Zoltán Nemes

Bricks and mortar of the epidermal barrier

A specialized tissue type, the keratinizing epithelium, protects terrestrial mammals from water loss and noxious physical, chemical and mechanical insults. This barrier between the body and the environment is constantly maintained by reproduction of inner living epidermal keratinocytes which undergo a process of terminal differentiation and then migrate to the surface as interlocking layers of dead stratum corneum cells. These cells provide the bulwark of mechanical and chemical protection, and together with their intercellular lipid surroundings, confer water-impermeability. Much of this barrier function is provided by the cornified cell envelope (CE), an extremely tough protein/lipid polymer structure formed just below the cytoplasmic membrane and subsequently resides on the exterior of the dead cornified cells. It consists of two parts: a protein envelope and a lipid envelope. The protein envelope is thought to contribute to the biomechanical properties of the CE as a result of cross-linking of specialized CE structural proteins by both disulfide bonds and N(ε)-(γ-glutamyl)lysine isopeptide bonds formed by transglutaminases. Some of the structural proteins involved include involucrin, loricrin, small proline rich proteins, keratin intermediate filaments, elafin, cystatin A, and desmosomal proteins. The lipid envelope is located on the exterior of and covalently attached by ester bonds to the protein envelope and consists of a monomolecular layer of ω-hydroxyceramides. These not only serve of provide a Teflon-like coating to the cell, but also interdigitate with the intercellular lipid lamellae perhaps in a Velcro-like fashion. In fact the CE is a common feature of all stratified squamous epithelia, although its precise composition, structure and barrier function requirements vary widely between epithelia. Recent work has shown that a number of diseases which display defective epidermal barrier function, generically known as ichthyoses, are the result of genetic defects of the synthesis of either CE proteins, the transglutaminase 1 cross-linking enzyme, or defective metabolism of skin lipids.

Transglutaminase induction by various cell death and apoptosis pathways.

Clarification of the molecular details of forms of natural cell death, including apoptosis, has become one of the most challenging issues of contemporary biomedical sciences. One of the effector elements of various cell death pathways is the covalent cross-linking of cellular proteins by transglutaminases. This review will discuss the accumulating data related to the induction and regulation of these enzymes, particularly of tissue type transglutaminase, in the molecular program of cell death. A wide range of signalling pathways can lead to the parallel induction of apoptosis and transglutaminase, providing a handle for better understanding the exact molecular interactions responsible for the mechanism of regulated cell death.

Cross-linking of ubiquitin, HSP27, parkin, and α-synuclein by γ-glutamyl-ε-lysine bonds in Alzheimer's neurofibrillary tangles

The accumulation of misfolded proteins in intracellular inclusions is a generic feature of neurodegenerative disorders. Although heavily ubiquitylated, the aggregated proteins are not degraded by the proteasomes. A possible reason for this phenomenon may be a modification of deposited proteins by transglutaminases forming γ‐glutamyl‐ε‐lysine (GGEL) cross‐links between distinct proteins. Here, we show that the frequency of GGEL cross‐links is an order of magnitude higher in Alzheimers brain cortex than in age‐matched or younger controls. This difference is due to the accumulation of GGEL cross‐links in ubiquitin‐immunopositive protein particles present in both Alzheimers brains and those from aged individuals. The highly cross‐ linked protein aggregates show immunoreactivity to antibodies against tau and neurofilament proteins, and partially also to α‐synuclein, indicating that these structures are inherent in Alzheimers neurofibrillary tangles and Lewy bodies. Using mass sequence analysis, we identified the same six pairs of peptide sequences cross‐linked in both senile and Alzheimers specimens: Gln31 and Gln190 of HSP27 protein are cross‐linked with Lys29 and Lys48 of ubiquitin and HSP27 therefore may cross‐link two (poly)ubiquitin chains. One lysine residue of parkin and one of α‐synuclein were also found to be cross‐linked. The data suggest that cross‐linking of (poly)ubiquitin moieties via HSP27 may have a role in the stabilization of the intraneuronal protein aggregates by interference with the proteasomal elimination of unfolded proteins.

