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L N Marekov

Publications and source records attributed to L N Marekov.

16 recordsLinked to original sources
PubMed1995

Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein.

Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a full-length cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no alpha or beta structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain cross-links involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.

Amino Acid Sequence↗
PubMed1995

The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.

The cornified cell envelope (CE) is a 15-nm thick layer of insoluble protein deposited on the intracellular side of the cell membrane of terminally differentiated stratified squamous epithelia. The CE is thought to consist of a complex amalgam of proteins cross-linked by isodipeptide bonds formed by the action of transglutaminases, but little is known about how or in which order the several putative proteins are cross-linked together. In this paper, CEs purified from human foreskin epidermis were digested in two steps by proteinase K, which released as soluble peptides about 30% and then another 35% of CE protein mass, corresponding to approximately the outer third (cytoplasmic surface) and middle third, respectively. Following fractionation, 145 unique peptides containing two or more sequences cross-linked by isodipeptide bond(s) were sequenced. Based on these data, most (94% molar mass) of the outer third of CE structure consists of intra- and interchain cross-linked loricrin, admixed with SPR1 and SPR2 proteins as bridging cross-links between loricrin. Likewise, the middle third of CE structure consists largely of cross-linked loricrin and SPR proteins, but is mixed with the novel protein elafin which also forms cross-bridges between loricrin. In addition, cross-links involving loricrin and keratins 1, 2e, and 10 or filaggrin were recovered in both levels. The data establish for the first time that these several proteins are indeed cross-linked protein components of the CE structure. In addition, the data support a model for the intermediate to final stages of CE assembly: the proteins elafin, SPR1 and SPR2, and loricrin begin to be deposited on a preformed scaffold; later, elafin deposition decreases as loricrin and SPR accumulation continues to effect final assembly. The recovery of cross-links involving keratins further suggests that the subjacent cytoplasmic keratin intermediate filament-filaggrin network is anchored to the developing CE during these events.

Amino Acid Sequence↗
PubMed1994

Mutations in the H1 and 1A domains in the keratin 1 gene in epidermolytic hyperkeratosis.

In the autosomal dominant disorder epidermolytic hyperkeratosis, the structural integrity of the keratin intermediate filaments is altered in the suprabasal layers of the epidermis. We and others have used genetic linkage studies and mutation analysis to establish that single amino acid substitutions in either the keratin 1 or keratin 10 chains can cause epidermolytic hyperkeratosis. However, a larger database of mutations is required to better understand the relationship between specific mutations in these keratin chains and their effect on keratin filament structure. A larger database will also provide a catalog that may be useful for genetic counseling purposes. In this paper, we report the identification of three new mutations of the keratin 1 chain of epidermolytic hyperkeratosis probands in highly conserved residues in the H1 or beginning of the 1A rod domain segments. These correspond to regions involved in molecular overlaps between neighboring molecules in keratin filaments. Using an in vitro assay, synthetic peptides bearing these substitutions show diminished capacity to disassemble preformed filaments in vitro in comparison to the wild type peptides. Moreover, analyses of all mutations in epidermolytic hyperkeratosis known to date demonstrate remarkable clustering in the molecular overlap region. We conclude that non-conservative substitutions in the overlap region are likely to interfere with normal keratin filament structure and function, leading to pathology.

Amino Acid Sequence↗
PubMed1993

Diversity of intermediate filament structure. Evidence that the alignment of coiled-coil molecules in vimentin is different from that in keratin intermediate filaments.

Although vimentin intermediate filaments (IF) are morphologically similar to all other IF types, cells have evolved different ways of manipulating vimentin and keratin IF. The structural basis for such differences is unknown. We have explored this by use of cross-linking experiments on vimentin oligomers, polymers, and intact IF to determine the axial length of vimentin molecules and the degrees to which neighboring molecules are aligned in IF. Our data reveal that the homodimer vimentin molecule (43.9 nm) is clearly shorter than a keratin heterodimer molecule (46.2 nm). Vimentin assemblies contain three modes of antiparallel molecular alignments: A11 and A22 in two-molecule or larger oligomeric assemblies, in which the two molecules are staggered so as to bring their 1B and 2B rod domain segments, respectively, into register; and A12 in higher order molecular assemblies in which the two neighboring molecules are largely overlapped. Since the repeat axial length of the vimentin assemblies (42.6 nm) is less than the molecular length, this means there is an overlap (designated as alignment ACN) of about 1 nm (5-10 residues) between the end of the 2B and beginning of the 1A rod domain segments of similarly directed molecules in the IF. Interestingly, these four modes of nearest neighbor molecular alignments also occur in keratin IF. However, the degree of stagger of alignments in the A11 and A22 modes is different (staggers of -19.5 for vimentin versus -16.6 nm for keratin, and 23.3 and 28.6 nm, respectively). Two-dimensional surface lattice maps of the two IF types are very similar, except for differences in molecule alignments and different axial repeats of 21.4 nm in vimentin and 22.6 nm in keratin IF. Although vimentin-keratin hybrid molecules can be induced to form in vitro, they do not assemble into higher order structures. The data suggest that vimentin and keratin are incapable of assembly into IF in vitro or in vivo simply because their molecules are of different axial lengths and because the exact axial alignments of neighboring molecules are different.

