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• W1997400162 abstract "glutathione S-transferase hemagglutinin indirect immunofluorescence immunoprecipitation keratin 85 keratin intermediate filament keratin-associated protein western blot Hair keratins and their associated proteins (keratin-associated proteins, KRTAPs) are the major structural components of the hair shaft (Shimomura and Ito, 2005Shimomura Y. Ito M. Human hair keratin-associated proteins.J Investig Dermatol Symp Proc. 2005; 10: 230-233Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar; Rogers et al., 2006Rogers M.A. Langbein L. Praetzel-Wunder S. et al.Human hair keratin-associated proteins (KAPs).Int Rev Cytol. 2006; 251: 209-263Crossref PubMed Scopus (174) Google Scholar). KRTAPs are distributed in the matrix between keratin intermediate filaments (KIFs). The human hair shaft consists of three layers: medulla, cortex, and cuticle. Hair keratins and KRTAPs have been shown to be expressed specifically in the respective layers of the hair shaft (Langbein and Schweitzer, 2005Langbein L. Schweitzer J. Keratins of the human hair follicle.Int Rev Cytol. 2005; 243: 1-78Crossref PubMed Scopus (199) Google Scholar; Rogers et al., 2006Rogers M.A. Langbein L. Praetzel-Wunder S. et al.Human hair keratin-associated proteins (KAPs).Int Rev Cytol. 2006; 251: 209-263Crossref PubMed Scopus (174) Google Scholar). KRTAPs are encoded by a large number of multigene families and are classified into three groups based on their amino acid composition: high sulfur (<30mol% cysteine content), ultrahigh sulfur (>30mol% cysteine content), and high glycine/tyrosine (Powell and Rogers, 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser, Basel1997: 59-148Crossref Google Scholar). We recently characterized the human hair shaft cortex–specific KRTAP2 family members at the DNA, RNA, and protein levels (Fujikawa et al., 2012Fujikawa H. Fujimoto A. Farooq M. et al.Characterization of the human hair keratin-associated protein 2 (KRTAP2) gene family.J Invest Dermatol. 2012; 132: 1806-1813Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Specifically, we showed that the KRTAP2 proteins interacted with each other and preferentially bound to hair keratins but not to epithelial keratins. However, the biophysical features of the other KRTAP families have remained largely unknown. In the present study, we focused on the hair shaft cuticle–specific KRTAP10 family members and analyzed their biological features, especially the interactions between KRTAP10 proteins and hair keratins. In humans, a total of 12 functional KRTAP10 genes (KRTAP10-1–KRTAP10-12) have been identified on chromosome 21q22.3 (Rogers et al., 2004Rogers M.A. Langbein L. Winter H. et al.Hair keratin associated proteins: characterization of a second high sulfur KAP gene domain on human chromosome 21.J Invest Dermatol. 2004; 122: 147-158Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar; Shibuya et al., 2004Shibuya K. Obayashi I. Asakawa S. et al.A cluster of 21 keratin-associated protein genes within introns of another gene on human chromosome 21q22.3.Genomics. 2004; 83: 679-693Crossref PubMed Scopus (31) Google Scholar). The identified KRTAP10 proteins belong to the high-sulfur group. As all of the 12 KRTAP10 members showed high homology in the amino acid sequence, we chose to investigate the KRTAP10-1 protein as a representative of this protein family. The detailed materials and methods are given elsewhere (Supplementary Materials and Methods online). Download .pdf (.1 MB) Help with pdf files Supplementary Information First, we investigated the expression of the KRTAP10 proteins in human hair shaft using western blot (WB) with an anti-KRTAP10 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), which is able to recognize several KRTAP10 members including KRTAP10-1. WB of the lysate from the hair shafts collected from a healthy Japanese individual showed a fragment of nearly the same molecular weight as that of the cell lysate from HEK293T cells overexpressing KRTAP10-1 protein (Figure 1a). Furthermore, indirect immunofluorescence (IIF) on human scalp skin sections revealed that the KRTAP10 proteins were predominantly expressed in the upper keratinizing zone of the hair shaft cuticle (Figure 1b), similar to other cuticle-specific KRTAPs, such as KRTAP24 (Rogers et al., 2007Rogers M.A. Winter H. Langbein L. et al.Characterization of human KAP24.1, a cuticular hair keratin-associated protein with unusual amino-acid composition and repeat structure.J Invest Dermatol. 2007; 127: 1197-1204Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and KRTAP26 (Rogers et al., 2008Rogers M.A. Langbein L. Praetzel-Wunder S. et al.Characterization and expression analysis of the hair keratin associated protein KAP26.1.Br J Dermatol. 2008; 159: 725-729Crossref PubMed Scopus (35) Google Scholar). Double IIF studies with the anti-KRTAP10 and anti-keratin 85 (K85) antibodies showed that KRTAP10 and K85 proteins were coexpressed in the hair shaft cuticle (Figure 1c). To investigate expression patterns of KRTAP10-1 and cuticle-specific hair keratins in cultured cells, we transfected expression vectors for hemagglutinin (HA)-tagged KRTAP10-1 (HA-KRTAP10-1) and Flag-tagged K82 (Flag-K82) into HaCaT (human epidermal keratinocyte) cells. When the vectors for both HA-KRTAP10-1 and Flag-K82 were co-transfected, KRTAP10-1 protein showed a granular expression pattern in the cytoplasm and appeared to colocalize with KIFs composed of K82 (Figures 1d–f). In contrast, when the expression vector for HA-KRTAP10-1 was transfected alone, KRTAP10-1 protein was expressed diffusely in the cytoplasm, and its relationship with endogenous K14 was not evident (Figures 1g–i). Next, to analyze biological features of KRTAP10 proteins, we performed a series of in vitro studies. First, we investigated self-interaction of the KRTAP10-1 proteins by co-immunoprecipitation (co-IP) assay between HA-KRTAP10-1 and Flag-KRTAP10-1 in HEK293T cells, which demonstrated that the KRTAP10-1 protein was able to interact with itself (Figure 2a). Subsequently, in order to investigate the physical interaction between KRTAP10-1 and hair keratins, we performed additional co-IP assays in HEK293T cells. Co-IP assay between HA-KRTAP10-1 and K85 showed that the KRTAP10-1 protein was able to bind to K85 (Figure 2b). Furthermore, we investigated the interactions between KRTAP10-1 and cuticle-specific hair keratins, K32 and K82, as well as the interaction between KRTAP10-1 and an epithelial keratin K10. Co-IP assays showed that KRTAP10-1 protein bound to both K82 and K32 (Figure 2c), but not to K10 (Figure 2d). We have recently reported that KRTAP2-1 protein specifically bound to the head domain of hair keratins (Fujikawa et al., 2012Fujikawa H. Fujimoto A. Farooq M. et al.Characterization of the human hair keratin-associated protein 2 (KRTAP2) gene family.J Invest Dermatol. 2012; 132: 1806-1813Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Here, we attempted to determine the domain of cuticle-specific hair keratins responsible for their interaction with KRTAP10-1. To this end, we generated expression constructs for the truncated K82 (Supplementary Table S1 online), and performed co-IP assays in HEK293T cells, together with glutathione S-transferase (GST) pull-down assays. The co-IP assays showed that only K82 proteins with the head domain were co-immunoprecipitated with HA-KRTAP10-1 (Figure 2e). For the GST pull-down assays, expression of the truncated K82 proteins with N-terminal GST and Flag tags was induced in DH10B-competent cells (Invitrogen, Carlsbad, CA) by the addition of 1.0mM isopropyl-β-D-thiogalactopyranoside at 37°C for 3 hours. The proteins were isolated from the bacterial lysates using Glutathione Sepharose beads (GE Healthcare, Piscataway, NJ), which were then incubated with lysate from HEK293T cells overexpressing HA-KRTAP10-1 protein. The results demonstrated that the head domain of K82 showed affinity to HA-KRTAP10-1, whereas the rod or the tail domain did not (Figure 2f). On the basis of these results, we conclude that K82 binds to KRTAP10-1 protein via its head domain.Figure 2Characterization of human KRTAP10-1 protein. Co-immunoprecipitation (co-IP) between hemagglutinin (HA)-KRTAP10-1 and Flag-KRTAP10-1. (b) Co-IP between HA-KRTAP10-1 and K85. (c) Co-IP between HA-KRTAP10-1 and Flag-K32, and between HA-KRTAP10-1 and Flag-tagged K82 (Flag-K82). The cell lysate was precipitated with anti-HA agarose beads (a–c). (d) Co-IP between HA-KRTAP10-1 and Flag-K10, and between HA-KRTAP10-1 and Flag-K32. (e) Co-IP between HA-KRTAP10-1 and Flag-tagged truncated K82 proteins. Full-length K82 (Flag-K82-F) was used as a positive control. Amino acid residues for each domain of K82 are shown on the top panel. The cell lysate was precipitated with anti-Flag agarose beads (d, e). (f) GST pull-down assays. GST-Flag fusion proteins for truncated K82 and HA-KRTAP10-1 proteins were overexpressed in competent cells and HEK293T cells, respectively. Schematic representation of each fusion protein is shown on the top panel. WB, western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we described the biological features of the hair shaft cuticle–specific KRTAP10 proteins, which displayed characteristics similar to the hair shaft cortex–specific KRTAP2 proteins investigated previously (Fujikawa et al., 2012Fujikawa H. Fujimoto A. Farooq M. et al.Characterization of the human hair keratin-associated protein 2 (KRTAP2) gene family.J Invest Dermatol. 2012; 132: 1806-1813Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). It has been established in previous studies that KRTAP10 proteins are abundantly expressed in the exocuticle of the keratinized cells, as well as in the keratinizing cells of the hair shaft cuticle (Jones et al., 2010Jones L. Rogers G. Rufaut N. et al.Location of keratin-associated proteins in developing fiber cuticle cells using immunoelectron microscopy.Int J Trichology. 2010; 2: 89-95Crossref PubMed Scopus (10) Google Scholar). Although the exact location of the KIFs in the cuticle cells remains to be elucidated (Takizawa et al., 1998Takizawa T. Takizawa T. Arai S. et al.Ultrastructure of human scalp hair shaft as revealed by freeze-substitution fixation.Anat Rec. 1998; 251: 406-413Crossref PubMed Scopus (22) Google Scholar), our data indicate that, similar to the hair shaft cortex, the close interaction between hair keratins and KRTAPs by disulfide bond cross-linking and also potentially by additional bonds, such as ionic and hydrophobic bonds, may have a crucial role in forming the rigid structure of the hair shaft cuticle. We thank Drs Satoshi Ishii (Akita University, Japan) and Junichi Miyazaki (Osaka University, Japan) for supplying the pCXN2.1 vector. We also thank Dr Lutz Langbein (German Cancer Research Center, Germany) for providing the anti-K85 antibody. This study was supported in part by the Special Coordination Funds for Promoting Science and Technology, the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a grant for the Promotion of Niigata University Research Projects (155451-522000) to Y.S. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid" @default.
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• W1997400162 title "Characterization of the Human Hair Shaft Cuticle–Specific Keratin-Associated Protein 10 Family" @default.
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