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1.
Neurochem Res ; 30(10): 1245-55, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16341586

ABSTRACT

Transglutaminase activity was found to be present in highly purified non-synaptosomal rat brain mitochondria. A 78-kDa protein in these organelles was shown to be a transglutaminase 2 substrate, and incubation of a non-synaptosomal mitochondrial lysate with transglutaminase 2 yielded high-Mr proteins. The 78-kDa protein was identified as mitochondrial aconitase by MALDI-TOF analysis. Aconitase activity was decreased in a dose-dependent manner when non-synaptosomal rat brain mitochondria were incubated with transglutaminase 2. Transglutaminase activity is increased about 2-fold in the mitochondrial fraction of HD caudate. Moreover, Western blotting of the mitochondrial fraction revealed that most of the mitochondrial aconitase in HD caudate is present as high-Mr aggregates. Aconitase activity was previously shown to be decreased in Huntington disease (HD) caudate (a region severely damaged by the disease). The present findings suggest that an increase of transglutaminase activity in HD caudate may contribute to mitochondrial dysfunction by incorporating aconitase into inactive polymers.


Subject(s)
Aconitate Hydratase , Brain/enzymology , GTP-Binding Proteins/metabolism , Huntington Disease/metabolism , Mitochondria/enzymology , Transglutaminases/metabolism , Aconitate Hydratase/chemistry , Aconitate Hydratase/metabolism , Animals , Brain/anatomy & histology , Brain/pathology , Humans , Huntington Disease/pathology , Mice , Molecular Weight , Peptides/genetics , Peptides/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , Rats , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Subcellular Fractions/metabolism
2.
J Cell Sci ; 118(Pt 4): 781-93, 2005 Feb 15.
Article in English | MEDLINE | ID: mdl-15671067

ABSTRACT

Epiplakin is a member of the plakin family with multiple copies of the plakin repeat domain (PRD). We studied the subcellular distribution and interactions of human epiplakin by immunostaining, overlay assays and RNAi knockdown. Epiplakin decorated the keratin intermediate filaments (IF) network and partially that of vimentin. In the binding assays, the repeat unit (PRD plus linker) showed strong binding and preferentially associated with assembled IF over keratin monomers. Epiplakin knockdown revealed disruption of IF networks in simple epithelial but not in epidermal cells. In rescue experiments, the repeat unit was necessary to prevent the collapse of IF networks in transient knockdown; however, it could only partially restore the keratin but not the vimentin IF network in stably knocked down HeLa cells. We suggest that epiplakin is a cytolinker involved in maintaining the integrity of IF networks in simple epithelial cells. Furthermore, we observed an increase of epiplakin expression in keratinocytes after the calcium switch, suggesting the involvement of epiplakin in the process of keratinocyte differentiation.


Subject(s)
Autoantigens/physiology , Intermediate Filaments/ultrastructure , Keratinocytes/ultrastructure , Keratins/analysis , Vimentin/analysis , Autoantigens/analysis , Autoantigens/chemistry , Cell Differentiation , Cells, Cultured , HeLa Cells , Humans , Intermediate Filaments/chemistry , Keratinocytes/cytology , Keratinocytes/metabolism , Protein Structure, Tertiary , RNA Interference
3.
J Mol Biol ; 340(1): 97-114, 2004 Jun 25.
Article in English | MEDLINE | ID: mdl-15184025

ABSTRACT

We have developed an assembly protocol for the intermediate filament (IF) protein vimentin based on a phosphate buffer system, which enables the dynamic formation of authentic IFs. The advantage of this physiological buffer is that analysis of the subunit interactions by chemical cross-linking of internal lysine residues becomes feasible. By this system, we have analyzed the potential interactions of the coiled-coil rod domains with one another, which are assumed to make a crucial contribution to IF formation and stability. We show that headless vimentin, which dimerizes under low salt conditions, associates into tetramers of the A(22)-type configuration under assembly conditions, indicating that one of the effects of increasing the ionic strength is to favor coil 2-coil 2 interactions. Furthermore, in order to obtain insight into the molecular interactions that occur during the first phase of assembly of full-length vimentin, we employed a temperature-sensitive variant of human vimentin, which is arrested at the "unit-length filament" (ULF) state at room temperature, but starts to elongate upon raising the temperature to 37 degrees C. Most importantly, we demonstrate by cross-linking analysis that ULF formation predominantly involves A(11)-type dimer-dimer interactions. The presence of A(22) and A(12) cross-linking products in mature IFs, however, indicates that major rearrangements do occur during the longitudinal annealing and radial compaction steps of IF assembly.


