STRUCTURAL FEATURES OF KERATIN AND RELATED INTERMEDIATE FILAMENTS

角蛋白及相关中间丝的结构特征

• 批准号: 6100520
• 负责人: PETER M STEINERT
• 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/6100520
• 关键词: X ray crystallography; cell differentiation; conformation; crosslink; cytoskeletal proteins; human tissue; intercellular connection; intermediate filaments; keratin; keratinocyte; protein sequence; protein structure function; skin; synthetic peptide; tissue /cell culture; yeast two hybrid system

Intermediate filaments (IF) are the ubiquitous constituents of the cytoskeletons of eukaryote cells. They consist of at least 6 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 in not only the structure, function and expression of keratin IF of human skin and their roles in keratinopathy diseases, but also of the related IF of other cell types in order to understand their roles in biology. Discovery of new mutations in keratinopathies We have discovered a variety of new mutations in some of the genes encoding keratin chains expressed in human epidermis. These occupy novel heretofore unreported residue positions near the beginning or end of the rod domain, and include: T109P of the 2B rod domain segment of the K2e gene in ichthyosis bullosa of Siemens; A12P of the 1A rod domain segment of the K10 gene in epidermolytic hyperkeratosis; and E106D of the 2B rod domain segment of the K1 gene in epidermolytic hyperkeratosis. Each of these mutations results in substitutions of inappropriate amino acids which result in defective keratin intermediate filaments in vivo (and hence result in clinical disease), and in filaments assembled in vitro. These data not only augment a growing database of keratin mutations, but also have provided useful clues on higher-order levels of structural information in intact filaments. Ongoing structural studies While the roles of the keratins in many genetic diseases are now well understood, further structural studies are necessary to develop rational approaches to therapy. We have initiated two types of structural/functional studies. In the first, we have developed synthetic peptides corresponding to the beginning of the 1A or end of the 2B rod domain segments. Several of these synthetic peptides have been injected into living cells to explore their dynamic behavior. Most function as very specific reagents for the disruption of all types of IF, but do not interfere with the state of assembly of microtubules or microfilaments. However, they do affect the supramolecular organization of them, thus indicating that all three components of the cytoskeleton function cooperatively in cells. On the other hand, we have discovered that an H1 peptide, a sequence which is specifically found only in type II keratin chains, is a very specific poison for the organization of keratin IF in epithelia. In those cultured cells which express both keratin IF and vimentin IF, only the keratin IF organization is disrupted. In a second experimental approach, we have also synthesized a series of peptides of sequence corresponding to keratin chains which are involved in the important overlap regions in assembled filaments, for biophysical structural studies, solution nmr, and for X-ray crystallography. To date, 2B peptides derived from the type III protein vimentin show simple dimerization, are essentially monodisperse in solution, and possess <90% a-helix, thus suggesting they will be useful to obtain detailed atomic-resolution structural information. The role of ionic interactions in IF structure The discovery that an E106D substitution causes disease has prompted us to re-evaluate the role of ionic interactions in the stability of the keratin chains in IF. We note that there are several potential pairs of charged residues which occupy e-f positions of the heptad repeat that have been precisely conserved in all IF chain types, including E106. Accordingly, using the type III vimentin and types I/II keratin 5/14 paradigms, we have expressed in bacteria a large number of mutant chains for use in in vitro assembly experiments to investigate the level of IF structural hierarchy at which the conserved charged pairs play important roles. The assays used will involve visual examination of the IF by negative staining, coiled-coil molecule stabilities as assessed by urea dissolution experiments, and isolation and characterization of a-helix-enriched proteolytic fragments to examine molecular alignments. Ongoing crosslinking studies In explorations of the structure and organization of the cornified cell envelope, we have discovered a large number of peptides involving crosslinks between a keratin chain and a variety of other envelope proteins. Notably, the vast majority of crosslinks involve a very specific and precisely conserved lysine residue located in the V1 region of the head domain of the type II keratins K1, K5 or K6. Interestingly, we have previously identified a case of non-epidermolytic palmaplantar keratoderma in which this conserved lysine residue of the K1 chain was substituted by isoleucine. Detailed ultrastructural analyses of the patient tissue at the level of the electron microscope revealed a severe discoordination between the cell periphery and the keratin IF cytoskeleton in the upper granular cells of the epidermis, proximal to the formation of the cornified cell envelope. Therefore, we believe this residue is critically involved in the structural organization of the cytoskeleton with the cornified cell envelope in terminally differentiated epidermis, and other related stratified squamous epithelia. Loss of this critical mode of organization results in a diminished barrier function for the epidermis. Future studies will involve attempts to ablate this lysine residue to create a mouse model for this disease and to further study the connection between cytoskeletal-cornified cell envelope coordination and barrier function. In addition, we have identified other crosslinks which reveal that the keratin IF are attached to the desmoplakin component of desmosomes indirectly through a series of related intermediate filament associated proteins. Further work will be directed to understand the complexity of these apparent connections and role in disease. The organization of molecules in various IF types We have previously demonstrated by detailed crosslinking experiments that pairs of epidermal keratin molecules are aligned in three basic modes termed A11, A22 and A12. When assimilated into IF, pairs of molecules in the same axial row adopt a fourth mode termed ACN, in which the end of one molecule overlaps the beginning of the adjacent molecule by about 1 nm. Interestingly, almost all known keratinopathy mutations/substitutions reside in this overlap 'window'.

