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.

中间丝（IF）是真核细胞骨架中普遍存在的成分。它们由五种不同的类型组成，其中数量最多和最复杂的是在上皮中广泛表达的I型和II型角蛋白。我们也对其他细胞类型的相关IF感兴趣，以便了解它们在生物学中的作用。我们与瑞士、德国和新西兰的其他研究人员合作，继续进行一项重大研究，以解决vimentin IF的三维结构。选择这些IF是因为：(a)它们是均聚物，因此可能比较容易解；(b)它们与角蛋白IF具有非常高的序列同源性，因此许多引自蛋白的结构原理应该适用于角蛋白IF。

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