Toward therapeutic targeting of liquid-liquid phase separation dynamics in skin

皮肤液-液相分离动力学的治疗靶向

• 批准号: 10679610
• 负责人: ALEXA REGINA CHUA AVECILLA
• 金额: $ 4.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-03 至 2026-04-02
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679610
• 关键词: Acceleration; Address; Adrenal Cortex Hormones; Adult; Affect; Architecture; Atopic Dermatitis; Behavior; Biotin; Biotinylation; Calcineurin inhibitor; Catalogs; Cell Nucleus; Characteristics; Child; Coupling; Cytoplasmic Granules; Cytosol; Defect; Destinations; Disease; Engineering; Epidermis; Event; Exons; Foundations; Future; Gene Delivery; Genes; Genetic Variation; Genetic study; Goals; Health; Histidine; Human; Human Genetics; Immune; Immunosuppression; Impairment; Inflammation; Inflammatory; Label; Length; Link; Liquid substance; Maps; Mass Spectrum Analysis; Membrane; Modeling; Molecular; Monoclonal Antibody Therapy; Mutation; Nature; Nonsense Mutation; Organelles; Pathogenesis; Pathologic; Patients; Phase; Physical condensation; Physiological; Process; Protein Engineering; Proteins; Proteomics; Recurrence; Research; Role; Skin; Skin repair; Stratification; Stratum Granulosum; Testing; Therapeutic; Tyrosine; Variant; Viscosity; Work; biochemical tools; chronic inflammatory skin; design; druggable target; experimental study; filaggrin; imaging approach; innovation; insight; keratinization; keratinocyte; keratinocyte differentiation; keratohyalin; live cell imaging; loss of function; loss of function mutation; novel; prevent; programs; restraint; skin barrier; skin disorder; standard of care; therapeutic target; tool

PROJECT SUMMARY Atopic dermatitis (AD) is a chronic inflammatory skin disorder that affects 15-20% of children and 1-3% of adults worldwide. The current standard-of-care for AD involves the use of calcineurin inhibitors, corticosteroids, and monoclonal antibody therapies that curb inflammation through immunosuppression. However, extensive genetic studies of AD patients suggest that AD is partly rooted in an epidermal differentiation defect: loss-of-function variants of filaggrin (FLG), an epidermal-specific protein that accumulates in membraneless organelles called keratohyalin granules (KGs). The role of KGs in epidermal differentiation has long remained elusive, preventing progress toward addressing FLG-linked skin barrier defects. Recent live cell- imaging of skin unearthed the assembly and pH-triggered disassembly of KGs as a crucial event in the process of skin barrier formation. Specifically, these studies demonstrated that FLG condenses into droplet-like KGs though a process of intracellular liquid-liquid phase separation (LLPS) — a key function that is impaired in truncated FLG variants. At the granular-to-corneum interface, abrupt intracellular acidification triggers partial KG disassembly to propel rapid enucleation in the terminal differentiation of keratinocytes to corneocytes. The novel finding that FLG-encoded LLPS dynamics drive terminal differentiation in the epidermis provides a new framework to dissect skin barrier formation in health and disease. Advancing toward addressing FLG-rooted skin barrier disorders, the long-term goal of this project is to program and rescue physiological KG dynamics. The central hypothesis is that dissecting the LLPS dynamics and composition of KGs will inform biomolecular approaches to overcome pathological loss of KG functionality. Crucially, still missing is a biomolecular catalog of KG components, and clues on their intracellular fate upon KG disassembly. A key underlying challenge is the inability to isolate and purify KGs. Using human epidermal equivalent models, this project will pursue biomolecular engineering approaches to (Aim 1) interrogate the biomolecular composition of human KGs through proximity proteomics and (Aim 2) rescue KG dynamics and functionality upon loss of FLG. The proposed research advances FLG variants integrated with biochemical tools to map the identity, release, and intracellular destination of KG components. Harnessing these fundamental insights and normal human genetic variation in FLG, this work will advance small FLG-like proteins (mini-FLG) capable of recapitulating the LLPS dynamics and composition of human KGs. Further testing if optimal mini- FLG-driven KGs functionally impact enucleation dynamics, these experiments will pioneer the direct programming of functional KG dynamics. The resulting molecular-level map of KGs may expose druggable targets to control KG dynamics and terminal differentiation, such as newly identified KG-residing proteins that cooperate to actuate rapid enucleation. Overall, the proposed research will lay a foundation for future therapeutic manipulation of epidermal LLPS dynamics in skin barrier disorders.

项目概要 特应性皮炎 (AD) 是一种慢性炎症性皮肤病，影响 15-20% 的儿童和 1-3% 目前的 AD 治疗标准涉及使用钙调神经磷酸酶抑制剂， 皮质类固醇和通过免疫抑制抑制炎症的单克隆抗体疗法。 然而，对 AD 患者的广泛遗传学研究表明，AD 部分根源于表皮分化 缺陷：丝聚合蛋白（FLG）的功能丧失变体，FLG是一种表皮特异性蛋白质，在 称为透明角质颗粒 (KG) 的无膜细胞器 KG 在表皮分化中发挥作用。 长期以来一直难以捉摸，阻碍了解决与 FLG 相关的皮肤屏障缺陷的进展。 皮肤成像发现 KG 的组装和 pH 触发的分解是该过程中的关键事件 具体来说，这些研究表明 FLG 凝结成液滴状 KG。 通过细胞内液-液相分离 (LLPS) 的过程——这是一个在细胞内受损的关键功能 截短的 FLG 变体在颗粒与角质层的界面处，突然的细胞内酸化会触发部分 KG。 分解以促进角质形成细胞终末分化为角质细胞的快速去核。 FLG 编码的 LLPS 动力学驱动表皮终末分化的发现提供了一种新的方法 剖析健康和疾病中皮肤屏障形成的框架。 为了推进解决 FLG 根源的皮肤屏障疾病，该项目的长期目标是 程序和救援生理 KG 动力学的中心假设是剖析 LLPS 动力学。 KG 的结构和组成将为克服 KG 功能的病理性丧失的生物分子方法提供信息。 至关重要的是，仍然缺少 KG 成分的生物分子目录，以及它们在 KG 上的细胞内命运的线索 一个关键的潜在挑战是无法使用人类表皮分离和纯化 KG。 等效模型，该项目将采用生物分子工程方法（目标 1）询问 通过邻近蛋白质组学研究人类 KG 的生物分子组成和（目标 2）拯救 KG 动力学和 拟议的研究进展了与生化工具集成的 FLG 变体。 利用这些基础知识来绘制 KG 成分的身份、释放和细胞内目的地。 深入了解 FLG 中的正常人类遗传变异，这项工作将推进小型 FLG 样蛋白（mini-FLG）的发展 能够概括 LLPS 动态和人类 KG 的组成，进一步测试是否是最佳的迷你模型。 FLG 驱动的 KG 在功能上影响去核动力学，这些实验将开创直接 功能 KG 动力学的编程所得到的 KG 的分子水平图可能会暴露可药物性。 控制 KG 动态和终末分化的目标，例如新发现的 KG 驻留蛋白 总体而言，拟议的研究将为未来的治疗奠定基础。 皮肤屏障疾病中表皮 LLPS 动力学的操纵。