中间丝（IF）是普遍存在的 真核细胞的细胞骨架的成分。它们包括 至少有 6 种不同类型，其中数量最多、最复杂 是广泛表达的 I 型和 II 型角蛋白 上皮细胞。我们感兴趣的不仅仅是结构、功能 人皮肤角蛋白IF的表达及其作用 角蛋白病疾病，以及其他细胞类型的相关 IF 以了解它们在生物学中的作用。发现新的 角蛋白病的突变 我们发现了多种新的 一些编码角蛋白链的基因发生突变 人类表皮。这些占据小说迄今未报道 靠近杆结构域的开始或末端的残基位置，以及 包括：K2e基因2B杆结构域片段的T109P 西门子大疱性鱼鳞病； 1A杆域段的A12P K10 基因在表皮松解性角化过度中的作用；和E106D的 表皮松解中 K1 基因的 2B 杆结构域片段 角化过度。这些突变中的每一个都会导致替换 不适当的氨基酸会导致角蛋白缺陷 体内的中间丝（并因此导致临床疾病）， 以及在体外组装的细丝。这些数据不仅增强了 不断增长的角蛋白突变数据库，而且还提供了 关于完整的高阶结构信息的有用线索 细丝。正在进行的结构研究而角蛋白的作用 许多遗传病现已得到充分了解，进一步 结构研究对于制定合理的方法是必要的 治疗。我们发起了两种类型的结构/功能 研究。首先，我们开发了合成肽 对应于1A杆的起点或2B杆的终点 域段。其中一些合成肽已被 注入活细胞以探索其动态行为。最多 作为非常特异的试剂，可破坏所有类型的 IF，但不干扰微管的组装状态 或微丝。然而，它们确实影响超分子 组织它们，从而表明所有三个组成部分 细胞骨架在细胞中协同发挥作用。另一方面， 我们发现了一个 H1 肽，该序列是 仅在 II 型角蛋白链中发现，是一种非常特殊的 对上皮细胞中角蛋白 IF 的组织具有毒害作用。在那些 仅表达角蛋白 IF 和波形蛋白 IF 的培养细胞 角蛋白 IF 组织被破坏。在第二次实验中 方法，我们还合成了一系列序列的肽 对应于参与重要的角蛋白链 组装丝中的重叠区域，用于生物物理结构 研究、溶液核磁共振和 X 射线晶体学。迄今为止，2B 来自 III 型波形蛋白的肽显示简单 二聚化，在溶液中基本上是单分散的，并且具有 <90% a-螺旋，因此表明它们将有助于获得详细的 原子分辨率的结构信息。离子的作用 IF 结构中的相互作用 E106D 的发现 替代导致疾病促使我们重新评估其作用 IF 中角蛋白链稳定性的离子相互作用。我们 请注意，有几个潜在的带电残基对 占据已精确确定的七肽重复序列的 e-f 位置 在所有 IF 链类型中都保守，包括 E106。因此，使用 III 型波形蛋白和 I/II 型角蛋白 5/14 范例，我们有 在细菌中表达大量突变链，用于 体外组装实验研究 IF 结构水平 保守带电对在其中发挥重要作用的层次结构 角色。使用的测定将涉及 IF 的目视检查 负染色，通过尿素评估卷曲螺旋分子稳定性 溶解实验以及分离和表征 富含α螺旋的蛋白水解片段用于检查分子 对齐。正在进行的交联研究 角质化细胞膜的结构和组织，我们有 发现了大量涉及之间交联的肽 角蛋白链和多种其他包膜蛋白。尤其， 绝大多数交联涉及非常具体且精确的 位于头部 V1 区域的保守赖氨酸残基 II型角蛋白K1、K5或K6的结构域。有趣的是，我们有 先前发现一例非表皮松解性掌跖病 角化病中 K1 链的保守赖氨酸残基 被异亮氨酸取代。详细的超微结构分析 电子显微镜下的患者组织显示出 细胞外周和角蛋白 IF 之间严重不协调 表皮上层颗粒细胞的细胞骨架，近端 至角质化细胞包膜的形成。因此，我们 相信这个残基在结构中起着至关重要的作用 细胞骨架与角化细胞膜的组织 终末分化表皮及其他相关分层 鳞状上皮。失去这种关键的组织模式 导致表皮屏障功能减弱。未来 研究将尝试消除这种赖氨酸残基以创建 这种疾病的小鼠模型并进一步研究其联系 细胞骨架-角化细胞包膜协调和 屏障功能。此外，我们还发现了其他交联 这表明角蛋白 IF 附着在桥粒斑蛋白上 桥粒的组成部分通过一系列相关的间接 中间丝相关蛋白。下一步的工作将是 旨在了解这些明显的复杂性 疾病中的联系和作用。分子的组织在 各种 IF 类型我们之前已经详细演示过 表皮角蛋白分子配对的交联实验 以称为 A11、A22 和 A12 的三种基本模式对齐。什么时候 同化到 IF 中，同一轴向行中的分子对采用 第四种模式称为 ACN，其中一个分子的末端 与相邻分子的起始部分重叠约 1 nm。 有趣的是，几乎所有已知的角蛋白病 突变/替换存在于这个重叠“窗口”中。