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Keratins are the major structural proteins of keratinocytes, which are the most abundant cell type in the mammalian epidermis. Mutations in epidermal keratin genes have been shown to cause severe blistering skin abnormalities. One such disease, epidermolytic hyperkeratosis (EHK), also known as bullous congenital ichthyosiform erythroderma, occurs as a result of mutations in highly conserved regions of keratins K1 and K10. Patients with EHK first exhibit erythroderma with severe blistering, which later is replaced by thick patches of scaly skin. To assess the effect of a mutated K1 gene on skin biology and to produce an animal model for EHK, we removed 60 residues from the 2B segment of HK1 and observed the effects of its expression in the epidermis of transgenic mice. Phenotypes of the resultant mice closely resembled those observed in the human disease, first with epidermal blisters, then later with hyperkeratotic lesions. In neonatal mice homozygous for the transgene, the skin was thicker, with an increased labeling index, and the spinous cells showed a collapse of the keratin filament network around the nuclei, suggesting that a critical concentration of the mutant HK1, over the endogenous MK1, was required to disrupt the structural integrity of the spinous cells. Additionally, footpad epithelium, which is devoid of hair follicles, showed blistering in the spinous layer, suggesting that hair follicles can stabilize or protect the epidermis from trauma. Blisters were not evident in adult mice, but instead they showed a thick, scaly hyperkeratotic skin with increased mitosis, resulting in an increased number of corneocytes and granular cells. Irregularly shaped keratohyalin granules were also observed. To date, this is the only transgenic model to show the typical morphology found in the adult form of EHK.
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PMID:A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis. 898 79

We report the presence of a novel filament system in goldfish xanthophores using a monoclonal antibody (A2) made against 40-70 kD proteins derived from cytoskeletal preparations. On Western blots, this antibody recognized a 45 kD protein in xanthophore cell extracts. In cells with dispersed pigment, immunofluorescence staining of xanthophores revealed a uniform distribution of A2-reactive filaments. In cells with aggregated pigment, these filaments assumed a distinctively radial orientation, such that filaments emanated from the central pigment mass (CPM). At the electron microscopic level, immunogold labeling identified a filament system with a diameter of 7 nm. Overall, the cellular distribution of A2-reactive filaments was distinctly different from that of the other known components of the cytoskeleton, such as intermediate filaments, actin filaments, and microtubules. A2-reactive filaments also appeared resistant to agents known to perturb the cytoskeleton such as cytochalasin B, which depolymerized the actin filaments. When xanthophores were treated with vinblastine, shown to depolymerize microtubules and induce the collapse of intermediate filaments (vimentin and keratin) in other cell types, no effect on the A2 filament distribution was observed. On the other hand, treatment with calyculin A, a phosphatase inhibitor, converted A2 filaments into a wavy bundles, the effect of which was completely reversible by the removal of the drug from culture medium. These novel properties of A2 filaments, together with their reorganization in response to pigment translocation suggest that A2 filaments might play a yet unidentified role in intracellular organelle transport in these cells.
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PMID:Characterization of a novel filament system in goldfish xanthophores. 906 17

The function of the human papillomavirus (HPV) E4 proteins is unknown. In cultured epithelial cells the proteins associate with the keratin intermediate filaments (IFs) and, for some E4 types, e.g., HPV type 16 (HPV-16), induce collapse of the keratin networks. An N-terminal leucine-rich motif (LLXLL) is a conserved feature of many E4 proteins. In a previous study we showed that deletion of this region from the HPV-1 and -16 E4 proteins abrogated the localization of the mutant proteins to the keratin cytoskeleton in a simian virus 40-transformed human keratinocyte cell line (S. Roberts, I. Ashmole, L. J. Gibson, S. M. Rookes, G. J. Barton, and P. H. Gallimore, J. Virol. 68:6432-6445, 1994). The E4 proteins of HPV-1 and -16 have little sequence homology except at the N terminus. Therefore, to establish the role of sequences other than those at the N terminus, we have performed a mutational analysis of the HPV-16 E4 protein. The results of the analysis were as follows: (i) similar to findings for the HPV-1 protein, no mutation of HPV-16 E4 sequences (other than the N-terminal leucine motif) results in a mutant protein which fails to colocalize to the keratin IFs; (ii) the C-terminal domain (residues 61 to 92) is not essential for association with the cytoskeleton; and (iii) deletion of C-terminal sequences (residues 84 to 92; LTVIVTLHP) corresponding to part of a domain conserved between mucosal E4 proteins affects the ability of the mutant protein to induce cytoskeletal collapse, despite colocalization with the keratin IFs. Further analysis of this region showed that conserved hydrophobic residues valines 86 and 88 are important. In addition, we show that the HPV-16 E4 protein is detergent insoluble and exists as several disulfide-linked, high-molecular-weight complexes which could represent homo-oligomers. The C-terminal sequences (residues 84 to 92), in particular valines 86 and 88, are important in the formation of these insoluble complexes. The results of this study support our postulate that the E4 proteins include functional domains at the N terminus and the C terminus, with the intervening sequences possibly acting as a flexible hinge.
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PMID:Mutational analysis of the human papillomavirus type 16 E1--E4 protein shows that the C terminus is dispensable for keratin cytoskeleton association but is involved in inducing disruption of the keratin filaments. 909 27

Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.
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PMID:The importance of intermediate filaments in the adaptation of tissues to mechanical stress: evidence from gene knockout studies. 935 Nov 89

Epidermolytic hyperkeratosis (EHK) is a congenital, autosomal dominant disorder of cornification characterized by hyperkeratosis and blister formation. The clinical manifestations are heterogeneous, with respect to the extent of body surface involvement, palmar and plantar hyperkeratosis and the presence of erythroderma. Point mutations in the genes encoding the suprabasal-specific keratins, keratins 1 and 10 have been identified in EHK patients. The inappropriate amino acid substitutions cause a collapse of the keratin filament network, resulting in cytolysis of the involved keratinocytes. We report a severe case of EHK with a single base pair mutation that causes a threonine for asparagine substitution in residue 8 (N8T) of the 1A region of the keratin 1 protein. This is the region involved in molecular overlaps between neighboring keratin heterodimers. These findings suggest that even conservative amino acid substitutions in overlap regions can cause tonofilament clumping.
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PMID:An asparagine to threonine substitution in the 1A domain of keratin 1: a novel mutation that causes epidermolytic hyperkeratosis. 1023 3

HSP27 and alphaB-crystallin are both members of the small heat shock protein family. alphaB-crystalllin has been proposed to modulate intermediate filaments and recently a mutation in alphaB-crystallin has been identified as the genetic basis of desmin related myopathy. This disease is characterised in its pathology by aggregates of intermediate filaments associated with alphaB-crystallin. Here we report that HSP27 like alphaB-crystallin is associated with glial fibrillary acidic protein and vimentin intermediate filament networks in unstressed U373MG astrocytoma cells. HSP27 is also associated with keratin filaments in MCF7 cells, indicating that this association is not restricted to a particular intermediate filament type. The association of sHSPs with both the soluble and filamentous intermediate filament fractions of U373 cells was demonstrated biochemically. Heat shock or drug treatments induced a co-collapse of intermediate filaments and associated small heat shock proteins. These data show that the presence of HSP27 or alphaB-crystallin could not prevent filament collapse and suggest that the purpose of this association is more than just filament binding. Indeed, in U373MG cells the intermediate filament association with small heat shock proteins is similar to that observed for another protein chaperone, HSC70. In order to discern the effect of different chaperone classes on intermediate filament network formation and maintenance, several in vitro assays were assessed. Of these, falling ball viscometry revealed a specific activity of small heat shock proteins compared to HSC70 that was apparently inactive in this assay. Intermediate filaments form a gel in the absence of small heat shock proteins. In contrast, inclusion of alphaB-crystallin or HSP27 prevented gel formation but not filament assembly. The transient transfection of GFAP into MCF7 cells was used to show that the induction of a completely separate network of intermediate filaments resulted in the specific association of the endogenous HSP27 with these new GFAP filaments. These data lead us to propose that one of the major functions of the association of small heat shock proteins with intermediate filaments is to help manage the interactions that occur between filaments in their cellular networks. This is achieved by protecting filaments against those non-covalent interactions that result when they come into very close proximity as seen from the viscosity experiments and which have the potential to induce intermediate filament aggregation as seen in some disease pathologies.
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PMID:Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin. 1036 40

Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. Profilaggrin is phosphorylated and contains multiple filaggrin repeats separated by a hydrophobic linker peptide. We have previously shown that filaggrin constructs containing the linker, when transiently transfected into epithelial cells, lead to expression of a protein that resembles keratohyalin (Dale et al. J Invest Dermatol 108:179-187 1997). To characterize further the region(s) of the linker and/or filaggrin that are necessary for granule formation, we generated several mutant constructs from Flag-FG-1, and generated fusions of filaggrin with green fluorescent protein. We also subjected profilaggrin to protein phosphatase 2A treatment and measured its subsequent solubility. We found that granular morphology is not dependent on the linker or conserved phosphorylation sites, nor is solubility affected by protein phosphatase 2A treatment. Granule morphology was abrogated only in a truncated construct, which still contains the linker. A construct consisting of 16 amino acids of filaggrin fused to green fluorescent protein led to rounded and bizarrely shaped transfected cells with compact keratin filaments, suggesting that very little filaggrin sequence is required for keratin filament interaction. Radiolabeled filaggrin-green fluorescent protein constructs specifically bound keratin in overlay assays confirming that the observed cytoskeletal collapse is due to filaggrin-keratin interaction. Our findings indicate that profilaggrin must be extensively processed before it loses both its granule forming ability as well as its insolubility, suggesting that granule formation in vivo correlates with insolubility in vitro. Further, filaggrin retains its ability to bind keratin as it is degraded to smaller peptides.
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PMID:Profilaggrin requires both linker and filaggrin peptide sequences to form granules: implications for profilaggrin processing in vivo. 1038 28

