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

项目总结 特应性皮炎(AD)是一种慢性炎症性皮肤疾病，影响15%-20%的儿童和1%-3%的儿童 全世界成年人的数量。目前AD的标准护理包括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抗疗法。 然而，对阿尔茨海默病患者的广泛遗传学研究表明，阿尔茨海默病部分源于表皮分化 缺陷：微丝蛋白(Flaggrin，Flg)功能缺失变体，一种表皮特异性蛋白质，聚集在 无膜细胞器称为角化透明质颗粒(KGs)。KGs在表皮分化中的作用有 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。最近的活细胞- 皮肤成像揭示了KGs的组装和pH触发的拆解是这一过程中的关键事件 皮肤屏障的形成。具体地说，这些研究表明，flg凝聚成液滴状的kgs。 通过细胞内液-液相分离(LLP)的过程-这是一种在 截断的FLG变体。在颗粒-角质层界面，突然的细胞内酸化触发部分KG 在角质形成细胞向角质形成细胞的终末分化过程中，拆解以推动快速去核。这部小说 发现FLG编码的LLP动态驱动表皮中的终末分化提供了一种新的 剖析健康和疾病中皮肤屏障形成的框架。 朝着解决源于FLG的皮肤屏障障碍的方向前进，该项目的长期目标是 规划和抢救生理性KG动态。中心假设是，剖析有限合伙人的动态 KG的组成将为克服KG功能病理性丧失的生物分子方法提供信息。 关键的是，KG组分的生物分子目录以及它们在KG上的细胞内命运的线索仍然缺失 拆卸。一个关键的潜在挑战是无法分离和提纯KGs。使用人的表皮 相当于模型，这个项目将寻求生物分子工程方法来(目标1)询问 通过邻近蛋白质组学和(AIM 2)挽救KG动力学和(AIM 2)拯救KG生物分子组成 在失去FLG时的功能。拟议的研究进展与生化工具整合的Flg变异体 以映射KG组分的身份、释放和细胞内目的地。利用这些基本原理 在Flg中的洞察力和正常的人类遗传变异，这项工作将推进小的Flg样蛋白(mini-Flg) 能够概括有限责任合伙人的动态和人类KG的组成。进一步测试是否最优迷你 FIG驱动的KGS功能影响去核动力学，这些实验将开创直接 功能KG动力学的编程。由此得到的KGs的分子水平图可能暴露出可用药 控制KG动态和末端分化的靶点，例如新发现的KG驻留蛋白 配合以启动快速眼球摘除。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动力学的操作。

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.