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Query: UNIPROT:P06889 (Mol)
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Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.
Mol Biol Cell 2004 Mar
PMID:Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits. 1466 78

Basal layers of stratified epithelia express keratins K5, K14, and K15, which assemble into intermediate filament networks. Mutations in K5 or K14 genes cause epidermolysis bullosa simplex (EBS), a disorder with blistering in the basal layer due to cell fragility. Nonkeratinizing stratified epithelia, e.g., in the esophagus, produce more keratin K15 than epidermis, which alleviates the esophageal symptoms in patients with K14 mutations. Hypothesizing that increasing the cellular content of K15 could compensate for the mutant K14 and thus ease skin blistering in K14 EBS patients, we cloned the promoter of the K15 gene and examined its transcriptional regulation. Using cotransfection, gel mobility shifts, and DNase I footprinting, we have identified the regulators of K15 promoter activity and their binding sites. We focused on those that can be manipulated with extracellular agents, transcription factors C/EBP, AP-1, and NF-kappaB, nuclear receptors for thyroid hormone, retinoic acid, and glucocorticoids, and the cytokine gamma interferon (IFN-gamma). We found that C/EBP-beta and AP-1 induced, while retinoic acid, glucocorticoid receptors, and NF-kappaB suppressed, the K15 promoter, along with other keratin gene promoters. However, the thyroid hormone and IFN-gamma uniquely and potently activated the K15 promoter. Using these agents, we could boost the amounts of K15 in human epidermis. Our findings suggest that treatments based on thyroid hormone and IFN-gamma could become effective agents in therapy for patients with EBS.
Mol Cell Biol 2004 Apr
PMID:Thyroid hormones and gamma interferon specifically increase K15 keratin gene transcription. 1506 Jan 41

Inherited mutations in the intermediate filament (IF) proteins keratin 5 (K5) or keratin 14 (K14) cause epidermolysis bullosa simplex (EBS), in which basal layer keratinocytes rupture upon trauma to the epidermis. Most mutations are missense alleles affecting amino acids located in the central alpha-helical rod domain of K5 and K14. Here, we study the properties of an unusual EBS-causing mutation in which a nucleotide deletion (1649delG) alters the last 41 amino acids and adds 35 residues to the C terminus of K5. Relative to wild type, filaments coassembled in vitro from purified K5-1649delG and K14 proteins are shorter and exhibit weak viscoelastic properties when placed under strain. Loss of the C-terminal 41 residues contributes to these alterations. When transfected in cultured epithelial cells, K5-1649delG incorporates into preexisting keratin IFs and also forms multiple small aggregates that often colocalize with hsp70 in the cytoplasm. Aggregation is purely a function of the K5-1649delG tail domain; in contrast, the cloned 109 residue-long tail domain from wild type K5 is distributed throughout the cytoplasm and colocalizes partly with keratin IFs. These data provide a mechanistic basis for the cell fragility seen in individuals bearing the K5-1649delG allele, and point to the role of the C-terminal 41 residues in determining K5's assembly properties.
Mol Biol Cell 2005 Mar
PMID:Defining the properties of the nonhelical tail domain in type II keratin 5: insight from a bullous disease-causing mutation. 1564 84

Epidermolysis bullosa (EB) is an inherited mechano-bullous disorder of the skin, and is divided into three major categories: EB simplex (EBS), dystrophic EB, and junctional EB (JEB). Mutations in the plectin gene (PLEC1) cause EBS associated with muscular dystrophy, whereas JEB associated with pyloric atresia (PA) results from mutations in the alpha6 and beta4 integrin genes. In this study, we examined three EB patients associated with PA from two distinct families. Electron microscopy detected blister formation within the basal keratinocytes leading to the diagnosis of EBS. Surprisingly, immunohistochemical studies using monoclonal antibodies to a range of basement membrane proteins showed that the expression of plectin was absent or markedly attenuated. Sequence analysis demonstrated four novel PLEC1 mutations. One proband was a compound heterozygote for a nonsense mutation of Q305X and a splice-site mutation of 1344G-->A. An exon-trapping experiment suggested that the splice-site mutation induced aberrant splicing of the gene. The second proband harbored a heterozygous maternal nonsense mutation, Q2538X and homozygous nonsense mutations R1189X. Analysis of the intragenic polymorphisms of PLEC1 suggested that R1189X mutations were due to paternal segmental uniparental isodisomy. These results indicate that PLEC1 is a possible causative gene in this clinical subtype, EBS associated with PA. Furthermore, two patients out of our three cases died in infancy. In terms of clinical prognosis, this novel subtype is the lethal variant in the EBS category.
J Mol Diagn 2005 Feb
PMID:Epidermolysis bullosa simplex associated with pyloric atresia is a novel clinical subtype caused by mutations in the plectin gene (PLEC1). 1568 71