Amino Acid Sequence↗
PubMed1993

Conservation of the structure of keratin intermediate filaments: molecular mechanism by which different keratin molecules integrate into preexisting keratin intermediate filaments during differentiation.

During development and differentiation, the intermediate filament component of the cytoskeleton of many cells and tissues is rebuilt by a dynamic exchange process in which one set of protein chains is replaced by another, without recourse to creation of a new network. One major example is the replacement of keratin 5/keratin 14 (K5/K14) keratin intermediate filaments (KIFs) by K1/K10 KIFs during terminal differentiation in the epidermis. The present work was undertaken to explore how this may occur. We have induced lysine-lysine cross-links with disulfosuccinimidyl tartrate in K5/K14 KIFs in order to determine the axial dimensions and relative axial alignments of the K5/K14 molecules. Many of the cross-links induced in subfilamentous oligomers containing one, two, or three molecules were also found in the intact KIF, indicating that the body of data thus generated provides physiologically relevant information on the structural organization in the KIF. A least-squares analysis using as data the positions of lysine residues involved in 23 induced cross-links has allowed the axial alignments of the various coiled-coil segments in the rod domain to be determined. Three modes of antiparallel alignment of two neighboring molecules were found: A11 (staggered by -16.7 nm), A22 (staggered by 28.8 nm), and A12 (almost in register; staggered by only 0.3 nm). Since the axial repeat length is about 1 nm less than the molecular length, the data require a fourth mode of molecule alignment, termed ACN, in which similarly directed molecules are overlapped by the equivalent of about 5-10 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.

Trichohyalin is an intermediate filament-associated protein that associates in regular arrays with keratin intermediate filaments (KIF) of the inner root sheath cells of the hair follicle and the granular layer of the epidermis and is a known substrate of transglutaminases. We have determined the full-length sequence of human trichohyalin by use of RNA-mediated anchored polymerase chain reaction methods and from a genomic clone and analyzed its potential secondary structure. We show here that trichohyalin may have at least three important functions in these cells. The protein of 248 kDa is unusual in that it contains one of the highest contents of charged residues of any protein. Of several defined domains, domains 2-4, 6, and 8 are almost entirely alpha-helical, configured as a series of peptide repeats of varying regularity, and are thought to form a single-stranded alpha-helical rod stabilized by ionic interactions between successive turns of the alpha-helix. Domain 6 is the most regular and may bind KIF directly by ionic interactions. Domains 5 and 7 are less well organized and may introduce folds in the molecule. Thus, human trichohyalin is predicted to be an elongated flexible rod at least 215 nm long and to function as a KIF-associated protein by cross-linking the filaments in loose networks. In addition, trichohyalin is similar to, but several times longer than, involucrin, a known cell envelope constituent, so that together, involucrin and trichohyalin may serve as scaffold proteins in the organization of the cell envelope of these cells or even anchor the cell envelope to the KIF network. Finally, trichohyalin possesses a pair of functional calcium-binding domains of the EF-hand type at its amino terminus that may be involved in its calcium-dependent postsynthetic processing during terminal differentiation.

Amino Acid Sequence↗
PubMed1993

Keratin intermediate filament structure. Crosslinking studies yield quantitative information on molecular dimensions and mechanism of assembly.

One of the major obstacles to solving the full three-dimensional structure of keratin intermediate filaments (KIF) is the determination of the exact mode(s) of alignment of nearest-neighbor molecules; this in turn requires precise information of the lengths of the non-alpha-helical linker segments within the coiled-coil alpha-helical heterodimer molecule. In this study, we have induced lysine-lysine and cysteine-cysteine crosslinks between keratin intermediate filament molecules in small assembly-competent oligomers, isolated them and then characterized the natures and locations of the crosslinks. Of more than 100 found, 21 quantitatively major crosslinks were used to obtain the relative axial alignments of rod domain segments by least-squares fitting methods. Three dominant modes of alignment were found. In each case the molecules are antiparallel with the first involving molecules in approximate register (stagger = -0.2 nm), the second involving molecules staggered so as to bring the 1B segments into approximate alignment (stagger = -16.1 nm), and the third involving molecules staggered so as to bring the 2B segments into approximate alignment (stagger = 28.2 nm). In addition, the data enable quantitative estimates to be made for the first time of the lengths of the non-coiled-coil segments (L1 = 2.5 nm, L12 = 1.6 nm, L2 = 0.8 nm), and the total length of the rod domain (46.0 nm). Alignment of molecules according to these parameters permits construction of a two-dimensional surface lattice which displays a 1.6 nm (10 or 11 residue) overlap between similarly directed molecules. Together, the data predict six important overlapping sequence regions that recur about 16 times per 46 nm of filament length. Interestingly, synthetic peptides corresponding to these sequences, singly or in combination, significantly interfere with keratin filament structural integrity. These results thus represent the most significant set of structural constraints for KIF yet available and provide insights into how disease-causing mutations disrupt filaments and their organization in cells.

