Structure-Function Analyses of a Keratin Heterotypic Complex Identify Specific Keratin Regions Involved in Intermediate Filament Assembly.

Intermediate filaments (IFs) provide vital mechanical support in a broad array of cell types. Interference with this role causes cell fragility and accounts for a large number of human diseases. Gaining an understanding of the structure of IFs is paramount to understanding their function and designing therapeutic agents for relevant diseases. Here, we report the 2.6-Å resolution crystal structure of a complex of interacting 2B domains of keratin 5 (K5) and K14. K5 and K14 form a long-range, left-handed coiled coil, with participating α helices aligned in parallel and in register. Follow-up mutagenesis revealed that specific contacts between interacting 2B domains play a crucial role during 10-nm IF assembly, likely at the step of octamer-octamer association. The resulting structural model represents an atomic-resolution visualization of 2B-2B interactions important to filament assembly and provides insight into the defects introduced by mutations in IF genes associated with human skin diseases.

Epithelial cell migration requires the interaction between the vimentin and keratin intermediate filaments.

Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

Complementary roles of specific cysteines in keratin 14 toward the assembly, organization, and dynamics of intermediate filaments in skin keratinocytes.

We recently showed that inter-keratin disulfide bonding plays an important role in the assembly, organization, and dynamics of keratin intermediate filaments in skin keratinocytes. In particular, cysteine 367 located in the central α-helical rod domain of keratin 14 is necessary for the formation of a stable perinuclear network of keratin filaments (with type II partner keratin 5) in skin keratinocytes analyzed by static and live cell imaging. Here, we show that two additional cysteine residues located in the non-helical head domain of K14, Cys-4 and Cys-40, also participate in inter-keratin disulfide bonding and tandemly play a key role complementary to that of Cys-367 in the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in primary culture. Analysis of K14 variants with single or multiple substitutions of cysteine residues points to a spatial and temporal hierarchy in how Cys-4/Cys-40 and Cys-367 regulate keratin assembly in vitro and filament dynamics in live keratinocytes in culture. Our findings substantiate the importance and complexity of a novel determinant, namely inter-keratin disulfide bonding, for the regulation of several aspects of keratin filaments in surface epithelia.

Novel KRT14 mutation causing epidermolysis bullosa simplex with variable phenotype.

About 75% of cases of epidermolysis bullosa simplex result from mutations in KRT5 and KRT14 genes. Here, we report a family with a novel heterozygous missense mutation p.Leu418Gln in the KRT14 gene causing EBS of phenotype varying from EBS-loc to EBS-gen intermed. To the best of our knowledge, the family reported by us is the largest one in which two different phenotypes can be distinguished. The molecular dynamics simulations show that p.Leu418Gln mutation results in clear disruption of intermolecular π-stacking between KRT14:Tyr415 and KRT5:Tyr470, which in turn may affect putative phosphorylation site at KRT14:Thr414. This study further supports the importance of the EIATYR/KLLEGE motif in maintaining structural stability of KRT14:KRT5 heterodimer and indicates that physical properties of introduced amino acid can modulate the disease severity. The results obtained indicate further need of genotype-phenotype studies in EBS. In conclusion, genotype-based prognosis should be given to patients with caution.

In silico analysis of all point mutations on the 2B domain of K5/K14 causing epidermolysis bullosa simplex: a genotype-phenotype correlation.

Epidermolysis bullosa simplex (EBS) is a genodermatosis caused by mutations in keratins 5 and 14 (K5 and K14), which leads to fragility of basal keratinocytes and eventually epidermal cytolysis and blistering. Depending upon the severity of symptoms, EBS is classified into three major subtypes. In order of increasing severity these classes are EBS, localized (EBS-loc), EBS, other generalized (EBS, gen-nonDM), and EBS, Dowling-Meara (EBS-DM). We have searched and assembled 36 previously reported point mutations located on the 2B domain of K5/K14 in order to investigate the effects of point mutations. By performing a comprehensive in silico analysis we determine the underlying relationship between the mutation and its phenotypic effects. Our result showed that all pathogenic point mutations exert their dominant negative effect on the K5/K14 coiled-coil heterodimer complex by altering interchain interaction, leading to the changes in stability and assembly competence of the heterodimer complex. The physico-chemical properties of substituted amino acid and location of the mutation are also deeply correlated with disease severity. In addition, we found a SNP previously reported as non-pathogenic (K14 p.M338R) that likely affects the stability of the dimer structure due to the loss of interchain interaction and steric clashes. Overall, our finding demonstrates the significance of in silico characterization of EBS severity and would allow for accurate genetic counseling and prenatal diagnosis.

Interaction of plectin with keratins 5 and 14: dependence on several plectin domains and keratin quaternary structure.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.

Isolation and in vitro characterization of basal and submucosal gland duct stem/progenitor cells from human proximal airways.

