Mouse Epidermal Keratin Research Articles

Identification of deiminated arginine residues in newborn mouse epidermal keratin K1

Identification of deiminated arginine residues in newborn mouse epidermal keratin K1

Organization of coiled-coil molecules in native mouse keratin 1/keratin 10 intermediate filaments: Evidence for alternating rows of antiparallel in-register and antiparallel staggered molecules

There is considerable diversity of opinion in the literature concerning the organization of two-chain coiled-coil molecules in intermediate filaments. I have reexplored this issue using the limited proteolysis paradigm with native mouse epidermal keratin intermediate filaments (KIF), consisting of keratins 1 and 10. KIF were harvested as cytoskeletal pellets, dissociated into subfilamentous forms at pH 9.8, 9.0, or 2.6, and were subjected to limited proteolytic digestion to recover α-helix-enriched particles that derived from the rod domains of the constituent chains, using conditions that do not promote reorganization of the constituent protein chains or coiled-coil molecules. The multichain particles were subjected to physicochemical analyses, amino acid sequencing, and electron microscopy in order to determine their composition, structure, and organization within the intact KIF. The results predict two principal modes of alignment: neighboring molecules may be aligned in register and antiparallel or staggered and antiparallel. From known structural constraints, this permits construction of a two-dimensional surface lattice for KIF which consists of alternating antiparallel rows of in-register and staggered molecules. These data establish the level of hierarchy at which the well-known antiparallelity and staggered features of KIF are introduced. This model supports the proposals of KIF structure based on theoretical considerations of ionic interactions scores (Crewther et al., 1983). When the KIF are dissociated at extremes of pH, this structural model allows for disruption along alternate axes; the in-register antiparallel alignment is seen only when KIF are dissociated at high pH values; below pH 9, only the staggered antiparallel alignment is seen. The process of molecule realignment especially in concentrated urea solutions indicates that the staggered antiparallel alignment is the more thermodynamically stable form in solution.

Solid-state NMR studies of the dynamics and structure of mouse keratin intermediate filaments

The molecular dynamics and structural organization of mouse epidermal keratin intermediate filaments (IF) have been studied via solid-state nuclear magnetic resonance (NMR) experiments performed on IF labeled both in vivo and in vitro with isotopically enriched amino acids. As a probe of the organization of the peripheral glycine-rich end domains of the IF, carbon-13 NMR experiments have been performed on subfilamentous forms (prekeratin) and on IF reassembled in vitro that had been labeled with either [1-13C]glycine or [2-13C]glycine, as more than 90% of the glycines of the keratins are located in the end domains. Although cross-labeling to seryl residues was observed, the proportion of serine located in the end domains is nearly the same as that for glycine. Measurements of carbon relaxation times, nuclear Overhauser enhancements, and signal intensities show that the motions of the peptide backbone in the end domains are effectively isotropic, with average correlation times distributed over the range of 0.2-20 ns. These results indicate that the end domains of IF are remarkably flexible and have little or no structural order. To probe the structural organization of the coiled-coil rod domains of the IF, separate samples of native keratin IF, raised in primary tissue culture, were labeled with L-[1-13C]leucine, L-[2H10]leucine, or L-[2,3,3-2H3]leucine, as greater than 90% of the leucyl residues of the keratin IF types studied are located in the coiled coils which form the central core of IF.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the expression of specific epidermal keratin markers in the hairless mouse by the topical application of the tumor promoters 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate

The potent toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes chloracne and acts as a tumor promoter in the hairless HRS/J mouse model. In the present study we characterized changes in mouse epidermal keratin expression following a single topical application of TCDD to the skin of hairless (hr/hr) and haired (hr/+) HRS/J mice. Morphologic changes and alterations in keratin biosynthesis following TCDD treatment were compared with those induced by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Both TPA and TCDD induced dose-dependent epidermal hyperplasia in hr/hr and hr/+ mice and this was associated with altered keratin subunit expression. In hr/hr mice TCDD caused a pattern of keratin expression that was similar to TPA, characterized by a marked decrease in the synthesis of the Mr 67,000 (basic) and 59,000 (acidic) keratins that are specific markers for suprabasal differentiation in the epidermis. In addition, the synthesis of an acidic keratin of Mr 48,000 and a basic keratin of Mr 62,000 was also decreased. Concomitantly, TCDD caused an increase in the synthesis of a basic keratin of Mr 60,000 and acidic keratins of Mr 54,000, 52,000 and 49,000 that are normally observed in proliferating basal cells and primary epidermal cell cultures. In contrast, while TPA induced similar changes in keratinization in both the hr/+ and hr/hr mice, TCDD-induced hyperplasia in hr/+ mice was only associated with changes in keratin synthesis reflecting increased basal cell proliferation. These results demonstrate that a single application of TCDD to the skin alters the normal pattern of epidermal differentiation in the hr/hr mouse. Molecular events influencing the expression of the keratin genes associated with this process may be linked to the strain- and/or species-specific toxicity of TCDD.

