Project Summary. Dental enamel comprises the outer layer of tooth crowns and is the hardest tissue of the human body. Enamel possesses a unique combination of high hardness and extreme mechanical resilience due to its intricate structural organization and graded chemical composition. Enamel is prone to dental caries, the most prevalent infectious chronic disease, affecting up to 90% of school children and the vast majority of adults. Caries is caused by bacteria producing lactic acid, which dissolves the enamel mineral. Caries is a complex multifactorial disease; with a significant genetic component. Recently several closely related epithelial keratins, K75, K6a, K6b and K6c, were discovered in ameloblasts and enamel. Importantly, several polymorphisms in these keratins, associated with hair and skin disorders, lead to structural and mechanical defects of enamel, and are linked to higher caries susceptibility. Notably, all these polymorphisms are contained in coding regions, which points to a direct structural effect. We identified K75 and K6b in forming murine and porcine enamel by mass spectrometry (MS). We further isolated an insoluble organic fraction in human enamel and MS analysis identified K6c and it strongly suggests the presence of K75 in this highly cross-linked matrix. Furthermore, a mouse model of pachyonychia congenita Krt75tm1Der knock-in (KI) with deletion of 159Asn displays major skin defects and microstructural changes in enamel. Building on our recent findings, we propose to identify the functional role of K75 in enamel and elucidate the link between mutations in keratins and caries susceptibility. We hypothesize that keratins in enamel form heavily cross-linked filament networks, which stabilize enamel structure and prevent accumulation of microdamage, contributing to its mechanical resilience. We further hypothesize that either structural and/or compositional changes in the protein network can lead to an increased enamel susceptibility to acid attack directly or through accumulation of mechanical damage, i.e. microcracks, which can serve as conduits for acid diffusion. Three aims were developed to test this hypothesis. Aim 1 is to assess structural and mechanical properties of enamel in Krt75tm1Der KI mice at different ages. In Aim 2 is to assess caries susceptibility of enamel in Krt75tm1Der KI mice, using a well-established murine cariogenic challenge protocol. Aim 3 is to assess mechanical properties of the insoluble extracellular enamel matrix from human enamel and Krt75tm1Der KI and WT mice. The data acquired in the proposed studies will serve as a basis for R01 application with the ultimate goal to gain a comprehensive understanding of how the genetic variations in keratins and other components of the insoluble enamel matric affect caries susceptibility, structural and mechanical properties of enamel. These studies will potentially lead to a paradigm shift in our understanding of the roles of the insoluble enamel matrix...