Subject(s)
Vimentin/chemistry , Buffers , Cross-Linking Reagents , Dimerization , Humans , Phosphates , Temperature , Ultracentrifugation , Vimentin/ultrastructure
4.
Proteins ; 55(4): 1043-52, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-15146501

ABSTRACT

The sequence of a protein chain determines both its conformation and its function in vivo. An attempt is made to gain an understanding of the classes of deformations that can arise in an important structural motif, the alpha-helical coiled coil, as a consequence of mutations occurring in its underlying heptad substructure. In order to do so we consider the model structure of segment 1A in intermediate filaments and then investigate the structures arising from each of the 22 mutations observed in cytokeratin K5/K14 molecules that lead to variants of epidermolysis bullosa simplex. These are refined separately using a molecular dynamics protocol. The mutations often result in a significant distortion of the backbone over a turn or so of the alpha helix in either the chain itself or its constituent partner, leading to the likelihood of impaired chain aggregation and hence molecular assembly. One mutant (K14-L143P; 1A-28) gave rise to structural distortion along almost the entire length of segment 1A. The remaining structures showed less deformation, and normal-looking intermediate filaments are likely in vivo. In addition, an identical mutation in the same position in each of the chains in the heterodimer did not necessarily give equivalent structural distortions. Although proline mutations frequently lead to the most severe structural deformations, a non-proline substitution (K14-R125S; 1A-10) gave rise to the largest local structural disruption that was observed. Unexpectedly, mutations in positions a and d were not always of the greatest structural significance, although three in position a were shown by AGADIR to result in a significant increase in alpha-helix stability.


Subject(s)
Epidermolysis Bullosa Simplex/genetics , Keratins/chemistry , Keratins/genetics , Models, Molecular , Mutation , Amino Acids/chemistry , Humans , Molecular Structure , Protein Structure, Secondary
5.
J Biol Chem ; 279(25): 26716-25, 2004 Jun 18.
Article in English | MEDLINE | ID: mdl-15084592

ABSTRACT

Epidermal-type Transglutaminase 3 (TGase 3) is a Ca(2+)-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 A resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (0.4 microm), GTP (0.6 microm), GDP (1.0 microm), GMP (0.4 microm), and ATP (28.0 microm). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest trade mark data base (Tripos Inc.) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (K(d) = 4.1 microm), TP395289 (K(d) = 38.5 microm), TP394305 (K(d) = 1.0 mm). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.


Subject(s)
Guanosine Monophosphate/chemistry , Adenosine Triphosphate/chemistry , Algorithms , Boron Compounds/pharmacology , Cell Membrane/metabolism , Crystallography, X-Ray , Dose-Response Relationship, Drug , Electrons , Guanine/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/chemistry , Guanosine Diphosphate/chemistry , Guanosine Triphosphate/chemistry , Humans , Hydrolysis , Kinetics , Ligands , Models, Chemical , Models, Molecular , Phosphates/chemistry , Protein Binding , Protein Conformation , Spectrometry, Fluorescence
6.
J Biol Chem ; 279(21): 22773-80, 2004 May 21.
Article in English | MEDLINE | ID: mdl-15033990

ABSTRACT

Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, although co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca(2+). These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca(2+)] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly.


Subject(s)
Calcium/metabolism , Cytoskeletal Proteins/chemistry , Epithelium/metabolism , Membrane Proteins/chemistry , Protein Precursors/chemistry , Anions/chemistry , Cell Differentiation , Cell Membrane/metabolism , Cells, Cultured , Circular Dichroism , Cross-Linking Reagents/pharmacology , Cytoplasm/metabolism , Cytoskeletal Proteins/metabolism , DNA, Complementary/metabolism , Dimerization , Dose-Response Relationship, Drug , Humans , Keratinocytes/cytology , Lipids/chemistry , Microscopy, Electron , Phospholipids/metabolism , Plakins , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry
7.
J Biol Chem ; 279(8): 7180-92, 2004 Feb 20.
Article in English | MEDLINE | ID: mdl-14645372

