Phosphatase-dependent regulation of desmosome intercellular junctions

桥粒细胞间连接的磷酸酶依赖性调节

基本信息

批准号：
10677182
负责人：
Abbey Leigh Perl
金额：
$ 7.18万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10677182
关键词：
Acantholysis Address Adhesions Adhesives Affect Affinity Apical Binding C-terminal Cadherins Cell Adhesion Cell division Cell-Cell Adhesion Cells Coupled Couples Cutaneous Cytoskeleton DNA Sequence Alteration Data Defect Deficiency Diseases Deletion Mutation Desmosomes Development Development Plans Disease Disease model Ectopic Expression Epidermis Epidermolysis Bullosa Equilibrium Event Exhibits Experimental Models Exposure to Family Dasypodidae Family member Foundations Funding Future Genetic Goals Human Immune In Vitro Intercellular Junctions Intermediate Filaments Keratin Label Maintenance Mechanical Stress Mechanics Mediating Membrane Microscopy Modeling Pemphigus Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Point Mutation Post-Translational Protein Processing Process Property Protein Dephosphorylation Protein Phosphatase Inhibitor Protein phosphatase Proteins Proteomics Regulation Research Personnel Role Signal Transduction Skin Stratification Stress Stretching Syndrome Tail Techniques Testing Tissues Training Work arrhythmogenic cardiomyopathy career career development desmoplakin desmoplakin II endosulfine genetic approach insight keratinocyte mechanical force mechanical properties mechanotransduction molecular phenotype mouse model mutant novel preservation protective effect recruit research and development response skin barrier superresolution microscopy trafficking training opportunity

项目摘要

Project Summary Intercellular junctions and their cytoskeletal connections are essential for maintaining tissue stability and function. This is particularly true of the multi-layered epidermis as it is exposed to high levels of mechanical stress while maintaining an essential physical and immune barrier. Critical contributors to the epidermis' ability to maintain the epidermal barrier while simultaneously adjusting to stress are attachments between keratin intermediate filaments (IF) and intercellular junctions called desmosomes. Within desmosomes, a cytoskeletal linker protein called desmoplakin (DP) is responsible for anchoring the IF to the junctions. DP is ubiquitously expressed in all desmosome-forming cells and is regulated by post-translational modification (PTM) of its C-terminal tail domain to control DP-IF affinity. Consistent with the importance of this phospho-regulatory region of DP for desmosome function, genetic deletions of the C-terminal region cause multiple diseases associated with severe cardio- cutaneous and lethal epidermal barrier defects. Functionally, when the C-terminal motif is in its hypo- phosphorylated state, DP exhibits its highest affinity towards IF. In experimental models, the increased DP-IF association had protective effects in mature cell sheets by generating stronger, more stable desmosomes. Conversely, during desmosome assembly hypo-phosphorylated DP accumulates on the IF network, disrupting it's trafficking to desmosome junctions, and interfering with the formation and maturation of new cell-cell desmosome junctions. Given the strong molecular phenotypes associated with DP's hypo-phosphorylated form in vitro, a coordinated process must exist to regulate DP phosphorylation for proper desmosome function. We previously identified GSK3 as the kinase responsible for phosphorylating DP; however, the protein phosphatase responsible for negatively regulating DP phosphorylation was previously unknown. My preliminary data identified PP2A-B55 as capable of binding to and dephosphorylating DP's C-terminus. This proposal will test the hypothesis that PP2A-B55 regulates DP phosphorylation during the dynamic process of desmosome assembly and in response to mechanical stress to allow cells to respond to the changing properties and specialized functions of the stratified epidermis. Aim 1 will employ cutting-edge microscopy and proteomics- based approaches to identify the mechanism by which PP2A regulates DP during desmosome assembly. Aim 2 will establish how PP2A phospho-regulation of DP impacts 1) desmosome-dependent cell adhesion and 2) the desmosomal response to mechanical stress using novel mechano-biology techniques. The proposed work will provide important insight into how phosphatase signaling controls desmosome assembly and function to maintain the human epidermal barrier and, therefore, how dysregulated phosphatase signaling could contribute to barrier-related diseases. Together, the proposed research and career development plan will provide a springboard for my development as an independent investigator and planned K99/R00 application.

项目摘要细胞间连接及其细胞骨架连接对于维持组织稳定性和功能至关重要。多层表皮尤其如此，因为它暴露于高水平的机械应力，而保持基本的物理和免疫障碍。表皮维护能力的关键贡献者表皮屏障同时调整应力是角蛋白中间体之间的附件细丝（if）和细胞间连接体称为脱骨小体。在脱粒体内，细胞骨架接头蛋白称为Desmoplakin（DP）负责将IF固定到交界处。 DP无处不在脱粒形成细胞，并由其C末端尾域的翻译后修饰（PTM）调节控制DP-如果亲和力。与DP的磷酸调节区域的重要性一致功能，C末端区域的遗传缺失会导致多种疾病与严重的心脏相关皮肤和致命的表皮屏障缺陷。在功能上，当C末端基序处于其低位磷酸化状态DP对IF表现出最高的亲和力。在实验模型中，DP-IF增加通过产生更强，更稳定的脱染色小体，关联在成熟的细胞表中受到保护。相反，在脱糖体组装期间，低磷酸化的DP在IF网络上积聚，破坏它正在贩运到脱骨头连接，并干扰新细胞细胞的形成和成熟轨道连接。考虑到与DP的低磷酸化形式相关的强分子表型在体外，必须存在一个协调的过程，以调节DP磷酸化以进行适当的脱骨功能。我们先前将GSK3识别为负责光元DP的激酶；然而，蛋白质光化负责对DP磷酸化负面控制的负责先前未知。我的初步数据已确定 PP2A-B55能够与DP的C末端结合和去磷酸化。该建议将测试 pp2a-b55在脱染色体动态过程中调节DP磷酸化的假设组装和对机械应力的响应，使细胞能够响应不断变化的性质和分层表皮的专门功能。 AIM 1将采用尖端的显微镜和蛋白质组学 - 基于识别PP2A在脱骨组装过程中调节DP的机制的方法。目标2 将确定DP的PP2A磷酸调节如何影响1）脱骨体依赖性细胞粘合剂和2）使用新型机械生物学技术对机械应力的脱骨反应。拟议的工作将提供有关磷酸酶信号如何控制脱骨组组装和功能的重要见解保持人类表皮屏障，因此，光蛋白酶信号传导如何促进与障碍有关的疾病。拟议的研究和职业发展计划一起将提供我作为独立调查员的发展板，并计划了K99/R00应用程序。