Basal cells and submucosal gland (SMG) duct cells have been isolated and shown to be stem/progenitor cell populations for the murine airway epithelium. However, methods for the isolation of basal and SMG duct cells from human airways have not been defined. We used an optimized two-step enzyme digestion protocol to strip the surface epithelium from tracheal specimens separate from SMG cells, and we then sorted the basal and duct stem/progenitors using fluorescence-activated cell sorting. We used nerve growth factor receptor, as well as a combination of CD166 and CD44, to sort basal cells and also used CD166 to isolate SMG duct cells. Sorted stem/progenitor cells were cultured to characterize their self-renewal and differentiation ability. Both basal and SMG duct cells grew into spheres. Immunostaining of the spheres showed mostly dense spheres with little to no central lumen. The spheres expressed cytokeratins 5 and 14, with some mucus- and serous-secreting cells. The sphere-forming efficiency and the rate of growth of the spheres varied widely between patient samples and correlated with the degree of hyperplasia of the epithelium. We found that only aldehyde dehydrogenase (ALDH)(hi) basal and duct cells were capable of sphere formation. Global inhibition of ALDH, as well as specific inhibition of the ALDH2 isoform, inhibited self-renewal of both basal and duct cells, thereby producing fewer and smaller spheres. In conclusion, we have developed methods to isolate basal and SMG duct cells from the surface epithelium and SMGs of human tracheas and have developed an in vitro model to characterize their self-renewal and differentiation.

The mechanical behavior of mutant K14-R125P keratin bundles and networks in NEB-1 keratinocytes.

Epidermolysis bullosa simplex (EBS) is an inherited skin-blistering disease that is caused by dominant mutations in the genes for keratin K5 or K14 proteins. While the link between keratin mutations and keratinocyte fragility in EBS patients is clear, the exact biophysical mechanisms underlying cell fragility are not known. In this study, we tested the hypotheses that mutant K14-R125P filaments and/or networks in human keratinocytes are mechanically defective in their response to large-scale deformations. We found that mutant filaments and networks exhibit no obvious defects when subjected to large uniaxial strains and have no negative effects on the ability of human keratinocytes to survive large strains. We also found that the expression of mutant K14-R125P protein has no effect on the morphology of the F-actin or microtubule networks or their responses to large strains. Disassembly of the F-actin network with Latrunculin A unexpectedly led to a marked decrease in stretch-induced necrosis in both WT and mutant cells. Overall, our results contradict the hypotheses that EBS mutant keratin filaments and/or networks are mechanically defective. We suggest that future studies should test the alternative hypothesis that keratinocytes in EBS cells are fragile because they possess a sparser keratin network.

The yin and the yang of keratin amino acid substitutions and epidermolysis bullosa simplex.

Mutations that change the same amino acid can result in different clinical phenotypes. Through in silico modeling and keratin filament assessment of genetically engineered HaCaT cells, Natsuga et al., as reported in this issue, have demonstrated how changes in charge and structure of a replacement amino acid in keratin 14 can cause disease (KRT14pA413P, EB simplex) or no clinical effect (KRT14pA413T, polymorphism).

Integrating chemistry and immunology in allergic contact dermatitis: more questions than answers?

In this issue, Simonsson and colleagues shed light on the chemical mechanisms determining hapten formation in the skin, which precede the elicitation of an antigen-specific immune response in allergic contact dermatitis. Combining fluorescence microscopy, proteomics, and mass spectrometry, the investigators identified keratins K5 and K14, particularly cysteine 54 of K5, in the human basal epidermal layer as the major molecular targets of caged thiol-reactive fluorescent haptens (i.e., bromobimanes). Anti-keratin antibody responses in mice exposed to bromobimanes suggest the generation of immunogenic epitopes by cysteine-reactive haptens. Although many issues await further investigation, Simonsson and co-workers' observations advance our understanding of the molecular basis of hapten-protein complex formation in skin.

Consequences of two different amino-acid substitutions at the same codon in KRT14 indicate definitive roles of structural distortion in epidermolysis bullosa simplex pathogenesis.

Numerous inherited diseases develop due to missense mutations, leading to an amino-acid substitution. Whether an amino-acid change is pathogenic depends on the level of deleterious effects caused by the amino-acid alteration. We show an example of different structural and phenotypic consequences caused by two individual amino-acid changes at the same position. Epidermolysis bullosa simplex (EBS) is a genodermatosis resulting from KRT5 or KRT14 mutations. Mutation analysis of an EBS family revealed that affected individuals were heterozygous for a, to our knowledge, previously unreported mutation of c.1237G>C (p.Ala413Pro) in KRT14. Interestingly, 2 of 100 unrelated normal controls were heterozygous, and 1 of the 100 was homozygous for a different mutation in this position, c.1237G>A (p.Ala413Thr). In silico modeling of the protein demonstrated deleterious structural effects from proline substitution but not from threonine substitution. In vitro transfection studies revealed a significantly larger number of keratin-clumped cells in HaCaT cells transfected with mutant KRT14 complementary DNA (cDNA) harboring p.Ala413Pro than those transfected with wild-type KRT14 cDNA or mutant KRT14 cDNA harboring p.Ala413Thr. These results show that changes in two distinct amino acids at a locus are destined to elicit different phenotypes due to the degree of structural distortion resulting from the amino-acid alterations.

Autoantibodies in scurfy mice and IPEX patients recognize keratin 14.