Non-Herlitz junctional epidermolysis bullosa (nH-JEB) disease manifests with skin blistering, atrophy and tooth enamel hypoplasia. The majority of patients with nH-JEB harbor mutations in COL17A1, the gene encoding type XVII collagen. Heterozygotes with a single COL17A1 mutation, nH-JEB defect carriers, may exhibit only enamel hypoplasia. In this study, to further elucidate COL17A1 mutation phenotype/ genotype correlations, we examined two unrelated families with nH-JEB. Furthermore, we hypothesized that COL17A1 mutations might underlie or worsen the enamel hypoplasia seen in amelogenesis imperfecta (AI) patients that are characterized by defects in tooth enamel formation without other systemic manifestations. We therefore conducted COL17A1 mutational analysis in three patients from two AI families. One nH-JEB patient showed no COL17A1 expression and was a compound heterozygote for the novel premature termination codon (PTC) mutations 1285delA and Q1387X. In addition, reduced COL17A1 expression was found in a second nH-JEB patient who was homozygous for the novel PTC mutation 4335delC, the most carboxyl terminal PTC mutation thus far identified. Due to nonsense mediated mRNA decay, the position of these PTC mutations is thought not to influence the effect of COL17A1 transcript loss and hence the severity of the nH-JEB phenotype. This study is the first to suggest that type XVII collagen carboxyl PTC mutations lead to restoration of truncated polypeptide expression and to a milder clinical disease severity in nH-JEB. Conversely, we failed to detect any pathogenic COL17A1 defects in AI patients, in either exon or within the intron-exon borders of AI patients. This study furthers the understanding of mutations in COL17A1 causing nH-JEB, and clearly demonstrates that the mechanism of enamel hypoplasia differs between nH-JEB and AI diseases.
Int J Mol Med 2006 Aug
PMID:Analysis of the COL17A1 in non-Herlitz junctional epidermolysis bullosa and amelogenesis imperfecta. 1682 Sep 43

Epidermolysis bullosa (EB) and associated skin-fragility syndromes are a group of inherited skin diseases characterised by trauma-induced blistering of the skin and mucous membranes. Mutations in at least 14 distinct genes encoding molecular components of the epidermis or the dermal-epidermal junction (DEJ) can cause blistering skin diseases that differ by clinical presentation and severity of the symptoms. Despite great advances in discerning the genetic basis of this group of diseases, the molecular pathways leading to symptoms are not yet fully understood. Unravelling these pathways by molecular analysis of the structure and in vitro assessment of functional properties of the human proteins involved, combined with genetic models in lower organisms, should pave the way for specific cures for inherited skin fragility.
Expert Rev Mol Med 2006 Oct 13
PMID:Molecular basis of inherited skin-blistering disorders, and therapeutic implications. 1704 May 78