Amino Acid Sequence↗
PubMed1992

Identification and purification of a novel serine/threonine messenger-independent growth-related protein kinase from lactating goat mammary gland.

A second messenger-independent serine/threonine protein kinase from lactating goat mammary gland is purified and characterized. The purification steps include: homogenization, ultracentrifugation, ammonium sulphate precipitation, DEAE-Sepharose, phosphocellulose, hydrophobic and Mono Q columns. On the final step of purification the enzyme is revealed as a single band of mol wt 45,000 on silver-stained SDS-PAGE. Mg2+ and K+ are necessary for its optimum activity. Phosvitin and casein are substrates for the enzyme but kemptide, RRREEETEEE, protamine and histone mixture are all poorly phosphorylated. The kinase is inhibited by quercetin, heparin, random tyrosine- and glutamic acid-containing polymers, Ca2+, NaF, 2,3-bis-phosphoglycerate. 1 mM Mn2+ affects positively the basal level of the kinase activity but 5 mM Mn2+ completely suppress the effect of 10 mM Mg2+. Km of this enzyme for ATP is 1.57 microM and pH optimum is from 6 to 7. Isolation of this kinase is facilitated by its unusually high affinity for phosphocellulose.

Amino Acid Sequence↗
PubMed1990

Characterization of the murine corticosteroid binding globulin: variations between mammalian forms.

Corticosteroid binding globulin (CGB) from term-pregnant mouse serum was isolated and characterized by peptide analysis after treatment with CNBr and Lys-specific protease, respectively. Amino acid sequence analysis of six segments, covering 189 of 383 positions in different regions of the protein, showed unexpectedly low overall homology (60%) to the indirectly deduced human amino acid sequence previously reported. However, some segments displayed a greater resemblance to their human counterparts. Differences were observed in at least two of six potential glycosylation sites. The nature of electrophoretic CBG variants and their immunological properties are described.

Amino Acid Sequence↗
PubMed1986

Protein HMG1 is different from a DNA helix unwinding protein in calf thymus.

A number of criteria were used--chromatography on columns with single-stranded and double-stranded DNA, electrophoresis, peptide analysis, immunological tests and thermal denaturation of DNA--to show that protein (high mobility group) HMG1 and an unwinding protein from calf thymus are two distinct, unrelated proteins. While both proteins are thought to be related to DNA replication this might involve different mechanisms of action.

Animals↗
PubMed1984

Involvement of protein HMG1 in DNA replication.

Antibodies against HMG1 inhibit the incorporation of [3H]thymidine in Ehrlich ascites cell nuclei. By the use of specific inhibitors it is shown that HMG1 is needed for the action of the replicative DNA polymerase and not for the reparative one. This is supported by the fact that the addition of exogenous HMG1 to the nuclei enhances the replication process.

Animals↗
PubMed1984

Isolation of high-mobility-group proteins HMG1 and HMG2 in non denaturing conditions and comparison of their properties with those of acid-extracted proteins.

We describe a method for isolation and purification of the chromosomal proteins HMG1 and HMG2 in non-denaturing conditions which overcomes the difficulties of the published methods concerning yield and purity. The method is based on salt extraction, selective precipitation with ammonium sulfate and DEAE-cellulose chromatography. All studied properties of these proteins (formation of protein tetramers, enhancement of micrococcal nuclease digestion of DNA and chromatin, and protection of 165-basepair DNA in chromatosome) differ significantly from the properties of HMG1 and 2 isolated under denaturing conditions.

Animals↗
PubMed1984

High mobility group proteins HMG1 and HMG2 do not decrease the melting temperature of DNA.

High mobility group proteins 1 and 2 isolated in non-denaturing conditions cannot decrease the temperature of denaturation of DNA. When they are isolated or treated with tricloroacetic acid a hyperchromic peak below the melting temperature of free DNA appears in agreement with previous data ( Javaherian et al. (1979) Nucl . Acids Res. 6, 3569-3580). We show that this is due to light scattering of aggregated protein at submelting temperatures and not to melting of DNA.

Animals↗