Phosphatase-dependent regulation of desmosome intercellular junctions

桥粒细胞间连接的磷酸酶依赖性调节

• 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
• 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

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.

项目概要 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。 对于多层表皮来说尤其如此，因为它在暴露于高水平的机械应力的情况下 维持重要的身体和免疫屏障。表皮维持能力的关键因素 表皮屏障同时调节压力是角蛋白中间体之间的附着物 细丝 (IF) 和称为桥粒的细胞间连接。在桥粒内，细胞骨架连接蛋白 桥粒斑蛋白 (DP) 负责将 IF 锚定到连接处。 DP 普遍存在于所有 桥粒形成细胞，并受其 C 端尾部结构域的翻译后修饰 (PTM) 调节 控制 DP-IF 亲和力。与 DP 磷酸化调节区域对于桥粒的重要性一致 C 末端区域的基因缺失会导致多种与严重心脏病相关的疾病 皮肤和致命的表皮屏障缺陷。从功能上讲，当 C 端基序处于其低位时 磷酸化状态下，DP 对 IF 表现出最高的亲和力。在实验模型中，增加的 DP-IF 通过产生更强、更稳定的桥粒，这种关联对成熟细胞片具有保护作用。 相反，在桥粒组装过程中，低磷酸化的 DP 在 IF 网络上积聚，破坏 它运输到桥粒连接处，并干扰新细胞间的形成和成熟 桥粒连接。鉴于与 DP 低磷酸化形式相关的强分子表型 在体外，必须存在一个协调过程来调节 DP 磷酸化以实现适当的桥粒功能。我们 先前确定 GSK3 是负责磷酸化 DP 的激酶；然而，蛋白磷酸酶 负向调节 DP 磷酸化的作用此前尚不清楚。我的初步数据确定 PP2A-B55 能够结合 DP 的 C 末端并使其去磷酸化。该提案将测试 假设 PP2A-B55 在桥粒动态过程中调节 DP 磷酸化 组装和响应机械应力，使细胞能够响应不断变化的特性和 分层表皮的特殊功能。目标 1 将采用尖端显微镜和蛋白质组学—— 基于方法来确定 PP2A 在桥粒组装过程中调节 DP 的机制。目标2 将确定 DP2A 磷酸化调节如何影响 1) 桥粒依赖性细胞粘附和 2) 使用新颖的机械生物学技术对桥粒响应机械应力。拟议的工作将 提供有关磷酸酶信号传导如何控制桥粒组装和功能的重要见解 维持人类表皮屏障，因此，磷酸酶信号传导失调如何发挥作用 与屏障相关的疾病。总之，拟议的研究和职业发展计划将提供 我作为一名独立研究者的发展和计划的 K99/R00 应用的跳板。