Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins.

BACKGROUND: Profilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 ("A") domain and a unique "B" domain with a nuclear localization sequence. OBJECTIVE: To determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal "AB" domain and compare it to other S100 fused-type proteins. METHODS: We used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins. RESULTS: Comparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins. CONCLUSION: The N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions.

Lamin A molecular compression and sliding as mechanisms behind nucleoskeleton elasticity.

Lamin A is a nuclear intermediate filament protein critical for nuclear architecture and mechanics and mutated in a wide range of human diseases. Yet little is known about the molecular architecture of lamins and mechanisms of their assembly. Here we use SILAC cross-linking mass spectrometry to determine interactions within lamin dimers and between dimers in higher-order polymers. We find evidence for a compression mechanism where coiled coils in the lamin A rod can slide onto each other to contract rod length, likely driven by a wide range of electrostatic interactions with the flexible linkers between coiled coils. Similar interactions occur with unstructured regions flanking the rod domain during oligomeric assembly. Mutations linked to human disease block these interactions, suggesting that this spring-like contraction can explain in part the dynamic mechanical stretch and flexibility properties of the lamin polymer and other intermediate filament networks.

Genes encoding cytoplasmic intermediate filament proteins of vertebrates revisited: identification of a cytoplasmic intermediate filament protein in the sea anemone Nematostella.

The cytoskeleton is crucial in determining cell architecture, division, motility, transport processes and in local control of signal transduction. Relatives of actin and tubulin are expressed in all phyla, underlining the fundamental importance of conserved cytoskeletal functions. Intermediate filament proteins have evolved in parallel with tissue diversity in the animal kingdom, likely from the demand to adapt one class of cytoskeletal proteins to cell type-restricted functions. Up to now, the evolutionary origin of cytoplasmic intermediate filament proteins remains unknown. Using a known gene encoding a cytoplasmic intermediate filament protein from the hemichordate Saccoglossus kowalevskii, we have identified the first corresponding gene in the sea anemone Nematostella, tentatively named cytovec. Our data reveal a relationship of cytovec with Hydra vulgaris nematocilins A and B that also lack a CAAX box. In light of additional recent findings, our data show that cytoplasmic intermediate filament genes are present in the common ancestor of Cnidaria and Bilateria.

An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis.

We report a technique for isolation and solubilization of intermediate filament (IF) proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography "shotgun" proteomics using mass spectrometry (MS). This is important because changes in the IF proteome, particularly in keratin expression and modification, are noted in colonic mucosa of patients with colorectal cancer. Though keratins have traditionally been dissolved in high concentration of urea, the latter solvent precludes efficient proteolytic digestion by trypsin prior to gel-free LC-MS/MS approaches. The extraction of cytoskeletal proteins was initially evaluated using MCF-7 cancer cell lines using a published, differential detergent solubilization protocol. IF proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between urea and guanidine hydrochloride (GuHCl) in triethylammonium bicarbonate buffer, isolated proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved proteins were tryptically digested and peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches.

Investigations into charge heterogeneity of wool intermediate filament proteins.

Genomic studies have shown that there are four abundant type I and type II intermediate filament proteins (IFPs) in wool. When separated using 2D-PAGE, the type I IFPs separated into four clearly defined major rows. The type II IFPs separated into two distinct staggered rows. The large number of spots seen by 2D-PAGE has previously been attributed to charge heterogeneity caused by post-translational modification of the protein. However, analysis of wool IFPs by 2D-PAGE techniques and mass spectrometry suggested an absence of phosphorylation or glycosylation modifications. Investigations with both the type I and type II IFPs showed that when single protein spots from a 2D-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. Amino acid analysis, mass spectrometry and Ellman's assay support the hypothesis that the proteins have the same sequence but vary in isoelectric charge, due to differences in exposure of charged residues on the molecular surface. The cause of IFP charge heterogeneity is thus proposed to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension.

[Identification and tissue localization of intermediate filament protein in Angiostrongylus cantonensis].

OBJECTIVE: To identify the type of the intermediate filament (IF) protein of Angiostrongylus cantonensis and analyze its tissue localization. METHODS: Recombinant pET-IF of antigen IF was expressed in E.coli with IPTG induction, and the expression products were purified by His.Bind column and identified for determining the type of the IF protein by Western blotting. Anti-IF antibody was prepared by multi-spot subcutaneous injection into mouse and used to detect the tissue slices of A. cantonensis by immunohistochemical analysis. RESULTS: The antigen IF were correctly expressed and purified, and identified as a keratin located in the intestine wall and cytoplusma. CONCLUSION: The antigen IF is distributed in the intestine wall of A. cantonensis.

