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...