Mouse keratin 6a (MK6a) is constitutively expressed in a single cell layer of the outer root sheath (ORS) of hair follicles, but its synthesis can be induced in interfollicular epidermis including the basal cell layer in response to perturbing stimuli. A basally inducible human K6 (HK6) isoform has not been described, and it is not clear which of the known HK6 isoforms is expressed in the ORS. In this study we show that expression of a dominant-negative MK6a construct (Delta2B-P) in the interfollicular epidermis caused severe blistering and neonatal lethality, suggesting that mutations in a yet to be identified basally expressed HK6 isoform might result in a severe blistering phenotype. Surviving Delta2B-P animals showed transgene expression only in isolated epidermal cells and not in all cells of the ORS, but nevertheless developed severe alopecia. Expression of two different C-terminal mutant transgenes also caused alopecia while a third C-terminal mutant had no phenotypic conse- quences. Electron microscopy revealed that Delta2B-P expression resulted in the collapse of keratin filaments, while destruction of hair follicles in the two phenotypic C-terminal mutant lines occurred in the absence of filament abnormalities. The latter finding indicates that the innermost ORS cells are uniquely sensitive to expression of even slightly altered K6 proteins, suggesting that mutations affecting an HK6 isoform expressed in this cell layer could result in alopecia in humans as well.
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PMID:Expression of MK6a dominant-negative and C-terminal mutant transgenes in mice has distinct phenotypic consequences in the epidermis and hair follicle. 1055 May 43

Mutations in keratin genes give rise to a number of inherited skin fragility disorders, demonstrating that the intermediate filament cytoskeleton has an essential function in maintaining the structural integrity of epidermis and its appendages. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder caused by mutations in keratins K5 or K14, which are expressed in the basal layer of stratified epithelia. Using a keratinocyte cell line established from an EBS patient, we investigated whether the muscle-specific intermediate filament protein desmin would be able to functionally complement a mutant keratin 14 in cultured keratinocytes. We show that in stably transfected EBS cells, desmin forms an extended keratin-independent cytoskeleton. Immunogold-EM analysis demonstrated that in the presence of numerous keratin filaments attached to desmosomes, desmin could nevertheless interact with desmosomes in the same cell, indicating the dynamic nature of the filament-desmosome association. When desmin-transfected cells were subjected to heat shock, the mutant keratin filaments showed a transient collapse while desmin filaments were maintained. Thus the defective keratin filaments and the wild-type desmin filaments appear to coexist in cells without interference. Expression of a type III intermediate filament protein like desmin may offer a strategy for the treatment of patients suffering from epidermal keratin mutations.
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PMID:Supplementation of a mutant keratin by stable expression of desmin in cultured human EBS keratinocytes. 1106 68

Keratin 8 (K8) serine 73 occurs within a relatively conserved type II keratin motif ((68)NQSLLSPL) and becomes phosphorylated in cultured cells and organs during mitosis, cell stress, and apoptosis. Here we show that Ser-73 is exclusively phosphorylated in vitro by p38 mitogen-activated protein kinase. In cells, Ser-73 phosphorylation occurs in association with p38 kinase activation and is inhibited by SB203580 but not by PD98059. Transfection of K8 Ser-73 --> Ala or K8 Ser-73 --> Asp with K18 generates normal-appearing filaments. In contrast, exposure to okadaic acid results in keratin filament destabilization in cells expressing wild-type or Ser-73 --> Asp K8, whereas Ser-73 --> Ala K8-expressing cells maintain relatively stable filaments. p38 kinase associates with K8/18 immunoprecipitates and binds selectively with K8 using an in vitro overlay assay. Given that K1 Leu-160 --> Pro ((157)NQSLLQPL --> (157)NQSPLQPL) leads to epidermolytic hyperkeratosis, we tested and showed that the analogous K8 Leu-71 --> Pro leads to K8 hyperphosphorylation by p38 kinase in vitro and in transfected cells, likely due to Ser-70 neo-phosphorylation, in association with significant keratin filament collapse upon cell exposure to okadaic acid. Hence, K8 Ser-73 is a physiologic phosphorylation site for p38 kinase, and its phosphorylation plays an important role in keratin filament reorganization. The Ser-73 --> Ala-associated filament reorganization defect is rescued by a Ser-73 --> Asp mutation. Also, disease-causing keratin mutations can modulate keratin phosphorylation and organization, which may affect disease pathogenesis.
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PMID:Keratin 8 phosphorylation by p38 kinase regulates cellular keratin filament reorganization: modulation by a keratin 1-like disease causing mutation. 1178 83


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