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.

A novel de novo mutation p.Ala428Asp in KRT5 gene as a cause of localized epidermolysis bullosa simplex.

Epidermolysis bullosa is a group of inherited blistering skin diseases resulting in most cases from missense mutations in KRT5 and KRT14 genes encoding the basal epidermal keratins 5 and 14. Here, we present a patient diagnosed with a localized subtype of epidermolysis bullosa simplex caused by a heterozygous mutation p.Ala428Asp in the KRT5 gene, that has not been previously identified. Moreover, a bioinformatic analysis of the novel mutation was performed, showing changes in the interaction network between the proteins. Identification of novel mutations and genotype-phenotype correlations allow to better understanding of underlying pathophysiologic bases and is important for genetic counselling, patients' management, and disease course prediction.

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.

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.

Elucidated lipid structures of various human stratum corneum investigated by electron paramagnetic resonance spectroscopy.

BACKGROUND: Electron paramagnetic resonance (EPR) in conjunction with a slow-tumbling simulation is useful for defining stratum corneum (SC) lipid structure. The objectives of this investigation were to evaluate and elucidate the lipid structure of various human SC as a function of the depth. METHODS: The SC from mid-volar forearms of human volunteers and a cadaver was stripped consecutively from one to three times using a glass plate coated with a cyanoacrylate resin. Spin probes were used to monitor SC ordering. EPR spectra were analyzed by an EPR slow-tumbling simulation. Keratin solution from human epidermis was mixed with 5-doxylstearic acid (5-DSA) aqueous solution. RESULTS: EPR analyses of 5-DSA and CHL probes showed immobilized and mobile spectra, respectively. The simulation for 5-DSA spectra showed differences in ordering values of the SC as a function of depth. EPR of keratin/5-DSA showed mobile spectral pattern. In addition, EPR of CHL showed a weak and a broad signal. CONCLUSIONS: The keratin/5-DSA results imply that the most likely location of 5-DSA probe is in SC lipid. CHL probe may not easily permeate the SC. The EPR results along with the simulation analysis provide elucidated SC lipid structure.

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.