Molecular function of an intermediate filament assembly mechanism in epidermal protein complexes and cell migration

项目总结 在分子水平上，人们在理解中间丝蛋白是如何作为蛋白质的基础上存在着根本的鸿沟。 形成成熟的细丝。这种差距的持续存在是一个重要的问题，因为在这一需要之前 是满足的，理解中间丝如何在大分子络合物中发挥作用，以及如何通过路径- 改变细丝组装的基因突变仍然是难以捉摸的。长期目标是智能地操纵和 用于预防和治疗人类疾病的靶向中间纤维系统。这件事的目的是 其应用是确定使中间丝蛋白能够组装成 成熟的细丝，并与大分子复合体中的表皮蛋白相互作用。中央高铁- 假说是中间纤维中的球袋四聚机制驱动成熟的纤维 形成并促进对建立皮肤屏障至关重要的皮肤蛋白质复合体的组装。 莱蒂。这一假设是从申请人实验室产生的初步数据中产生的。该计划的基本原理 建议的研究是，一旦中间丝组装和蛋白质相互作用机制被分子化- 最终表征的中间丝网络可以被特定地靶向并在药理学上进行调节。 为目前没有适当治疗的患者的临床利益而延迟。由稳健的初步支持 数据，这一假设将通过追求两个具体目标来检验：1)确定1B旋钮与口袋的相互作用 调节成熟中间纤维的组装；以及2)阐明角蛋白相互作用的分子基础 含有桥粒蛋白和丝状蛋白。在第一个目标下，诱变、光散射、灯丝作为- 半定量结合负染电子显微镜、x射线结晶学和瞬时转染法 细胞生物学将被用来建立“旋钮口袋”四聚化机制的分子功能 位于中间细丝的1B结构域。在第二个目标下，结合分析、诱变、异构体- 热滴定量热法、x射线结晶学、电子显微镜和细胞黏附和迁移 分析将被用来表征驱动角蛋白中间丝形成复合角蛋白的分子界面。 人类表皮中含有桥粒粘连蛋白和丝状丝蛋白的网状结构。这种方法是创新的，因为它需要一种新的 在申请人的实验室中发现了中间纤维组装机制，并检查了以前未见过的 对这种机制如何通过蛋白质复合体促进人类皮肤屏障完整性的问题进行了深入探讨 在表皮中形成，并通过调节细胞迁移。它也是创新的，因为它利用了小说 在申请人的实验室中开发的用来探测细胞中中间丝功能的多肽。建议的重行- 搜索具有重要意义，因为它将推进作为一种新分子的旋钮口袋四聚机理 开发预防或治疗人类疾病的中间纤维特异性疗法的目标。

PROJECT SUMMARY There exists a fundamental gap at the molecular level in understanding how intermediate filament proteins as- semble into mature filaments. Continued existence of this gap is an important problem because, until this need is met, understanding of how intermediate filaments function in large macromolecular complexes and how path- ologic mutations alter filament assembly will remain elusive. The long-term goal is to intelligently manipulate and target intermediate filament systems for the prevention and treatment of human diseases. The objective in this application is to define the molecular mechanisms that enable intermediate filament proteins to assemble into mature filaments and to interact with epidermal proteins in large macromolecular complexes. The central hy- pothesis is that the knob-pocket tetramerization mechanism in intermediate filaments drives mature filament formation and facilitates the assembly of cutaneous protein complexes critical for establishing skin barrier integ- rity. This hypothesis is generated from preliminary data produced in the applicant’s lab. The rationale for the proposed research is that, once intermediate filament assembly and protein interaction mechanisms are molec- ularly characterized, intermediate filament networks can be specifically targeted and pharmacologically regu- lated for the clinical benefit of patients currently without adequate therapies. Supported by robust preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine how 1B knob-pocket interactions regulate assembly of mature intermediate filaments; and 2) Elucidate the molecular basis of keratin interactions with desmoplakin and filaggrin. Under the first aim, a combination of mutagenesis, light scattering, filament as- sembly assays coupled with negative stain electron microscopy, x-ray crystallography, and transient transfection cell biology will be used to establish the molecular functions of the “knob-pocket” tetramerization mechanism located in the 1B domain of intermediate filaments. Under the second aim, binding assays, mutagenesis, iso- thermal titration calorimetry, x-ray crystallography, electron microscopy, and cellular adhesion and migration assays will be used to characterize the molecular interfaces driving keratin intermediate filaments to form com- plexes with desmoplakin and filaggrin in human epidermis. The approach is innovative because it takes a new intermediate filament assembly mechanism discovered in the applicant’s lab and examines previously unex- plored questions about how this mechanism contributes to human skin barrier integrity through protein complex formation in the epidermis and through regulation of cell migration. It is also innovative because it utilizes novel peptides developed in the applicant’s lab to probe intermediate filament functions in cells. The proposed re- search is significant because it will advance the knob-pocket tetramerization mechanism as a novel molecular target for developing intermediate filament-specific therapies that prevent or treat human diseases.