The intermediate filament (IF) cytoskeleton of mammalian epithelia is generated from pairs of type I and type II keratins that are encoded by two large gene families, made up of 54 genes in humans and the mouse. These genes are expressed in a spatiotemporal and tissue-specific manner from the blastocyst stage onward. Since the discovery of keratin mutations leading to epidermolysis bullosa simplex, mutations in at least 18 keratin genes have been identified that result in keratinopathies of the epidermis and its appendages. Recently, noncanonical mutations in simple epithelial keratins were associated with pancreatic, liver, and intestinal disorders, demonstrating that keratins protect epithelia against mechanical and other forms of stress. In recent years, animal models provided novel insight and significantly improved understanding of IF function in tissue homeostasis and its role in disease. Pathological phenotypes detected in mutant mice generated so far range from embryonic lethality to tissue fragility to subtlety, which often depends on their genetic background. This range implies at least a partial influence of yet unidentified modifier genes on the phenotype after the ablation of the respective keratin. To date, nearly all available keratin mouse models were generated by taking advantage of conventional gene-targeting strategies. To reveal their cell type-specific functions and the mechanisms by which mutations lead to disease, it will be necessary to use conditional gene-targeting strategies and the introduction of point-mutated gene copies. Furthermore, conditional strategies offer the possibility to overcome embryonic or neonatal lethality in some of the keratin-deficient mice.
Methods Mol Biol 2007
PMID:Keratin transgenic and knockout mice: functional analysis and validation of disease-causing mutations. 1717 32

Patients with dystrophic epidermolysis bullosa (DEB) have incurable skin fragility, blistering, and multiple skin wounds because of mutations in the gene that encodes for type VII collagen (C7), which holds together the epidermal and dermal layers of human skin. The intradermal injection of gene-corrected DEB fibroblasts, recombinant C7 protein, or lentiviral vectors expressing C7 is a potential therapy for DEB. Nevertheless, severe DEB causes widespread wounds and treatment would require multiple injections. An alternative strategy might be to inject genetically engineered cells into the patient's circulation that home to the skin wounds and deposit the transgene product. In this study, we demonstrated that intravenously (IV) injected, molecularly engineered DEB fibroblasts (overexpressing human C7) homed to murine skin wounds and continuously delivered C7 at the wound site, where it incorporated into the skin's basement membrane zone and formed anchoring fibril structures. Wounds made on murine or grafted human skin demonstrated accelerated healing when the animals were IV injected with gene-corrected DEB fibroblasts. Our data demonstrate that abundant C7 promotes wound healing. This is also the first evidence that IV injected, molecularly engineered skin fibroblasts can deliver C7 to skin wounds. This strategy could be useful for treating DEB patients.
Mol Ther 2007 Mar
PMID:Intravenously injected human fibroblasts home to skin wounds, deliver type VII collagen, and promote wound healing. 1724 57

A recent publication that describes gene therapy treatment of a patient with an inherited blistering skin disease, epidermolysis bullosa, demonstrates for the first time that gene therapy can cure a disease of solid tissue. The treatment relies on ex vivo transduction of autologous epidermal stem cells with a normal copy of the defective gene, followed by reconstitution of the patient's skin with epithelial sheets that are grown from these genetically corrected cells. This approach holds promise for treatment not only of inherited disorders of the skin but also of other solid tissues that are becoming amenable to tissue engineering.
Trends Mol Med 2007 Jun
PMID:Ex vivo gene therapy cures a blistering skin disease. 1741 53

Autoimmune bullous diseases are associated with autoimmunity against structural components maintaining cell-cell and cell matrix adhesion in the skin and mucous membranes. Pemphigus diseases are characterized by autoantibodies against the intercellular junctions and intraepithelial blisters. In pemphigoid diseases and epidermolysis bullosa acquisita, sub-epidermal blistering is associated with autoantibodies targeting proteins of the hemidesmosomal anchoring complex. The autoantigens in autoimmune blistering diseases have been extensively characterized over the past three decades. In general, the pathogenicity of autoantibodies, already suggested by clinical observations, has been conclusively demonstrated experimentally. Detection of tissue-bound and circulating serum autoantibodies and characterization of their molecular specificity is mandatory for the diagnosis of autoimmune blistering diseases. For this purpose, various immunofluorescence methods as well as immunoassays, including immunoblotting, enzyme-linked immunosorbent assay and immunoprecipitation have been developed. This review article describes the immunopathological features of autoimmune bullous diseases and the immunological and molecular tests used for their diagnosis and monitoring.
J Cell Mol Med
PMID:Immunopathology and molecular diagnosis of autoimmune bullous diseases. 1752 73


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