Recombinant Human Hair Keratin Nanoparticles Accelerate Dermal Wound Healing.

In recent years, favorable enhanced wound-healing properties and excellent biocompatibility of keratin derived from human hair have attracted considerable attention. Recombinant keratin proteins can be produced by recombinant DNA technology and have higher purity than extracted keratin. However, the wound-healing properties of recombinant keratin proteins remain unclear. Herein, two recombinant trichocyte keratins including human type I hair keratin 37 and human type II hair keratin 81 were expressed using a bacterial expression system, and recombinant keratin nanoparticles (RKNPs) were prepared via an ultrasonic dispersion method. The molecular weight, purity, and physicochemical properties of the recombinant keratin proteins and nanoparticles were assessed using gel electrophoresis, circular dichroism, mass spectrometry, and scanning electron microscope analyses. The RKNPs significantly enhanced cell proliferation and migration in vitro, and the treatment of dermal wounds in vivo with RKNPs resulted in improved wound healing associated with improved epithelialization, vascularization, and collagen deposition and remodeling. In addition, the in vivo biocompatibility test revealed no systemic toxicity. Overall, this work demonstrates that RKNPs are a promising candidate for enhanced wound healing, and this study opens up new prospects for the development of keratin biomaterials.

Localisation of keratin K78 in the basal layer and first suprabasal layers of stratified epithelia completes expression catalogue of type II keratins and provides new insights into sequential keratin expression.

Among the 26 human type II keratins, K78 is the only one that has not yet been explored with regard to its expression characteristics. Here, we show that, at both the transcriptional and translational levels, K78 is strongly expressed in the basal and parabasal cell layers with decreasing intensity in the lower suprabasal cells of keratinising and non-keratinising squamous epithelia and keratinocyte cultures. The same pattern has been detected at the transcriptional level in the corresponding mouse epithelia. Murine K78 protein, which contains an extraordinary large extension of its tail domain, which is unique among all known keratins, is not detectable by the antibody used. Concomitant studies in human epithelia have confirmed K78 co-expression with the classical basal keratins K5 and K14. Similarly, K78 co-expression with the differentiation-related type I keratins K10 (epidermis) and K13 (non-keratinising epithelia) occurs in the parabasal cell layer, whereas that of the corresponding type II keratins K1 (epidermis) and K4 (non-keratinising epithelia) unequivocally starts subsequent to the respective type I keratins. Our data concerning K78 expression modify the classical concept of keratin pair K5/K14 representing the basal compartment and keratin pairs K1/K10 or K4/K13 defining the differentiating compartment of stratified epithelia. Moreover, the K78 expression pattern and the decoupled K1/K10 and K4/K13 expression define the existence of a hitherto unperceived early differentiation stage in the parabasal layer characterized by K78/K10 or K78/K13 expression.

In vitro assembly properties of human type I and II hair keratins.

Multiple type I and II hair keratins are expressed in hair-forming cells but the role of each protein in hair fiber formation remains obscure. In this study, recombinant proteins of human type I hair keratins (K35, K36 and K38) and type II hair keratins (K81 and K85) were prepared using bacterial expression systems. The heterotypic subunit interactions between the type I and II hair keratins were characterized using two-dimensional gel electrophoresis and surface plasmon resonance (SPR). Gel electrophoresis showed that the heterotypic complex-forming urea concentrations differ depending on the combination of keratins. K35-K85 and K36-K81 formed relatively stable heterotypic complexes. SPR revealed that soluble K35 bound to immobilized K85 with a higher affinity than to immobilized K81. The in vitro intermediate filament (IF) assembly of the hair keratins was explored by negative-staining electron microscopy. While K35-K81, K36-K81 and K35-K36-K81 formed IFs, K35-K85 afforded tight bundles of short IFs and large paracrystalline assemblies, and K36-K85 formed IF tangles. K85 promotes lateral association rather than elongation of short IFs. The in vitro assembly properties of hair keratins depended on the combination of type I and II hair keratins. Our data suggest the functional significance of K35-K85 and K36-K81 with distinct assembly properties in the formation of macrofibrils.

Purification of porcine hair keratin subunits and their immobilization for use as cell culture substrates.

We purified both the type I subunit and type II subunit of porcine hair keratin and compared their ability to form a uniform film of reconstituted keratin on a culture plate, and their effect on a model of neural cells. We observed the surface of the keratin-immobilized plate using a scanning electron microscope (SEM) and measured water contact angles to characterize the surface. We cultured PC12 cells on plates on which crude keratin, the type I subunit, or the type II subunit were immobilized. The water contact angles were slightly different from each other. The cells proliferated well on all three keratin-immobilized plates. The type II subunit showed a tendency to inhibit the differentiation of PC12 cells significantly as an extension of the cell shapes and neurite outgrowth in comparison with the crude extract and the type I subunit. The type I subunit and the type II subunit showed slight differences in cell differentiation, but not in cell proliferation.

