Structural Features Of Keratin And Related Intermediate

角蛋白及相关中间体的结构特征

• 批准号: 6501625
• 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/6501625
• 关键词: X ray crystallography; cell component structure /function; conformation; crosslink; cytoskeletal proteins; disulfide bond; fibrous protein; gene mutation; intercellular connection; intermediate filaments; keratin; 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 eukaryote cells. They consist of six 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. 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 a major study in collaboration with other investigators in Switzerland, Germany and New Zealand to solve the three-dimensional structure of vimentin IF by use of Xray crystallographic techniques. 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 used for vimentin should be applicable to keratin IF. To date, we have solved the structure of the last 35 residues of the 2B rod domain segment, which encompasses the helix-termination motif, and the likely trigger motif required for successful IF assembly. Work is now in progress on numerous other constructs have been made which cumulatively cover the entire rod domain portion of vimentin. The role of ionic interactions in IF structure: the presence of trigger motifs Current evidence suggests that the packing of protein molecules within IF is in part governed by favorable ionic interactions between charged residues along the rod domain segments of the IF chains. Using the type I/II keratin 5/14 paradigm, we have identified two regions along the chains that are absolutely required for the formation of a two-chained coiled-coil molecule. These are residues 100-113 of the 2B rod domain segment, and residues 79-93 of the 1B rod domain segment. These probably form triggers which are necessary to stabilize and/or initiate coiling of the two chains to form a coiled-coil molecule. Also residues Glu106 and Lys23 of the 2B rod domain segment are required to specify and stabilize the A22 mode of alignment of a pair of molecules. Likewise, residue 10 of the 1A rod domain segment, and residues 4-7 of the L2 linker are required to specify and stabilize the A11 alignment mode of two molecules. These studies have now defined key residues required for the first two hierarchical levels of keratin IF structure. Parallel studies are now in progress on the type III vimentin system: preliminary data documents that different residues are in fact required for maintenance of the A11 and A22 molecular alignments. The organization of molecules in trichocyte keratin IF We have previously demonstrated by detailed cross-linking experiments that pairs of epidermal keratin molecules are aligned in three 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. We also have shown that the molecules of type III IF adopt the same basic four modes, but the alignments of the former three are slightly offset. This adequately explains why type III and types I/II keratin chains cannot and do not coassemble in vivo or in vitro. We have now re-examined these questions in trichocyte IF, and have found that the alignments for reduced trichocyte keratin IF are exactly the same as for cytokeratin IF. However, when trichocyte keratins are oxidized, a series of disulfide bonds form that shift the A11 alignment mode, with the net result of a 1 nm gap between the end and beginning of adjacent molecules. Interestingly, several of the disulfide bonds occurred between head domain and rod domain cysteines, which suggests that the head domain is intimately involved in the molecular shifts and is essential for the maintenance of stability of the trichocyte IF structure. The only way this could occur is if the head domain sequences fold back over the rod domain. This may explain why head domain sequences are essential for successful IF assembly. Tests of this hypothesis will be extremely difficult for trichocyte IF, however, due to the probability of random disulfide interchange. Therefore, we plan to explore this issue with both type III vimentin and types I/II K5/K14 keratin filaments. Initially, we will use deletion cloning experiments coupled with more cross-linking studies to define precisely (a) which residues are essential for successful IF assembly; (b) which residues if any interact with which residues of the 1A and 1B rod domain segments; and (c) how. Structure of the end domains We have discovered a novel mutation in the gene encoding keratin 1 which causes a frameshift and change in the coding sequence of the tail domain. In the wildtype protein, such sequences are enriched in glycines, but in the mutant, the frame has been changed to alanine-rich instead. The net result is a severe case of ichthyosis hystrix Curth-Macklin. It appears the abnormal keratin tail allows IF to form, but severely interferes with keratin IF function. In particular, the distribution of loricrin to the cell periphery is prevented, resulting in a severe barrier function defect. This is not only the first mutation reported for a keratin end domain in disease, but now allows for new insights into the structure and function of the tail domains. Searches for high molecular weight IF associated proteins When type III vimentin IF are passaged through cycles of assembly and disassembly in vitro, certain high molecular weight proteins always co-cycle. We have purified one of these from BHK-21 fibroblasts, and shown that it is the type VI IF protein nestin. This protein can be incorporated in small amounts into vimentin IF and its long tail is thought to serve as an interfilamentous spacer to separate individual IF and affect supramolecular organization in cells. We believe that functionally similar molecules should exist for keratin IF. Keratin IF in epithelia usually occur in tonofibril bundles containing 100s of individual IF loosely separated by 1-3 filament diameters from each other. Ongoing experiments are designed to look for keratin IF organizing or spacer proteins in epithelial cells.