Differentiation markers in hemangiopericytoma

Normal pericytes were found to express factor XIIIa (F-XIII) and histocompatibility antigen HLA-DR. These markers were studied in 15 hemangiopericytomas (HPC) and 16 other tumors with an HPC-like pattern. A subpopulation of tumor cells expressing F-XIII and HLA-DR antigens was a constant feature of HPC and supported their pericytic origin. Meningeal HPC did not differ in phenotype from peripheral soft tissue HPC. Most tumors with an HPC-like pattern (including synovial sarcomas, malignant schwannomas, leiomyosarcomas, and liposarcomas) were negative for F-XIII and HLA-DR. Malignant fibrous histiocytomas, however, invariably contained a subpopulation expressing these antigens. Therefore, F-XIII can be considered as a marker of fibrohistiocytic differentiation in HPC. Individual tumor cells in HPC occasionally were positive for factor VIII-related antigen (F-VIII-R-Ag), suggesting that the spectrum of differentiation in HPC may include the endothelial cell line. The differentiation characteristics of HPC support the concept that pericytes are primitive cells that may act as precursors to other mesenchymal cells.

Three-dimensional structure of the human transglutaminase 3 enzyme: binding of calcium ions changes structure for activation.

Transglutaminase (TGase) enzymes catalyze the formation of covalent cross‐links between protein‐bound glutamines and lysines in a calcium‐dependent manner, but the role of Ca2+ ions remains unclear. The TGase 3 isoform is widely expressed and is important for epithelial barrier formation. It is a zymogen, requiring proteolysis for activity. We have solved the three‐dimensional structures of the zymogen and the activated forms at 2.2 and 2.1 Å resolution, respectively, and examined the role of Ca2+ ions. The zymogen binds one ion tightly that cannot be exchanged. Upon proteolysis, the enzyme exothermally acquires two more Ca2+ ions that activate the enzyme, are exchangeable and are functionally replaceable by other lanthanide trivalent cations. Binding of a Ca2+ ion at one of these sites opens a channel which exposes the key Trp236 and Trp327 residues that control substrate access to the active site. Together, these biochemical and structural data reveal for the first time in a TGase enzyme that Ca2+ ions induce structural changes which at least in part dictate activity and, moreover, may confer substrate specificity.

Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes

MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.

Involucrin Cross-linking by Transglutaminase 1 BINDING TO MEMBRANES DIRECTS RESIDUE SPECIFICITY

The transglutaminase 1 (TGase 1) enzyme is essential for the assembly of the cell envelope barrier in stratified squamous epithelia. It is usually bound to membranes, but to date most studies with it have involved solution assays. Here we describe anin vitro model system for characterizing the function of TGase 1 on the surface of synthetic lipid vesicles (SLV) of composition similar to eukaryote plasma membranes. Recombinant baculovirus-expressed human TGase 1 readily binds to SLV and becomes active in cross-linking above 10 μm Ca2+, in comparison to above 100 μm in solution assays, suggesting that the membrane surface is important for enzyme function. Involucrin also binds to SLV containing 12–18% phosphatidylserine and at Ca2+ concentrations above 1 μm. In reactions of involucrin with TGase 1 enzyme in solution, 80 of its 150 glutamines serve as donor residues. However, on SLV carrying both involucrin and TGase 1, only five glutamines serve as donors, of which glutamine 496 was the most favored. As controls, there was no change in specificity toward the glutamines of other substrates used by free or SLV-bound TGase 1 enzyme. We propose a model in which involucrin and TGase 1 bind to membranes shortly after expression in differentiating keratinocytes, but cross-linking begins only later as intracellular Ca2+levels increase. Furthermore, the data suggest that the membrane surface regulates the steric interaction of TGase 1 with substrates such as involucrin to permit specific cross-linking for initiation of cell envelope barrier formation.