ABSTRACT

Transglutaminase 3 (TGase 3) is a member of a family of Ca2+-dependent enzymes that catalyze covalent cross-linking reactions between proteins or peptides. TGase 3 isoform is widely expressed and is important for effective epithelial barrier formation in the assembly of the cell envelope. Among the nine TGase enzyme isoforms known in the human genome, only TGase 2 is known to bind and hydrolyze GTP to GDP; binding GTP inhibits its transamidation activity but allows it to function in signal transduction. Here we present biochemical and crystallographic evidence for the direct binding of GTP/GDP to the active TGase 3 enzyme, and we show that the TGase 3 enzyme undergoes a GTPase cycle. The crystal structures of active TGase 3 with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) and GDP were determined to 2.1 and 1.9 A resolution, respectively. These studies reveal for the first time the reciprocal actions of Ca2+ and GTP with respect to TGase 3 activity. GTPgammaS binding is coordinated with the replacement of a bound Ca2+ with Mg2+ and conformational rearrangements that together close a central channel to the active site. Hydrolysis of GTP to GDP results in two stable conformations, resembling both the GTP state and the non-nucleotide bound state, the latter of which allows substrate access to the active site.


Subject(s)
Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , Calcium/chemistry , Gene Expression Regulation, Enzymologic , Magnesium/chemistry , Transglutaminases/biosynthesis , Transglutaminases/genetics , Amino Acid Sequence , Binding Sites , Calorimetry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Genome , Guanosine 5'-O-(3-Thiotriphosphate)/chemistry , Guanosine Diphosphate/chemistry , Guanosine Triphosphate/chemistry , Humans , Hydrolysis , Ions , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Isoforms , Recombinant Proteins/chemistry , Signal Transduction , Substrate Specificity , Thermodynamics , Time Factors
8.
Exp Dermatol ; 12(6): 876-81, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14675368

ABSTRACT

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant disease characterized clinically by localized palmoplantar thickening and histopathologically by granular degeneration of the epidermis. Recent molecular biological studies have revealed that EPPK is caused by mutations of the keratin 9 gene in sequences mainly encoding the highly conserved 1 A rod domain. Here we demonstrate a novel mutation of N160H (position 8 of the 1 A domain) and two other previously reported mutations, R162W and N160S, in five unrelated Korean families with EPPK. The three-dimensional structure of the 1 A domain of the related vimentin intermediate filament protein chain is now known. Based on its likely similarity to the keratin 9 chain, we predict that inappropriate amino acid substitutions in position 10 of 1 A will likely interfere with coiled-coil dimer stability, and those in position 8 will interfere with tetramer stability. Accordingly, these mutations compromise the structural integrity of the keratin intermediate filaments leading to the pathology of EPPK.


Subject(s)
Hyperkeratosis, Epidermolytic/genetics , Keratins/genetics , Keratoderma, Palmoplantar/genetics , Mutation , Adolescent , Adult , Child , DNA/chemistry , Dimerization , Family Health , Female , Humans , Korea , Male , Pedigree , Peptides/chemistry , Protein Structure, Tertiary
9.
Exp Mol Med ; 35(4): 228-42, 2003 Aug 31.
Article in English | MEDLINE | ID: mdl-14508061

ABSTRACT

Transglutaminase enzymes (TGases) catalyze the calcium dependent formation of an isopeptide bond between protein-bound glutamine and lysine substrates. Previously we have shown that activated TGase 3 acquires two additional calcium ions at site two and three. The calcium ion at site three results in the opening of a channel. At this site, the channel opening and closing could modulate, depending on which metal is bound. Here we propose that the front of the channel could be used by the two substrates for enzyme reaction. We propose that the glutamine substrate is directed from Trp236 into the enzyme, shown by molecular docking. Then a lysine substrate approaches the opened active site to engage Trp327, leading to formation of the isopeptide bond. Further, direct comparisons of the structures of TGase 3 with other TGases have allowed us to identify several residues that might potentially be involved in generic and specific recognition of the glutamine and lysine substrates.