中间丝（IF）是真核细胞骨架中普遍存在的成分。它们由六种不同的类型组成，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。我们不仅对人类皮肤角蛋白IF的结构、功能和表达及其在角变性疾病中的作用感兴趣，而且对其他细胞类型的相关IF感兴趣，以了解它们在生物学中的作用。正在进行的结构研究虽然角蛋白在许多遗传疾病中的作用现在已经被很好地理解，但进一步的结构研究对于制定合理的治疗方法是必要的。我们与瑞士、德国和新西兰的其他研究人员合作开展了一项重大研究，利用x射线晶体学技术解决了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，因此用于波形蛋白的许多结构原理应该适用于角蛋白IF。到目前为止，我们已经解决了2B棒结构域段最后35个残基的结构，其中包括螺旋终止基序，以及成功组装IF所需的可能触发基序。目前，许多其他结构的工作正在进行中，这些结构累积覆盖了vimentin的整个杆状结构域部分。离子相互作用在中频结构中的作用：触发基序的存在目前的证据表明，蛋白质分子在中频内的包装部分是由沿中频链棒域段的带电残基之间的有利离子相互作用所控制的。使用I/II型角蛋白5/14范式，我们已经确定了沿着链的两个区域，这两个区域对于形成双链卷曲卷曲的分子是绝对必需的。这些是2B棒状结构域段的残基100-113和1B棒状结构域段的残基79-93。这些可能形成触发器，这些触发器是稳定和/或启动两个链的卷曲以形成卷曲的线圈分子所必需的。2B棒结构域段的Glu106和Lys23残基也需要指定和稳定一对分子的A22排列模式。同样，1A棒结构域段的残基10和L2连接体的残基4-7也需要指定和稳定两个分子的A11排列模式。这些研究现在已经确定了角蛋白IF结构的前两个层次所需的关键残基。目前对III型静脉蛋白系统的平行研究正在进行中：初步数据文件表明，实际上需要不同的残基来维持A11和A22分子序列。我们之前已经通过详细的交联实验证明，表皮角蛋白分子对以A11、A22和A12三种模式排列。当同化成中频时，同一轴列的分子对采用第四种称为ACN的模式，其中一个分子的末端与相邻分子的开始重叠约1nm。有趣的是，几乎所有已知的角变性突变/替换都位于这个重叠窗口。我们还表明，III型中频分子采用相同的基本四种模式，但前三种模式的排列略有偏移。这充分解释了为什么III型和I/II型角蛋白链在体内或体外不能也不能聚集。我们现在在毛细胞IF中重新检查了这些问题，并发现毛细胞角蛋白IF的还原排列与细胞角蛋白IF完全相同。然而，当毛细胞角蛋白被氧化时，形成了一系列的二硫键，改变了A11的排列模式，最终结果是相邻分子的末端和开始之间有1nm的间隙。有趣的是，一些二硫键发生在头部结构域和杆状结构域半胱氨酸之间，这表明头部结构域与分子转移密切相关，对维持毛细胞IF结构的稳定性至关重要。唯一可能发生这种情况的方法是头结构域序列折叠回杆结构域。这也许可以解释为什么头部结构域序列对于IF的成功组装至关重要。然而，由于随机二硫化物交换的可能性，对毛细胞IF的检验将是极其困难的。因此，我们计划用III型角蛋白和I/II型K5/K14角蛋白丝来探讨这个问题。最初，我们将使用缺失克隆实验加上更多的交联研究来精确定义(a)哪些残基是成功的IF组装所必需的；(b)哪些残基（如果有的话）与1A和1B棒状结构域段的哪些残基相互作用；(c)如何。我们在编码角蛋白1的基因中发现了一个新的突变，该突变引起了尾部结构域编码序列的移码和改变。在野生型蛋白中，这些序列富含甘氨酸，但在突变体中，框架已改为富含丙氨酸。最终的结果是一个严重的子宫肌瘤鱼鳞病。似乎异常的角蛋白尾部允许IF形成，但严重干扰角蛋白IF的功能。特别是，loricrin向细胞周围的分布被阻止，导致严重的屏障功能缺陷。这不仅是在疾病中报道的第一个角蛋白末端结构域突变，而且现在允许对尾部结构域的结构和功能有新的认识。搜索高分子量IF相关蛋白