Unusual surface and solution behaviour of keratin polypeptides

Keratins are the major structural proteins of the epidermis. Analyzing keratin gene sequences, appreciating the switch in keratin gene expression that takes place as epidermal cells commit to terminally differentiate, and elucidating how keratins assemble into 10 nm filaments, have provided the foundation that has led to the discoveries of the genetic bases of two major classes of human skin diseases, epidermolysis bullosa simplex (EBS) and epidermolytic hyperkeratosis (EH). These diseases involve point mutations in either the basal epidermal keratin pair, K5 and K14 (EBS), or the suprabasal pair, K1 and K10 (EH). In severe cases of EBS and EH, mutations are found in the highly conserved ends of the alpha-helical rod domain, regions that, by random mutagenesis, had already been found to be important for 10 nm filament assembly. In order to identify regions of the keratin polypeptides that might be more subtly involved in 10 nm filament assembly and to explore the diversity in mutations within milder cases of these diseases, we have focused on Weber-Cockayne EBS, where mild blistering occurs primarily on the hands and feet in response to mechanical stress. In this report, we show that affected members of two different W-C EBS families have point mutations within 1 residue of each other in the non-helical linker segment of the K5 polypeptide. Genetic linkage analyses, the absence of this mutation in > 150 wild-type alleles and filament assembly studies suggest that these mutations are responsible for the W-C EBS phenotype. These findings provide the best evidence to date that the non-helical linker region in the middle of the keratin polypeptides plays a subtle but significant role in intermediate filament structure and/or intermediate filament cytoskeletal architecture.

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Keratins Turn over by Ubiquitination in a Phosphorylation-Modulated Fashion

The Journal of Cell Biology ◽

10.1083/jcb.149.3.547 ◽

2000 ◽

Vol 149 (3) ◽

pp. 547-552 ◽

Cited By ~ 122

Author(s):

Nam-On Ku ◽

M. Bishr Omary

Keyword(s):

Intermediate Filament ◽

Liver Diseases ◽

Direct Evidence ◽

Proteasome Inhibition ◽

Type I ◽

Ubiquitinated Proteins ◽

Antibody Staining ◽

Keratin Polypeptides ◽

Proteasome Pathway ◽

Turn Over

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.

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Paracrystalline Aggregates of Psoriatic Keratin and Their Relation to Normal Keratin Structure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120096 ◽

1985 ◽

Vol 43 ◽

pp. 680-681

Author(s):

S. Trachtenberg ◽

P.M. Steinert ◽

B.L. Trus ◽

A.C. Steven

Keyword(s):

Cell Death ◽

Stratum Corneum ◽

Intermediate Filament ◽

Skin Disease ◽

Amorphous Matrix ◽

Terminal Differentiation ◽

Proteolytic Degradation ◽

Horny Layer ◽

Chronic Skin ◽

Chronic Skin Disease

During terminal differentiation of vertebrate epidermis, certain specific keratin intermediate filament (KIF) proteins are produced. Keratinization of the epidermis involves cell death and disruption of the cytoplasm, leaving a network of KIF embedded in an amorphous matrix which forms the outer horny layer known as the stratum corneum. Eventually these cells are shed (desquamation). Normally, the processes of differentiation, keratinization, and desquamation are regulated in an orderly manner. In psoriasis, a chronic skin disease, a hyperkeratotic stratum corneum is produced, resulting in abnormal desquamation of unusually large scales. In this disease, the normal KIF proteins are diminished in amount or absent, and other proteins more typical of proliferative epidermal cells are present. There is also evidence of proteolytic degradation of the KIF.

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An Early Developmental Marker for Radial Glia in Rat Spinal Cord

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162648 ◽

1996 ◽

Vol 54 ◽

pp. 36-37

Author(s):

V. Kriho ◽

H.-Y. Yang ◽

C.-M. Lue ◽

N. Lieska ◽

G. D. Pappas

Keyword(s):

Spinal Cord ◽

Intermediate Filament ◽

Radial Glia ◽

Rat Spinal Cord ◽

Future Studies ◽

Spinal Cord Development ◽

Median Septum ◽

Differentiation Marker ◽

Early Developmental ◽

Developing Rat

Radial glia have been classically defined as those early glial cells that radially span their thin processes from the ventricular to the pial surfaces in the developing central nervous system. These radial glia constitute a transient cell population, disappearing, for the most part, by the end of the period of neuronal migration. Traditionally, it has been difficult to definitively identify these cells because the principal criteria available were morphologic only.Using immunofluorescence microscopy, we have previously defined a phenotype for radial glia in rat spinal cord based upon the sequential expression of vimentin, glial fibrillary acidic protein and an intermediate filament-associated protein, IFAP-70/280kD. We report here the application of another intermediate filament-associated protein, IFAP-300kD, originally identified in BHK-21 cells, to the immunofluorescence study of radial glia in the developing rat spinal cord.Results showed that IFAP-300kD appeared very early in rat spinal cord development. In fact by embryonic day 13, IFAP-300kD immunoreactivity was already at its peak and was observed in most of the radial glia which span the spinal cord from the ventricular to the subpial surfaces (Fig. 1). Interestingly, from this time, IFAP-300kD immunoreactivity diminished rapidly in a dorsal to ventral manner, so that by embryonic day 16 it was detectable only in the maturing macroglial cells in the marginal zone of the spinal cord and the dorsal median septum (Fig. 2). By birth, the spinal cord was essentially immuno-negative for this IFAP. Thus, IFAP-300kD appears to be another differentiation marker available for future studies of gliogenesis, especially for the early stages of radial glia differentiation.

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