Bidirectional binding property of high glycine-tyrosine keratin-associated protein contributes to the mechanical strength and shape of hair.

Since their first finding in wool 50years ago, keratin-associated proteins (KAPs), which are classified into three groups; high sulfur (HS) KAPs, ultra high sulfur (UHS) KAPs, and high glycine-tyrosine (HGT) KAPs, have been the target of curiosity for scientists due to their characteristic amino acid sequences. While HS and UHS KAPs are known to function in disulfide bond crosslinking, the function of HGT KAPs remains unknown. To clarify the function as well as the binding partners of HGT KAPs, we prepared KAP8.1 and other KAP family proteins, the trichocyte intermediate filament proteins (IFP) K85 and K35, the head domain of K85, and the C subdomain of desmoplakin C-terminus (DPCT-C) and investigated the interactions between them in vitro. Western blot analysis and isothermal titration calorimetry (ITC) indicate that KAP8.1 binds to the head domain of K85, which is helically aligned around the axis of the intermediate filament (IF). From these results and transmission electron microscopy (TEM) observations of bundled filament complex in vitro, we propose that the helical arrangement of IFs found in the orthocortex, which is uniquely distributed on the convex fiber side of the hair, is regulated by KAP8.1. Structure-dependent binding of DPCT-C to trichocyte IFP was confirmed by Western blotting, ITC, and circular dichroism. Moreover, DPCT-C also binds to some HGT KAPs. It is probable that such bidirectional binding property of HGT KAPs contribute to the mechanical robustness of hair.

Electrophoretic mapping of highly homologous keratins: a novel marker peptide approach.

Identification of the intermediate filament proteins (IFPs) in the wool proteome has formerly been hampered by limited sequence information, the high degree of IFP homology and their close proximity on 2-DE maps. This has been partially rectified by the recent acquisition of four new Type I and two Type II wool IFP sequences. Among closely migrating proteins, such as IFP clusters in a 2-DE map, proteins with higher sequence coverage will be assigned higher scores, but the identification of unique peptides in such tight clusters may distinguish these closely migrating proteins. Two approaches were adopted for the study of wool IFPs. In the first, searches were conducted for peptides known to be unique to each member of the family in each spot. In the second, MALDI imaging was employed to examine peptides bound to a PVDF membrane from a poorly resolved part of the Type I IFP region of the 2-DE map. As a result, a distinct picture has emerged of the distribution of the six Type I and four Type II IFPs across the 2-DE wool protein map.

Mice expressing a mutant Krt75 (K6hf) allele develop hair and nail defects resembling pachyonychia congenita.

KRT75 (formerly known as K6hf) is one of the isoforms of the keratin 6 (KRT6) family located within the type II cytokeratin gene cluster on chromosome 12 of humans and chromosome 15 of mice. KRT75 is expressed in the companion layer and upper germinative matrix region of the hair follicle, the medulla of the hair shaft, and in epithelia of the nail bed. Dominant mutations in members of the KRT6 family, such as in KRT6A and KRT6B cause pachyonychia congenita (PC) -1 and -2, respectively. To determine the function of KRT75 in skin appendages, we introduced a dominant mutation into a highly conserved residue in the helix initiation peptide of Krt75. Mice expressing this mutant form of Krt75 developed hair and nail defects resembling PC. This mouse model provides in vivo evidence for the critical roles played by Krt75 in maintaining hair shaft and nail integrity. Furthermore, the phenotypes observed in our mutant Krt75 mice suggest that KRT75 may be a candidate gene for screening PC patients who do not exhibit obvious mutations in KRT6A, KRT6B, KRT16, or KRT17, especially those with extensive hair involvement.

Sequence analyses of Type I and Type II chains in human hair and epithelial keratin intermediate filaments: promiscuous obligate heterodimers, Type II template for molecule formation and a rationale for heterodimer formation.

Sequence comparisons have been undertaken for all hair and epithelial keratin IF chains from a single species--human. The results lead to several new proposals. First, it is clear that not only is the chain structure of the molecule an obligate heterodimer but promiscuous association of Type I and Type II chains must occur in vivo. Second, the higher predicted content of alpha-helix in Type II chains in solution relative to that expected for Type I chains suggests that it is the Type II chains that precede their Type I counterparts and that they may serve as templates for molecule formation. Third, heterodimer formation leads naturally to greater structural and functional specificity, and this may be required not only because keratin IF have more interacting partners in its cell type than other types of IF have in theirs but also because hair and skin IF have two distinct structures that relate to the "reducing" or "oxidizing" environment in which they can find themselves. The transition between the two forms may require specific head/tail interactions and this, it is proposed, would be more easily accomplished by a heterodimer structure with its greater in-built specificity.