Bcl-XL modulates the differentiation of immortalized human neural stem cells.

Understanding basic processes of human neural stem cell (hNSC) biology and differentiation is crucial for the development of cell replacement therapies. Bcl-X(L) has been reported to enhance dopaminergic neuron generation from hNSCs and mouse embryonic stem cells. In this work, we wanted to study, at the cellular level, the effects that Bcl-X(L) may exert on cell death during differentiation of hNSCs, and also on cell fate decisions and differentiation. To this end, we have used both v-myc immortalized (hNS1 cell line) and non-immortalized neurosphere cultures of hNSCs. In culture, using different experimental settings, we have consistently found that Bcl-X(L) enhances neuron generation while precluding glia generation. These effects do not arise from a glia-to-neuron shift (changes in fate decisions taken by precursors) or by only cell death counteraction, but, rather, data point to Bcl-X(L) increasing proliferation of neuronal progenitors, and inhibiting the differentiation of glial precursors. In vivo, after transplantation into the aged rat striatum, Bcl-X(L) overexpressing hNS1 cells generated more neurons and less glia than the control ones, confirming the results obtained in vitro. These results indicate an action of Bcl-X(L) modulating hNSCs differentiation, and may be thus important for the future development of cell therapy strategies for the diseased mammalian brain.

Generation and characterization of mouse parthenogenetic embryonic stem cells containing genomes from non-growing and fully grown oocytes.

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem cells. Unfortunately, differentiation capacity of the parthenogenetic embryonic stem cells was rather lower than fertilized embryos derived ES cells, which might be the result of the absence of male genome. It had been found that some maternally expressed genes were repressed and some paternally expressed genes were expressed in the non-growing oocytes. Therefore, maternal genome from non-growing oocytes can partially act as "sperm genome". In the present study, parthenogenetic blastocysts containing genome from non-growing and fully grown oocytes (named as NF-pBlastocysts) were produced by germinal vesicle transfer, and three newly established parthenogenetic embryonic stem (named as NF-pES) cell lines were derived from the resulting parthenogenetic blastocysts. All three NF-pES cell lines were positive for ES cell markers, such as alkaline phosphatase (AKP), stage-specific embryonic antigen 1 (SSEA-1) and octamer-binding transcription factor (Oct-4). They have a normal chromosome karyotype (40) and can be maintained in an undifferentiated state for extended periods of time. When NF-pES cells were injected into severe combined immunodeficient mice, teratomas with all three embryonic germ layers were obtained. The in vitro differentiation potential of NF-pES cells was analyzed by embryonic bodies (EB) formation. The expression of germ layer markers, such as nestin (ectoderm), desmin (mesoderm), and alpha-fetoprotein (endoderm) demonstrated that the NF-pES cells can differentiate into all three germ layers.

Generation and expansion of multipotent mesenchymal progenitor cells from cultured human pancreatic islets.

Cellular models and culture conditions for in vitro expansion of insulin-producing cells represent a key element to develop cell therapy for diabetes. Initial evidence that human beta-cells could be expanded after undergoing a reversible epithelial-mesenchymal transition has been recently negated by genetic lineage tracing studies in mice. Here, we report that culturing human pancreatic islets in the presence of serum resulted in the emergence of a population of nestin-positive cells. These proliferating cells were mainly C-peptide negative, although in the first week in culture, proliferating cells, insulin promoter factor-1 (Ipf-1) positive, were observed. Later passages of islet-derived cells were Ipf-1 negative and displayed a mesenchymal phenotype. These human pancreatic islet-derived mesenchymal (hPIDM) cells were expanded up to 10(14) cells and were able to differentiate toward adipocytes, osteocytes and chondrocytes, similarly to mesenchymal stem/precursor cells. Interestingly, however, under serum-free conditions, hPIDM cells lost the mesenchymal phenotype, formed islet-like clusters (ILCs) and were able to produce and secrete insulin. These data suggest that, although these cells are likely to result from preexisting mesenchymal cells rather than beta-cells, hPIDM cells represent a valuable model for further developments toward future replacement therapy in diabetes.

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (alpha-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.

Interactions of intermediate filament protein synemin with dystrophin and utrophin.

Synemin is a unique, very large intermediate filament (IF) protein present in all types of muscle cells, which forms heteropolymeric intermediate filaments (IFs) with the major IF proteins desmin and/or vimentin. We show herein that tissue-purified avian synemin directly interacts with both dystrophin and utrophin, and that specific expressed regions of both of the mammalian (human) synemin isoforms (alpha-synemin and beta-synemin) directly interact with specific expressed domains/regions of the dystrophin and utrophin molecules. Mammalian synemin is also shown to colocalize with dystrophin within muscle cell cultures. These results indicate that synemin is an important IF protein in muscle cells that helps fortify the linkage between the peripheral layer of cellular myofibrils and the costameric regions located along the sarcolemma and the sarcolemma region located within the neuromuscular and myotendinous junctions (NMJs and MTJs).

