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

项目摘要 在分子水平上存在着一个根本的差距，在理解中间丝蛋白是如何作为- 长成成熟的花丝。这一差距的持续存在是一个重要问题，因为在这一需要之前， 满足，了解中间丝如何在大分子复合物中发挥作用，以及如何通过 改变丝装配的逻辑突变仍然是难以捉摸的。长期目标是智能地操纵和 靶向中间丝系统用于预防和治疗人类疾病。在这方面的目标 应用是定义分子机制，使中间丝蛋白组装成 成熟的花丝，并与大分子复合物中的表皮蛋白相互作用。中央卫生- 假设是中间丝的节袋四聚机制驱动成熟丝 形成并促进皮肤蛋白质复合物的组装， rity。该假设是根据申请人实验室产生的初步数据得出的。的理由 建议的研究是，一旦中间丝组装和蛋白质相互作用机制是分子， 具有特殊特征的中间丝网络可以被特异性地靶向和非特异性地靶向， 旨在为目前没有充分治疗的患者提供临床益处。由强大的初步支持 数据，这一假设将通过追求两个具体目标进行测试：1）确定1B旋钮-口袋相互作用 调节成熟中间丝的组装; 2）阐明角蛋白相互作用的分子基础 桥粒斑蛋白和丝聚蛋白。在第一个目标下，诱变，光散射，细丝作为- 结合负染电子显微镜、X射线晶体学和瞬时转染的sweetness测定 细胞生物学将被用来建立“节袋”四聚化机制的分子功能 位于中间丝的1B域中。在第二个目标下，结合测定、诱变、异源- 热滴定量热法、X射线晶体学、电子显微镜和细胞粘附和迁移 测定将用于表征驱动角蛋白中间丝形成复合物的分子界面， 人表皮桥粒斑蛋白和丝聚蛋白复合体。这种方法是创新的，因为它需要一个新的 在申请人的实验室中发现的中间细丝组装机构，并检查了先前未被发现的中间细丝组装机构。 探索了关于这种机制如何通过蛋白质复合物促进人类皮肤屏障完整性的问题 在表皮中形成并通过调节细胞迁移。它也是创新的，因为它利用了新颖的 在申请人的实验室中开发的用于探测细胞中的中间丝功能的肽。拟议的重新- 这一研究具有重要意义，因为它将推进knob-pocket四聚机制作为一种新的分子机制 用于开发预防或治疗人类疾病中间体特异性疗法的靶点。

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.