Alterations in Mouse Epidermal Keratin Production Induced by Dietary Vitamin A Deficiencya

Alterations in Mouse Epidermal Keratin Production Induced by Dietary Vitamin A Deficiency^a

The coiled-coil molecules of intermediate filaments consist of two parallel chains in exact axial register

Amino acid sequence studies of helical particles derived from proteolytic digests of mouse epidermal keratin intermediate filaments (IF) have shown that their coiled-coil molecules are heterodimers of Type I and Type II keratins, with a parallel arrangement of the two chains. From a reappraisal of published chemical cross-linking data, it is concluded that the coiled-coil molecules in all IF consist of pairs of parallel chains in precise axial register.

The complete cDNA and deduced amino acid sequence of a type II mouse epidermal keratin of 60,000 Da: analysis of sequence differences between type I and type II keratins.

We present the complete nucleotide and deduced amino acid sequences of a mouse epidermal keratin subunit of 60,000 Da. The keratin possesses a central alpha-helical domain of four tracts (termed 1A, 1B, 2A, and 2B) that can form coiled-coils, interspersed by short linker sequences, and has non-alpha-helical terminal domains. This pattern of secondary structure is emerging as common to all intermediate filament subunits. The alpha-helical sequences conform to the type II class of keratins. Accordingly, this is the first type II keratin for which complete sequence information is available, and thus it facilitates elucidation of the fundamental distinctions between type I and type II keratins. It has been observed that type I keratins are acidic and type II keratins are neutral--basic in charge. We suggest that the basis for this empirical correlation between type and charge resides in the respective net charges of the 1A and 2B tracts. Calculations on interchain interactions between charged residues in the alpha-helical domains indicate that this keratin prefers to participate in dimers according to an in-register parallel arrangement. The terminal domains of this keratin possess characteristic glycine-rich sequences, and the carboxyl-terminal domain is highly homologous to that of a human epidermal keratin of 56,000 Da. According to the hypothesis that end-domains are located on the periphery of keratin filaments, we conclude that the corresponding mouse and human keratins are closely related, both structurally and functionally.

Structural Features of Epidermal Keratin Filaments Reassembled in Vitro

We have studied the structure of epidermal keratin filaments polymerized in vitro, addressing two different levels of organization. First, we have determined the amino acid sequence of a mouse epidermal keratin subunit from the nucleotide sequence of a cDNA clone. The subunit contains a large central region, representing about 50 percent, whose sequence strongly suggests that it assumes a coiled-coil alpha-helical conformation. This is flanked on the amino and carboxyl terminals by long glycine-rich sequences. Second, we have used scanning transmission electron microscopy to study the structure of frozen, unstained filaments. Analyses of such images provides information on the mass per unit length and on the distribution of mass within the filament. These data impose rigorous constraints on possible models for the packing of protofilaments within the filament. Epidermal keratin filaments assembled in vitro are polymorphic; however, the majority of bovine filaments weigh about 37 kD/nm, but most human filaments have masses of only about 27 kD/nm. The filament width is at least 15 nm, substantially more than the generally accepted value of 8 to 10 nm, owing to the existence of low-density mass at the periphery that has not been visualized by conventional microscopic methods. We currently postulate that the alpha-helical regions of the subunits comprise the structural core or backbone of the filament from which at least some of the glycine-rich sequences protrude.

Complete amino acid sequence of a mouse epidermal keratin subunit and implications for the structure of intermediate filaments.

We have determined the complete primary structure of an intermediate filament subunit, the 59,000 molecular weight subunit of mouse epidermal keratin, from the nucleotide sequence of cDNA clones. The central portion of the sequence forms extended tracts of a coiled-coil alpha-helical conformation. This is flanked at both termini by similar non-alpha-helical sequences that are extremely rich in glycine residues, frequently configured in tandem peptide repeats. Limited chymotryptic digestion of keratin filaments containing this protein suggests a structural organization whereby the terminal glycine-rich sequences protrude from a conserved core structure into which the coiled-coil alpha-helical segments are packed.

Specific quantification of mouse and human keratin proteins by radioimmunoassay.

Mouse epidermal keratin proteins were purified and labelled with 125I by chemical techniques. A radioimmunoassay method was established with a rabbit antibody elicited against the mouse keratin. This assay method was utilized to quantify keratin proteins of mouse and human epidermal extracts, both from intact tissues and cells in culture. As little as 30 ng of mouse keratin (0.5 pmol) was quantifiable.

Subunit structure of the mouse epidermal keratin filament

The two proteins which are the subunits of mouse epidermal keratin filaments have been isolated from fully differentiated epidermis (stratum corneum), viable differentiating cells and cells grown in culture. The proteins have molecular weights of 68 000 and 60 000, consist of families of very similar species, have common N-terminal ( N-acetylserine) and C-terminal (glycine) residues, contain 35–40% α-helix and are immunologically cross-reacting. In mixtures, the two proteins polymerize in vitro into native-type keratin filaments that are 70–80 Å in diameter, up to 30 μm long, possess a characteristics α-type X-ray diffraction pattern and contain the subunits in the precise molar ratio of 1 : 2 or 2 : 1.