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

项目摘要 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%的儿童。 全世界的成年人。目前AD的护理标准包括使用钙调磷酸酶抑制剂， 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：丝聚蛋白（FLG）的功能丧失变体，FLG是一种表皮特异性蛋白， 称为透明角质颗粒（KG）的无膜细胞器。KGs在表皮分化中的作用， 长期以来一直难以捉摸，阻碍了解决FLG相关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触发的分解是这个过程中的一个关键事件 皮肤屏障的形成。具体地说，这些研究表明，FLG凝聚成液滴状KG 虽然细胞内液-液相分离（LLPS）的过程-一个关键的功能， 截短的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中，细胞解体以推动快速去核。小说 发现FLG编码的LLPS动力学驱动表皮中的终末分化提供了一个新的 框架，剖析健康和疾病中的皮肤屏障形成。 为了解决FLG根源的皮肤屏障疾病，该项目的长期目标是 程序和拯救生理KG动力学。中心假设是， KG的组成将为克服KG功能的病理性丧失提供生物分子方法。 至关重要的是，仍然缺少的是KG组分的生物分子目录，以及KG后它们在细胞内命运的线索 拆卸一个关键的潜在挑战是无法分离和纯化KG。使用人类表皮 等效模型，该项目将追求生物分子工程方法（目标1）询问 通过邻近蛋白质组学和（目的2）拯救KG动力学研究人类KG的生物分子组成， 失去FLG后的功能。拟议的研究将FLG变体与生化工具相结合 绘制KG组分的身份、释放和细胞内目的地。利用这些基本的 的见解和正常的人类遗传变异FLG，这项工作将推进小FLG样蛋白（迷你FLG） 能够重现LLPS动力学和人类KG的组成。进一步测试，如果最佳迷你- 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去核的先河。 功能KG动力学的编程。由此产生的KG分子水平图谱可能会暴露出可药用的 控制KG动力学和终末分化的靶点，例如新鉴定的KG驻留蛋白， 协同作用以启动快速摘除。总的来说，这项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。

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.