Scurfy mice have a deletion in the Foxp3 gene, resulting in a failure to generate Foxp3(+) regulatory T cells, and they subsequently develop severe CD4(+) T-cell-mediated autoimmune inflammation. Multiple organs are involved, but the skin is one of the main organs affected. During the course of disease, Scurfy mice develop autoantibodies; however, the targeted antigens are unknown. In this study, we show that Scurfy mice develop autoantibodies directed against skin antigens. Using western blot analysis, we found that Scurfy serum reacted with proteins in total skin lysate, as well as in a keratinocyte lysate. Most of the Scurfy sera tested identified a major band at 50 kDa. Transfer of Scurfy CD4(+) T cells into nu/nu mice yielded autoantibodies with similar reactivity. Further analysis using 2D western blots, followed by peptide mass fingerprinting, identified several keratins as targets. To confirm this observation, we chose one of the identified targets, keratin 14, and prepared recombinant proteins encompassing the N-terminal, middle, and C-terminal portions of the keratin 14 protein. Scurfy serum predominantly recognized the C-terminal fragment. Sera from patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, the human disease resulting from FOXP3 mutations, also recognized skin antigens, including keratin 14. Thus, the results of our study indicate that autoantibodies in Scurfy mice and patients with IPEX target keratins.

[Interaction of human cytokeratins with isatin analogues].

Using an optical biosensor Biacore 3000 the interaction of human recombinant cytokeratins (CK) with isatin analogues (5-aminocaproyl-isatin and 5-aminoisatin) immobilized on the CM-5 chip has been investigated. CK-14 effectively interacted with 5-aminocaproyl-isatin immobilized on the carboxymethyl dextran chip surface, but not with a "shorter" analogue (5-aminoisatin). In contrast to CK14 CK8 effectively interacted only with 5-aminoisatin. In both cases cytokeratin binding with the immobilized isatin analogues was characterized by rather high affinity (Kd of 0.7 microM for the pair CK14/immobilized 5-aminocaproylisatin and 1.7 microM for the pair CK8/immobilized 5-aminoisatin). CK20 did not interact with both immobilized isatin analogues. Taking into consideration non-specific binding of mouse CK14 and rat CK8 with 5-aminocaproyl-Sepharose we have performed comparative analysis of amino acid sequences of human, mouse, and rat CK8 and CK14. The data obtained suggest that in the case of human, mouse, and rat CK14 the N-terminal domain is the most variable among these species, whereas the major differences between amino acid sequences of human, mouse, and rat CK8 have been found both in N-terminal and C-terminal regions.

Mass spectrometric identification of covalent adducts of the skin allergen 2,4-dinitro-1-chlorobenzene and model skin proteins.

A large proportion of allergic skin reactions are considered to be the result of skin exposure to small organic chemicals that possess the intrinsic ability to covalently modify skin proteins, either directly or following activation. In the absence of information about specific skin protein targets, studies of chemical modifications are limited to the use of model proteins. We have previously demonstrated that selected well known skin sensitizers (2,4-dinitro-1-chlorobenzene and phenyl salicylate) have the ability to covalently modify residues selectively on the model protein, human serum albumin. In the present work, we focus on the differences in covalent binding observed for two additional model proteins, human cytokeratin 14 and human cofilin, both constituent proteins of skin. Using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano LC-MS and -MS/MS strategies, the amino acid residues targeted by 2,4-dinitro-1-chlorobenzene on the two model proteins have been identified. In contrast, a structurally related non-sensitiser (2,4-dichloro-1-nitrobenzene) and a non-sensitising irritant (benzalkonium chloride) did not covalently modify the model proteins. Detailed examination of the results for the sensitizers indicate that reactive chemicals target nucleophilic amino acids residing in specific microenvironments of the 3D protein structure that are conducive to reactivity. This observation has important implications for the development of hapten-peptide binding assays. It is envisaged that the data from such assays will be integrated with outputs from other in vitro assays in the future to give a prediction of the sensitisation potential of novel chemicals.

Identification of a keratin-associated protein with a putative role in vesicle transport.

Protection of skin against UV light requires a coordinated interaction between melanocytes and keratinocytes. Melanosomes are lysosome-related organelles that originate in melanocytes and are transferred into keratinocytes where they form a supranuclear cap. The mechanism responsible for melanosome transfer into keratinocytes and their intracellular distribution is poorly understood. Recently, we reported for the first time that loss-of-function mutations in the keratin K5 gene affect melanosome distribution in keratinocytes and results in a reticulate hyperpigmentation disorder, called Dowling-Degos disease. Here, we characterise the distribution and behaviour of individual K5 and K14 domains following transient and stable transfection into cells. We report that the K5 head domain is considerably more stable than the K14 head. Moreover, the distribution of the K5 head domain is altered following depolymerisation of microtubules. Following co-immunoprecipitation, we verified a specific interaction between the head domain of K5 with Hsc70, a chaperone also involved in vesicle uncoating. We hypothesise that this interaction is involved in melanosome formation or transport in keratinocytes. Alternatively, it may have a general function in the regulation of keratin assembly.