Subject(s)
Calcium-Binding Proteins/metabolism , Transglutaminases/metabolism , Animals , Binding Sites , Calcium/metabolism , Glutamine/metabolism , Humans , Lysine/metabolism , Models, Chemical , Models, Molecular , Protein Binding , Protein Structure, Tertiary
10.
J Biol Chem ; 278(42): 41409-19, 2003 Oct 17.
Article in English | MEDLINE | ID: mdl-12853460

ABSTRACT

Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks.


Subject(s)
Hair Follicle/metabolism , Protein Precursors/pharmacology , Amino Acid Sequence , Animals , Autoantigens/chemistry , Chromatography, High Pressure Liquid , Citrulline/chemistry , Cross-Linking Reagents/pharmacology , Intermediate Filament Proteins , Keratins/chemistry , Mice , Microscopy, Electron , Microscopy, Fluorescence , Molecular Sequence Data , Peptides/chemistry , Proline/chemistry , Protein Precursors/chemistry , Protein Structure, Tertiary , S100 Proteins/chemistry , Time Factors
11.
J Biol Chem ; 278(26): 23834-41, 2003 Jun 27.
Article in English | MEDLINE | ID: mdl-12679341

ABSTRACT

The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca2+ ions, which in site one is constitutively bound, while those in sites two and three are acquired during activation and are required for activity. In particular, binding at site three opens a channel through the enzyme and exposes two tryptophan residues near the active site that are thought to be important for enzyme reaction. In this study, we have solved the structures of three more forms of this enzyme by x-ray crystallography in the presence of Ca2+ and/or Mg2+, which provide new insights on the precise contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty, and it has a binding affinity of Kd = 0.3 microm (DeltaH = -6.70 +/- 0.52 kcal/mol), which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca2+ of the Group 2 family of alkali earth metals, and its occupancy are required for activity. Site three can be occupied by some lanthanides, Ca2+,or Mg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site.


Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/physiology , Transglutaminases/metabolism , Binding Sites , Calcium/chemistry , Calcium-Binding Proteins/chemistry , Crystallization , Crystallography, X-Ray , Humans , Lanthanoid Series Elements/chemistry , Magnesium/chemistry , Molecular Structure , Protein Conformation , Recombinant Proteins , Transglutaminases/chemistry
12.
J Mass Spectrom ; 38(4): 373-7, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12717748

ABSTRACT

High-sensitivity, rapid identification of proteins in proteomic studies normally uses a combination of one- or two-dimensional electrophoresis together with mass spectrometry. The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have increased its application in recent years. The most common method of 'peptide fingerprinting' often may not provide robust identification. Normally additional sequence information by post-source decay (PSD) MALDI-TOFMS provides additional constraints for database searches to achieve highly confident results. Here we describe a derivatization procedure to facilitate the acquisition of such sequence information. Peptide digests from a skin-expressed protein were modified with 4-sulfophenyl isothiocyanate. The resulting peptides carry a fixed negative charge at the N-terminal end and the resulting PSD spectrum is dominated by C-terminal y-type ions. The sequence information in most cases can be obtained manually or with simple programming tools. Methods of optimizing the procedure and increasing the sensitivity are discussed.


Subject(s)
Benzenesulfonates/chemistry , Calcium-Binding Proteins/chemistry , Isothiocyanates/chemistry , Peptide Fragments/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transglutaminases/chemistry , Amino Acid Sequence , Calcium-Binding Proteins/metabolism , Humans , Molecular Sequence Data , Sequence Analysis, Protein , Transglutaminases/metabolism , Trypsin/metabolism
13.
J Struct Biol ; 142(2): 266-71, 2003 May.
Article in English | MEDLINE | ID: mdl-12713954

ABSTRACT

The so-called hard alpha-keratins, such as quill and hair, have a composite structure in which intermediate filaments (IF) are embedded in a sulfur-rich matrix. Recent studies of these trichocyte keratin IF have revealed that substantial changes in the molecular architecture take place when oxidation of the cysteine residues occurs as part of the terminal differentiation/keratinization process. Recent cryoelectron microscope studies suggest that the IF has a tubular structure prior to keratinization, but transmission electron micrographs of thin sections of fully keratinized fibers exhibit a "ring-core" structure. In the present contribution we develop a generic model for the IF in the reduced state based on cross-linking studies and discuss two possibilities for the way in which this structure may be modified during the keratinization process.


