Keratins, together with microtubules and microfilaments, are cytoskeletal filament-forming proteins that are critical for cytoarchitecture and composition of skin and other epithelia . The type I/type II keratin heterodimers are expressed in highly specific pairs (e.g., KRT1/KRT10, KRT3/KRT12, KRT4/KRT13, KRT5/KRT14, KRT6A/KRT16, KRT6B/KRT17) depending on tissue type and differentiation stage [27, 28]. A growing number of keratin gene mutations have been found to be causative factors for a wide range of skin, hair, and mucosal diseases .
EPPK is characterized by diffuse severe hyperkeratosis of palms and soles, whose pathogenic genes are identified as KRT9 and KRT1 . At present, 29 KRT9 and a few KRT1 mutations identified in EPPK have been documented in the HIFD and Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk) . These mutations are disruptive to type I/type II filament assembly via dominant negative effect, causing abnormal perinuclear tonofilament clumping and large distorted keratohyalin granules .
PC is characterized by hypertrophic nail dystrophy, painful palmoplantar keratoderma, oral leukokeratosis, pilosebaceous cysts, and follicular keratoses . The reported causative genes for PC are the keratin 6A gene (KRT6A, OMIM: 148041), the keratin 6B gene (KRT6B, OMIM: 148042), KRT6C, the keratin 16 gene (KRT16, OMIM: 148067), and the keratin 17 gene (KRT17, OMIM: 148069) . Based on the pathogenic genetic factor, PC is classified as PC-K6a (OMIM: 615726), PC-K6b (OMIM: 615728), PC-K6c, PC-K16 (OMIM: 167200), and PC-K17 (OMIM: 167210) . Data in the International PC Research Registry (IPCRR, www.pachyonychia.org/) shows that only 3% (22/774) of PCs are attributed to pathogenic KRT6C mutations, while KRT6A, KRT6B, KRT16 and KRT17 mutations account for 39% (304/774), 9% (70/774), 32% (247/774), and 17% (130/774) of PCs, respectively. Although the clinical diagnostic criterion for PC is the triad of toenail thickening, plantar keratoderma, and plantar pain . In the PC-K6c patients, only 59% (13/22) of them had mild toenail dystrophy, while 96%~99% of PC patients carrying mutations in the other four keratin genes had toenail thickened .
To date, most identified type I/type II keratin heterodimers were discovered by conforming empirical data with co-localization assay or using yeast-two hybrid analysis [32, 33]. In this study, we discovered that KRT6C is the type II keratin-binding partner for KRT9 in human sole skin by using co-immunoprecipitation coupled with mass spectroscopy. The KRT6C gene, belonging to the type II keratins clustered in chromosome 12q13, consists of nine exons, and encodes 564 amino acids (NCBI GenBank database). The three KRT6 isoforms (KRT6A, KRT6B and KRT6C), encoded by three functional isogenes (KRT6A, KRT6B and KRT6C), only differ by 7 separated amino acid substitutions. This fact has enhanced the difficulty in distinguishing them using effective antibodies . Therefore, the keratin pair remains unidentified until now. By using co-immunoprecipitation coupled with mass spectroscopy, we identified KRT6C/KRT9 interaction in a native endogenous environment. In this way, this method functions as a sensitive and powerful way to identify novel protein interactions with physiological importance. By performing in silico model for KRT6C/KRT9 interaction and multiple sequence alignment of several related intermediate filament proteins, we propose that such interaction is significant for the formation of highly organized KRT6C/KRT9 heterodimers and tetramers.
Traditional classification of keratin-associated cutaneous disorders was generally based on morphologic appearance of skin lesions and presence or absence of extracutaneous symptoms and signs . Growing evidence of pathogenic keratin mutations for hereditary cutaneous disorders and their overlapping phenotypes have facilitated a molecular-based classification of hereditary cutaneous disorders. For instance, mutations in either of the keratin pair KRT5/KRT14 are causative for epidermolysis bullosa simplex (EBS); KRT6A/KRT16 and KRT6B/KRT17 for PC; KRT1/KRT10 for epidermolytic ichthyosis (EI, OMIM: 113800) [27, 36].
In the case for keratin pair KRT6C/KRT9, underlying trace evidence for functional correlation between the two keratin proteins can be discovered by carefully reviewing previous studies. In the Krt9+/mut, Krt9mut/mut and Krt9−/− mice, KRT6 increased significantly in response to KRT9 dysfunction in the footpad [17, 20]. In comparison, KRT1 showed no obvious expression level or location alteration in the Krt9+/−, Krt9+/mut and Krt9−/− mice and increased in the Krt9mut/mut mice [17, 20]. KRT5 showed no obvious expression alteration in the Krt9+/− and Krt9+/mut mice and decreased in the Krt9−/− and Krt9mut/mut mice [17, 20]. In the PC-K6a patients, whose KRT6A, KRT6B and KRT6C transcripts were increased, KRT9 showed dramatic decrease . The relationship between KRT9 and its putative partner KRT1 or KRT5 seems ambiguous. The interesting reciprocal correlation between KRT9 and KRT6 expression suggest that the keratin pair KRT6C/KRT9 may be collectively involved in EPPK and PC-K6c pathogenesis.
The functional correlation between KRT9 and KRT6C may be used to explain the phenotypic overlap in EPPK and PC-K6c. EPPK is characterized by palmplantar hyperkeratosis, and PC-K6c by focal palmplantar hyperkeratosis with minor nail involvement [2, 38]. For EPPK kindreds, whose causative mutations were not identified in KRT9 or KRT1, it is reasonable to carefully examine KRT6C to seek for potential mutations. Nonetheless, KRT9 seems to function as a more important cytoskeletal protein in keratinocytes to maintain cell structure and function than KRT6C. More KRT9 mutations (about 29 mutations) have been identified than KRT6C mutations (about 4 mutations), indicating that occurrence of KRT9 mutations in the conservative sites are more likely to cause disease phenotypes. KRT6C gene variants were also identified in participants with no clinical symptoms or signs of genodermatoses, implying that these variants were not severe enough to produce a clinical phenotype [22, 31].
Combined with the proteomics analysis result of the KRT9-related differential protein data produced by Metascape, which integrated a broad set of current biological datasets, we realized that the keratin pair KRT6C/KRT9 together with the keratin 19 (KRT19, OMIM: 148020) play an important role in intermediate filament-based cytoskeleton organization, keratinization, and formation of the cornified envelope. The dysfunction of KRT6C/KRT9 may trigger an imbalance in a subset of closely related keratin family and disrupt epidermal desquamation homeostasis. The produced structurally weakened keratinocytes and compromised epidermis may thus more likely prone to external stimuli, stress, and injury. Due to different KRT6C/KRT9 mutation type and different degree of conservativeness in various mutation sites, a certain range of subtly different phenotypes may be produced in the context of various genetic and environmental backgrounds [17, 36]. Therefore, it may be more reasonable and useful to consider disorders caused by the KRT6C/KRT9 keratin pair as a group of hereditary defects, as the role of KRT5/KRT14 for EBS and KRT1/KRT10 for EI mentioned above.