related IF of other cell types in order to understand their roles in biology. We continued a major study in collaboration with other investigators in Switzerland, Germany and New Zealand to solve the three-dimensional structure of vimentin IF. These IF have been chosen because: (a) they are homopolymeric, and therefore likely to be somewhat simpler to solve; and (b) they have a very high sequence homology with keratin IF, and thus many of the structural principles adduced for vimentin should be applicable to keratin IF. (1) Work was completed on numerous deletion / mutation constructs which cumulatively cover the entire portion of vimentin. Chemical sequencing of these constructs have revealed interactions of the N- and C- terminal 100 amino acids with the coiled-coiled region of the molecule and revealed the role of these regions in the early (monomer to tetramer) stages of the filament assembly. The results were published. A second paper including the full filament growth from early subunits is in preparation. It may also include a refinement of the final, mature IF structure by spectroscopic (EPR) data from a collaborating laboratory (University of California at Davis) leading to completion of this study. (2) A similar project on the role of the head domain of hair keratins in the assembly of hair keratin intermediate filaments was initiated. Thirty two mutant and deletion constructs were created and the proteins have been purified. Initial experiments, with two of the constructs, revealed specific amino acid interactions between the head and rod domains. Work was suspended because of the lack of human resources. (3) Characterization of human epiplakin as a cytolinker: RNAi-mediated epiplakin depletion leads to the disruption of keratin and vimentin IF networks. Epiplakin is a member of the plakin family with multiple copies of the plakin repeat domain (PRD). We studied the subcellular distribution and interactions of human epiplakin by immunostaining, overlay assays, and RNAi knockdown. Epiplakin decorated the keratin IF network and partially that of vimentin. In the binding assays, the repeat unit (PRD plus linker) showed strong binding and preferentially associated with assembled IF over keratin monomers. Epiplakin knock-down revealed disruption of IF networks in simple epithelial but not in epidermal cells. In rescue experiments, the repeat unit was necessary to prevent the collapse of IF networks in transient knock-down; however, it could only partially restore the keratin but not the vimentin IF network in stably knocked-down HeLa cells. We infer that epiplakin is a versatile cytolinker with functions involved in maintaining the integrity of IF networks in simple epithelial cells. Furthermore, we observed an increase of epiplakin expression in keratinocytes after the calcium switch suggesting the involvement of epiplakin in the process of keratinocyte differentiation. The work was completed and submitted for publication. This project will not be further pursued in the LSB but certain ongoing lines of investigation will be continued in NIAMS by Dr L. Marekov.

中间细丝（如果）是真核细胞细胞骨架的无处不在成分。它们由五种不同类型组成，其中最多和复杂的是I型和II型角蛋白，它们在上皮中广泛表达。我们也对其他细胞类型的相关性感兴趣，以了解它们在生物学中的作用。我们继续与瑞士，德国和新西兰的其他研究人员合作进行了一项重大研究，以解决波形蛋白的三维结构。如果之所以选择这些，是因为：（a）它们是均一的，因此可能更简单地解决； （b）如果IF，它们与角蛋白具有很高的序列同源性，因此，为波形蛋白添加的许多结构原理应适用于角蛋白。 （1）完成了许多缺失 /突变构建体的工作，这些缺失 /突变构建体累计覆盖了波形蛋白的整个部分。这些构建体的化学测序揭示了N-末端和C-末端100氨基酸与分子盘绕的区域的相互作用，并揭示了这些区域在细丝组件的早期（单体至四聚体）阶段的作用。 结果发表了。第二篇论文包括早期亚基的全部细丝生长。它还可能包括对合作实验室（加利福尼亚大学戴维斯大学）的光谱（EPR）数据进行最终成熟的IF结构的改进，从而完成了这项研究的完成。 （2）启动了角膜蛋白在头发角蛋白中间丝组装中的头部结构域的作用类似的项目。创建了32个突变体和缺失构建体，并纯化了蛋白质。具有两个构建体的初始实验揭示了头部和杆域之间的特定氨基酸相互作用。由于缺乏人力资源，工作被暂停。 （3）将人类表蛋白作为细胞链链接的表征：RNAi介导的表皮耗竭会导致角蛋白和波形蛋白（如果网络）的破坏。 Epiplakin是Plakin家族的成员，具有多个Plakin重复域（PRD）的副本。我们通过免疫染色，覆盖分析和RNAi敲低研究了人类表皮蛋白的亚细胞分布和相互作用。 Epiplakin如果网络和波形蛋白的部分进行了装饰。在结合测定中，重复单元（PRD Plus链接器）表现出很强的结合，并且优先与组装蛋白单体相关。表皮敲低揭示了在简单上皮中而不是表皮细胞中IF网络的破坏。在救援实验中，必须进行重复单位，以防止IF网络在瞬时敲低中的崩溃；但是，如果在稳定敲击的HELA细胞中网络，它只能部分恢复角蛋白，而不能部分恢复波形蛋白。我们推断Epiplakin是一种多功能的细胞链接器，其功能涉及维持简单上皮细胞中IF网络的完整性。此外，我们观察到钙开关后角质形成细胞中表达的增加，这表明表皮在角质形成细胞分化过程中参与。这项工作已完成并提交出版。 LSB将不会在LSB中进一步追求该项目，但L. Marekov博士将在NIAMS继续进行某些持续的调查。

项目成果

Structural changes in trichocyte keratin intermediate filaments during keratinization.

角化过程中毛细胞角蛋白中间丝的结构变化。

• DOI: 10.1016/s1047-8477(02)00636-6
• 发表时间: 2003
• 期刊: Journal of structural biology
• 影响因子: 3
• 作者: Fraser,RDBruce;Steinert,PeterM;Parry,DavidAD
• 通讯作者: Parry,DavidAD

In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding.

毛细胞角蛋白中间丝的体外组装和结构：二硫键稳定的新作用。

• DOI: 10.1083/jcb.151.7.1459
• 发表时间: 2000
• 期刊: The Journal of cell biology
• 作者: Wang,H;Parry,DA;Jones,LN;Idler,WW;Marekov,LN;Steinert,PM
• 通讯作者: Steinert,PM

Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments.

1B 和 2B 杆结构域片段中的卷曲螺旋触发基序对于角蛋白中间丝的稳定性是必需的。

• DOI: 10.1091/mbc.11.10.3539
• 发表时间: 2000
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Wu,KC;Bryan,JT;Morasso,MI;Jang,SI;Lee,JH;Yang,JM;Marekov,LN;Parry,DA;Steinert,PM
• 通讯作者: Steinert,PM