Dermatophyte growth and degradation of human stratum corneum in vitro (pathogenesis of dermatophytosis).

BACKGROUND: This study was carried out to determine growth of dermatophytes using human stratum corneum in vitro and the degrading effect of Keratinases (Proteinases) on stratum corneum for a complete understanding of the host parasite relationship. METHOD: Trichophyton rubrum isolates derived from patients with tinea cruris infections were obtained from the Department of Medical Microbiology, University Hospital of Wales, U.K. Human stratum corneum sterilized with ethylene oxide was used as a nitrogen source in agar culture medium plates. RESULT: Fungal growth took place in plates which contained human stratum corneum particles while there was no growth in the plates without stratum corneum at three weeks after initiation. There was a gradual disappearance of the particles of stratum corneum from the plates at the end of the third week CONCLUSION: The growth of organisms in plates with human stratum corneum and their disappearance at third week suggested that stratum corneum was not only source of nutrition for the dermatophytes, but also the growing fungal mycelia and the proteinases induced by them were playing a part in the digestion of granules and thus may have an important role in the pathogenesis of dermatophyte infections.

Clinical significance of anti-filaggrin antibody recognizing uncitrullinated filaggrin in rheumatoid arthritis.

Filaggrin is expressed in the cornified layer of epidermis and known to be one of the antigenic targets in rheumatoid arthritis. Although the citrulline residue in filaggrin is thought to be an antigenic determinant recognized by autoantibodies, the diagnostic sensitivity of synthetic citrullinated peptide is variable. To investigate the implication of anti-filaggrin antibodies recognizing uncitrullinated filaggrin in rheumatoid arthritis, we assayed antibody titers using unmodified recombinant filaggrin in the sera from 73 patients with rheumatoid arthritis, 150 patients with other connective tissue diseases and 70 normal controls. We also performed the correlation analysis between antibody titers and the clinical variables in patients with rheumatoid arthritis. Titers of IgG anti-filaggrin antibodies were significantly higher in rheumatoid arthritis patients compared to normal controls (P=0.02), but not in patients with osteoarthritis, ankylosing spondylitis or systemic lupus erythematosus. IgG anti-filaggrin antibodies were more frequently found in patients with rheumatoid arthritis compared to normal controls (12.3% vs 1.4% respectively, P=0.04). An anti-filaggrin antibody titer was correlated with visual analogue scale of pain, tender joint count, Ritchie articular index or C-reactive protein, but not with anti-nuclear antibody or rheumatoid factor. These results suggest that anti-filaggrin antibody recognizes the uncitrullinated filaggrin as an antigen and its titer correlates with clinical parameters, explaining the variable sensitivity of anti-filaggrin antibody test.

Predicted and measured disorder in peripherin/rds, a retinal tetraspanin.

Vertebrate photoreceptor outer segment (OS) morphogenesis requires peripherin/rds (P/rds). We have characterized this protein's C-terminus and present evidence that suggests it is intrinsically disordered. We propose that structural flexibility may underlie the multifunctionality proposed for this domain previously. The extremely short C-termini present in other tetraspanin family members suggest that intrinsic disorder may also play a role for those proteins.

The mouse synemin gene encodes three intermediate filament proteins generated by alternative exon usage and different open reading frames.

We have previously cloned and characterized the human synemin gene, which encodes two intermediate filament proteins (IFPs). We now show that the mouse synemin gene encodes three different synemin isoforms through an alternative splicing mechanism. Two of them, synemin H and M are similar to human alpha and beta synemin, and the third isoform, L synemin, constitutes a new form of IFP. It has a typical rod domain and a short tail (49 residues) with a novel sequence that is produced by a different open reading frame. The synthesis of H/M synemins starts in the embryo, whereas the synemin L isoform is present in adult muscles. The H/M isoforms are bound to desmin or vimentin in the muscle cells of wild-type mice. Using desmin- and vimentin-deficient mice, we have obtained direct evidence that synemin is associated with muscle intermediate filaments in vivo. The organization of the synemin fibril is disrupted in skeletal and cardiac muscle when desmin is absent and in smooth muscle when vimentin is absent. The fact that the three synemin isoforms differ in the sequences of their tail domains as well as in their developmental patterns suggests that they fulfill different functions.