Keratins are the most abundant proteins in surface epithelia, in which they occur as a cytoplasmic network of 10 nm wide intermediate filaments (IFs). Keratins are encoded by an evolutionarily conserved family of 54 genes subdivided into two major types (I and II). Pairwise regulation of type I and type II keratin genes in epithelia reflects a strict heteropolymerization requirement during keratin intermediate filament assembly. Pairs of keratin genes are individually regulated in an epithelial tissue-type and differentiation-specific fashion, the functional basis of which is only partly understood. A major role fulfilled by keratin IFs is to act as resilient yet pliable scaffolds that endow epithelial cells and tissues with the ability to sustain various types of stress. Many additional functions that are non-mechanical in nature and manifested in a keratin protein-specific and context- dependent fashion, have been identified by us and other researchers in recent years. Mutations affecting the coding sequence of keratins account for a large number of genetically-determined epithelial disorders. In studies supported by this project, we recently discovered that keratin-dependent disulfide bonding plays an important role towards the intracellular organization and steady-state dynamics of keratin filaments in progenitor basal keratinocytes of epidermis, with an associated impact on the balance between proliferation and differentiation, and skin barrier function. The latter entails a powerful interplay between disulfide bonding mediated by residue cysteine 367 (C367) in keratin 14 (K14), the adaptor protein 14-3-3sigma, and YAP1, a terminal effector of Hippo signaling. Because residue C367 in human K14 is conserved in several additional type I keratins expressed in crucial cellular settings in skin epithelia we propose, as an overarching hypothesis, that keratins act as general regulators of Hippo signaling with an associated impact on skin tissue homeostasis and function.
In Aim 1, we will test the hypothesis that residue C401 in keratin 10 is responsible for proper regulation of YAP1 and Hippo signaling in the suprabasal differentiating layers of epidermis.
In Aim 2, we will test the hypothesis that residue C336 in keratin 17 regulates YAP1 and Hippo signaling and the balance between proliferation and differentiation in hair follicles, sweat glands, tooth, and possibly in other ectoderm- derived epithelial appendages.
In Aim 3 we will map the binding interface between keratins and 14-3-3sigma and define the molecular basis for the regulation the keratin/14-3-3 interactions. Finally, in Aim 4, we will test the possibility that keratin-dependent disulfide bonding fulfills additional roles in skin epithelia in vivo. The proposed body of work is highly original, lies on a robust premise supported by substantive evidence, and is poised to significantly advance our understanding of the significance of keratin proteins in vivo as well as the pathophysiology of keratin mutation-based skin diseases.
Keratins comprise a large family of cytoskeletal filament-forming proteins that are highly abundant in epidermis and skin epithelia, where they fulfill a broad range of important roles including structural support. Mutations affecting the coding sequence of keratin proteins account for a large number of individually rare but devastating skin diseases. This project is focused on the cellular and molecular mechanisms through which keratins ensure proper structure, function and homeostasis in skin tissue.