Subject(s)
Intermediate Filament Proteins/chemistry , Intermediate Filaments/ultrastructure , Keratins/chemistry , Animals , Cryoelectron Microscopy , Cysteine/metabolism , Disulfides , Intermediate Filaments/chemistry , Keratins/metabolism , Microscopy, Electron , Models, Biological , Oxidation-Reduction , Wool
14.
J Invest Dermatol ; 120(2): 224-8, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12542526

ABSTRACT

Spontaneous healing with no or only very mild ichthyosis distinguishes the "self-healing collodion baby" from other congenital ichthyoses. In two self-healing collodion baby siblings with markedly diminished epidermal transglutaminase 1 activity we found the compound heterozygous transglutaminase 1 mutations G278R and D490G. Molecular modeling and biochemical assays of mutant proteins under elevated hydrostatic pressure suggest significantly reduced activity in G278R and a chelation of water molecules in D490G that locks the mutated enzyme in an inactive trans conformation in utero. After birth these water molecules are removed and the enzyme is predicted to isomerize back to a partially active cis form, explaining the dramatic improvement of this skin condition.


Subject(s)
Ichthyosis/genetics , Infant, Newborn, Diseases/genetics , Point Mutation , Skin/enzymology , Transglutaminases/genetics , Child, Preschool , Female , Humans , Ichthyosis/pathology , Infant, Newborn , Infant, Newborn, Diseases/pathology , Models, Chemical , Phenotype , Protein Structure, Tertiary , Skin/pathology , Transglutaminases/chemistry , Transglutaminases/metabolism
17.
Bioessays ; 24(9): 789-800, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12210515

ABSTRACT

Terminally differentiating stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery termed the cornified cell envelope (CE). It is composed of numerous structural proteins that become cross-linked by several transglutaminase enzymes into an insoluble macromolecular assembly. Several proteins are involved in the initial stages of CE assembly, but only certain proteins from a choice of more than 20 different proteins are used in the final stages of CE reinforcement, apparently to meet tissue-specific requirements. In addition, a variable selection of proteins may be upregulated in response to genetic defects of one of the CE proteins or tissue injury, in an effort to maintain an effective barrier. Additionally, in the epidermis and hair fiber cuticle, a layer of lipids is covalently attached to the proteins, which provides essential water barrier properties. Here we describe our current understanding of CE structure, a possible mechanism of its assembly, and various disorders that cause a defective barrier.


Subject(s)
Epithelium/physiology , Animals , Cytoskeletal Proteins/chemistry , Epithelial Cells/cytology , Epithelium/metabolism , Humans , Lipids/chemistry , Membrane Proteins/chemistry , Models, Biological , Models, Chemical , Models, Molecular , Plakins , Proline/chemistry , Protein Precursors/chemistry , Transglutaminases/chemistry
18.
J Biol Chem ; 277(44): 42268-79, 2002 Nov 01.
Article in English | MEDLINE | ID: mdl-12200429

ABSTRACT

The major protein component of the cornified cell envelope barrier structure of the epidermis is loricrin, and it is expressed late during terminal differentiation in epidermal keratinocytes. We have previously shown that an AP1 site located in the proximal promoter region (position -55) is essential for human loricrin promoter activity (Rossi, A., Jang, S-I., Ceci, R., Steinert, P. M., and Markova, N. G. (1998) J. Invest. Dermatol. 110, 34-40). In this study we show that its regulation requires complex cooperative and competitive interactions between multiple transcription factors in keratinocytes located in different compartments of the epidermis. We show that as few as 154 base pairs of 5'-upstream sequences from the cap site can direct the keratinocyte-specific expression in cultured keratinocytes. Mutation and DNA-protein analyses show that Sp1, c-Jun, an unidentified regulator, and the co-activator p300/CREB-binding protein up-regulate whereas Sp3, CREB-1/CREMalpha/ATF-1, Jun B, and an AP2-like protein (termed the keratinocyte-specific repressor-1 (KSR-1)) suppress loricrin promoter activity. We show that CREB protein can compete with c-Jun for the AP1 site and repress loricrin promoter activity. We show here that the protein kinase A pathway can activate loricrin expression by manipulation of the Sp1, Sp3, and KSR-1 levels in the nucleus. Thus, in undifferentiated cells, loricrin expression is suppressed by Jun B, Sp3, and KSR-1 proteins. But in advanced differentiated cells, levels of Sp3, KSR-1, and CREB proteins are lower; the unidentified regulator protein can bind; Sp1 and c-Jun are increased; and then p300/CBP is recruited. Together, these events allow loricrin transcription to proceed. Indeed, the synergistic effects of the Sp1, c-Jun, and p300 factors indicate that p300/CBP might act as bridge to form an active transcription complex.


Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation , Keratinocytes/metabolism , Membrane Proteins/genetics , Nuclear Proteins/physiology , Sp1 Transcription Factor/physiology , Trans-Activators/physiology , Transcription Factor AP-1/physiology , Transcription Factors/physiology , Amino Acid Motifs , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP-Dependent Protein Kinases/physiology , Humans , Models, Genetic , Promoter Regions, Genetic , Protein Kinases/physiology , Sp3 Transcription Factor , Transcription Factor AP-2 , Transcription, Genetic
19.
J Invest Dermatol ; 119(2): 440-8, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12190868

ABSTRACT

E-cadherin-mediated adherens junction formation and maintenance are thought to involve actin filament rearrangements through the action of small GTPases. Recently, we demonstrated that microtubule disruption in normal human epidermal keratinocytes grown in low calcium media conditions induces cell-cell adhesion by redistribution of endogenous E-cadherin, and it promotes stress fiber formation. This actin rearrangement was apparently mediated by RhoA activation. This model system therefore provides a tool with which to dissect relationships between cell-cell adhesion and Rho-mediated stress fiber formation. In this study, we have demonstrated in normal human epidermal keratinocytes that disruption of actin structures including stress fibers does not interfere with E-cadherin redistribution during microtubule-induced cell-cell adhesion. Moreover, this cell-cell adhesion could not be blocked by RhoA inactivation at the level for inhibition of stress fiber formation. Additionally, in the immortalized HaCaT keratinocyte cell line, which does not undergo cell-cell adhesion after microtubule disruption in low calcium conditions, expression of dominant-active RhoA could induce stress fiber formation without inducing adhesion. On the other hand, a variant of the HaCaT cell line, HC-R1, showed microtubule-disruption-induced cell-cell adhesion without stress fiber formation. Together, our results suggest that, in keratinocytes, the process of cell adhesion can occur independently of RhoA-mediated stress fiber formation.


Subject(s)
Actins/metabolism , Cell Adhesion , Keratinocytes/physiology , Microtubules/physiology , rhoA GTP-Binding Protein/physiology , Cadherins/physiology , Cell Adhesion/drug effects , Cells, Cultured , Humans , Neomycin/pharmacology , Nocodazole/pharmacology , Polymers/metabolism , Stress Fibers/physiology
20.
J Interferon Cytokine Res ; 22(6): 677-82, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12162878

ABSTRACT

Transglutaminase 2 (tissue transglutaminase, TGase 2) was recently identified as an endomysial autoantigen in celiac disease (CD). Identification of how TGase 2 expression is increased may allow a better understanding of this autoimmune disease. Certain inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), and the Th type I cytokine interferon-gamma (INF-gamma) are abundant in CD. We have investigated whether these play a role in the regulation of TGase 2 expression in a model rat small intestinal epithelial cell line (IEC-6). After treatment for 24 h, TNF-alpha did not significantly alter TGase 2 mRNA or activity, but TGF-beta decreased mRNA and activity by 4-5-fold. IFN-gamma increased mRNA and TGase 2 activity by about 2-fold in 24 h and 5-fold by 5 days. Our new data suggest that increased TGase 2 expression in the upper small intestine of CD patients may be due to increased IFN-gamma expression, loss of TGF-beta signaling, or both.


Subject(s)
Epithelial Cells/enzymology , GTP-Binding Proteins/metabolism , Gene Expression Regulation, Enzymologic , Interferon-gamma/pharmacology , Intestine, Small/enzymology , Transglutaminases/metabolism , Animals , Cell Line , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , GTP-Binding Proteins/genetics , Humans , Interferon-gamma/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , RNA, Messenger/metabolism , Rats , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Time Factors , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology , Transglutaminases/genetics , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology
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