Cholesterol 3-Sulfate Interferes with Cornified Envelope Assembly by Diverting Transglutaminase 1 Activity from the Formation of Cross-links and Esters to the Hydrolysis of Glutamine

The loss of transglutaminase 1 enzyme (TGase 1) activity causes lamellar ichthyosis. Recessive X-linked ichthyosis (XI) results from accumulation of excess cholesterol 3-sulfate (CSO4) in the epidermis but the pathomechanism how elevated epidermal CSO4 causes ichthyosis is largely unknown. Here we provide evidence that XI is also a consequence of TGase 1 dysfunction. TGase 1 is a key component of barrier formation in keratinocytes: it participates in the cross-linking of cell envelope (CE) structural proteins, and also forms the lipid bound envelope by esterification of long chain ω-hydroxyceramides onto CE proteins. Using involucrin and an epidermal ω-hydroxyceramide analog as substrates, kinetic analyses revealed that at membrane concentrations above 4 mol %, CSO4 caused a marked and dose-dependent inhibitory effect on isopeptide and ester bond formation. Sequencing of tryptic peptides from TGase 1-reacted involucrin showed a large increase in deamidation of substrate glutamines. We hypothesize that supraphysiological levels of CSO4 in keratinocyte membranes distort the structure of TGase 1 and facilitate the access of water into its active site causing hydrolysis of substrate glutamine residues. Our findings provide further evidence for the pivotal role of the TGase 1 enzyme in CE formation.

Pathology of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease is one of the leading causes of death and morbidity worldwide. Despite intensive investigation, its pathology and pathophysiology are not well understood. The hallmarks of the disease are irreversible airflow limitation and chronic inflammation. Small airway obstruction due to progressive inflammation and fibrosis, and the loss of elastic recoil mediated by elastolysis and apoptosis equally contribute to pathologic changes. However, it is debated to what extent the obstruction of large airways leads to altered lung function. Three morphologic entities are described in the literature under one disease; chronic bronchitis, obstructive bronchiolitis and emphysema may appear in the same patient at the same time. The authors review pathologic changes observed in chronic obstructive pulmonary disease, including acute exacerbations and secondary pulmonary hypertension as severe but common complications of the disease. Furthermore, we detail recent scientific evidences for major cellular and molecular inflammatory pathway activation. These mechanisms result in accelerated apoptosis, remodeling and increased proinflammatory cytokine release. Targeting intracellular pathological changes may lead to the discovery of a new generation of drugs that could reduce chronic obstruction before airway irreversibility is established.

Changes in the expression and distribution of the inducible and endothelial nitric oxide synthase in mucosal biopsy specimens of inflammatory bowel disease

Objective The role of nitric oxide (NO) in the pathophysiology of inflammatory bowel disease (IBD) is controversial. The aim of this study was to investigate the expression and localization of nitric oxide synthase isoforms (iNOS, eNOS) in IBD colonic mucosa. Material and methods Forty-four patients with IBD (24 ulcerative colitis (UC), 20 Crohns disease (CD) and 16 controls) were investigated by colonoscopy. iNOS and eNOS in tissue sections was demonstrated by histochemistry (NADPH-diaphorase reaction) and immunohistochemistry. Cell type analysis and quantitative assessment of the iNOS immunoreactive (IR) cells and densitometry of iNOS in immunoblots were also performed. Results iNOS-IR cells were significantly numerous in inflamed mucosa of UC (64±4 cells/mm2) than in CD (4±2 cells/mm2). iNOS-IR/CD15-IR cells showed significant elevation in inflamed (i) versus uninflamed (u) UC mucosa (UCu 8±3%, UCi 85±10%) In CD, the percentage of iNOS-IR/CD68-IR cells was lower in inflamed sites (CDu 23±8%, CDi 4±3%). Immunoblot of biopsies revealed significant elevation of iNOS in active UC compared with uninflamed sites, whereas in CD no significant changes were detected. Differences were observed in eNOS and endothelial marker CD31 immunoreactivity. In patients with UC and in controls the ratios of eNOS/CD31-IR vessels were 82.3% and 92.0% respectively, whereas in CD the ratio was 8.3% with a concomitantly significant increase of CD31-IR vessels. The distribution and morphological characteristics of the NOS-IR inflammatory cells and endothelia were similar to those showing NADPH-diaphorase reactivity. Conclusions Differences observed in the expression and distribution of NOS isoforms in immune and endothelial cells may contribute to better understanding of the structural and physiological changes in UC and CD.