PROTEINS WITH A PREVIOUS LINK TO CURL IN HUMAN OR SHEEP HAIR Interpretation of the functions of structural proteins (especially keratins and KAPs) is hindered by the less differentiated cortical organization of human hair than that of sheep wool. All mammalian hair is made of modifi ed cell remnants, with the “cells” composed of structural components called macro-fi brils (keratin fi lament bundles with a KAP matrix), digested waste left over from the cytoplasm and nucleus, and sometimes melanin granules. The cells are glued to one another by a cell membrane complex that is composed of highly transformed plasma membranes, cell junction complexes, and extracellular matrix (22). In wool fi bers, cortical cell remnants form clusters of similar structural organization and protein chemistry. The orthocortex contains helically twisted macro-fi brils and is dominated by high–glycine–tyrosine KAP species, and the paracortex contains macro-fi brils with less twist and a matrix dominated by high-sulfur KAP species (23,24). The cortex characteristics of human hair cell types are less clear cut (25), macrofi bril helical twist varies across the cortex (26), and most cells have high sulfur content. Notable among the structural proteins found to be more abundant in the curly human hair samples is K38 (Figure 2, Table II). K38 has a history of association with hair curl in hu- mans and sheep. In follicles from straight human hair, its distribution across the hair shaft cortex is sporadic but uniform (27), but in Western blot studies of curly human hair, K38 was found on the concave side of the curvature and evenly distributed in straight hair (28). In sheep follicles, K38 expression is restricted to orthocortical cell remnants (21) and has been noted as potentially playing a key role in keratin polymerization and structure self- assembly (29). In sheep wool, the orthocortex is dominated by twisted fi bril architectures within which keratin fi laments are embedded in a mixture of high–glycine–tyrosine KAPs (21,24) and, when associated with one side of the cortex, is associated with high curvature fi bers (12,30,31). Because cortical cell types are less clear-cut in human hair than in wool (25), the association between K38 and different KAP families is unlikely to be the same. However, our fi nding does raise the intriguing prospect that K38 may be associated with a particular level of macrofi bril internal twist or with a particular set of KAP species. KAP4-2, KAP4-4, and KAP9-8 are all ultrahigh-sulfur proteins that, like other KAPs, are major constituents of the matrix between keratin fi laments in macro-fi brils. In wool, members of the KAP4 family have been found to be expressed primarily in the paracortex (21,30,32). Thus, the prominence of KAP4-2 and KAP9-8 in the straight hair fi bers would be consistent with them having some aspect in common with wool paracortex, but whether this is structural (e.g., less intense macrofi bril internal twist) or in terms of protein chemistry has yet to be established. KAP13-2, a high-sulfur family KAP, was found in higher amounts in curly hair (Figure 2, Table II). Although no direct link has been reported in the literature, another protein from the KAP13 family, KAP13-1, is present in signifi cantly higher amounts in curly sheep wool fi bers than in the same-diameter straight fi bers (Plowman et al. 2020 unpublished results). PROTEINS NOT PREVIOUSLY ASSOCIATED WITH HAIR CURL K34 and K81 are cortical proteins that have previously been observed to be uniformly distributed across the follicle in both human straight hair and sheep wool. K40 is notably one of the last keratins to be expressed during the hair formation process and is found HAIR SHAPE PROTEOMICS 259

only in the cuticle of beard and scalp hair (33). Other than K85, which is expressed in both cuticle and cortex, K40 is the only cuticle protein of interest. Interestingly, a recent microstructure study found statistically fewer cuticle layers in curly hair of individuals of African genetic ancestry than those with straight hair from European and East Asian genetic ancestries (9). K1, K2, and K10 are epithelial keratins, normally playing a role in the follicle root sheathes rather than the hair shaft (34). They are, however, found in the medulla of hair fi bers (35). It is likely that these are associated with the peculiar assembly process and structure of the medulla by which, possibly random, combinations of various keratin species (trichocyte and epithelial) are brought together to form the medulla structures (36). The two differential non-keratin proteins, SBP1 and ADAMTS-like protein 3 (ALT3), had higher abundance in the straight hair samples. Punctin (also known as ALT3), when it occurs in living tissue, is an extracellular matrix protein associated with epithelial cells (37). During hair development, the extracellular material is transformed into cell-type– specifi c continuous structured cement called the cell membrane complex (22,38). Although identifi ed as signifi cantly different between the two groups, it should be noted that this is based on just one peptide identifi cation, and hence, should be considered with extreme care. Nevertheless, one intriguing possibility is that the higher abundance observed in straight hair is an indication of differences in cortical cell lengths across the cortex be- tween straight and curly hair similar to that correlated to the extent of fi ber curvature in wool (39). Finally, methanethiol oxidase is a cellular selenium-binding protein (SBP1) expressed in multiple tissues, also previously found in hair samples (40), which is likely a leftover from fi ber development. However, its function such as interacting with thiol groups to poten- tially generate formaldehyde molecules (41) may indicate an unknown role in the fi nal stages of hair maturation, which is an environment in which sulfur chemistry is critical to the function. In conclusion, this proteome analysis between two extreme groups of hair shape indicates that differences at the protein abundance level can provide useful insights into the physiochemistry underpinning differences in hair shape and the relevant hair growth processes which should inform technological interventions for hair care. However, although differences between the very curly and the very straight hair sample groups could be identifi ed, the linkage between biogeographic genetic differences and curl phenotype is currently unknown and would require further controlled investigation. REFERENCES (1) R. D. Sinclair, Healthy hair: what is it? J. Invest. Dermatol. Symp. Proc., 12(2), 2–5 (2007). (2) R. De la Mettrie, D. Saint-Leger, G. Loussouarn, A. Garcel, C. Porter, and A. Langaney, Shape variability and classifi cation of human hair: a worldwide approach, Hum. Biol., 79(3), 265–281 (2007). (3) B. Lindelof, B. Forslind, M. A. Hedblad, and U. Kaveus, Human hair form. Morphology revealed by light and scanning electron microscopy and computer aided three-dimensional reconstruction. Arch. Dermatol., 124(9), 1359–1363 (1988). (4) R. E. Chapman, “The ovine arrector pili musculature and crimp formation in wool,” in Biology of the Skin and Hair Growth, A. G. Lyne, B. F. Short. Eds. (Angus and Robertson, Sydney, Australia, 1965), pp. 201–232. (5) G. E. Westgate, R. S. Ginger, and M. R. Green, The biology and genetics of curly hair. Exp. Dermatol., 26(6), 483–490 (2017). JOURNAL OF COSMETIC SCIENCE 260