Karin Aufenvenne

Loss of Corneodesmosin Leads to Severe Skin Barrier Defect, Pruritus, and Atopy: Unraveling the Peeling Skin Disease

Generalized peeling skin disease is an autosomal-recessive ichthyosiform erythroderma characterized by lifelong patchy peeling of the skin. After genome-wide linkage analysis, we have identified a homozygous nonsense mutation in CDSN in a large consanguineous family with generalized peeling skin, pruritus, and food allergies, which leads to a complete loss of corneodesmosin. In contrast to hypotrichosis simplex, which can be associated with specific dominant CDSN mutations, peeling skin disease is characterized by a complete loss of CDSN expression. The skin phenotype is consistent with a recent murine Cdsn knockout model. Using three-dimensional human skin models, we demonstrate that lack of corneodesmosin causes an epidermal barrier defect supposed to account for the predisposition to atopic diseases, and we confirm the role of corneodesmosin as a decisive epidermal adhesion molecule. Therefore, peeling skin disease will represent a new model disorder for atopic diseases, similarly to Netherton syndrome and ichthyosis vulgaris in the recent past.

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains.

Abstract Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.

Transglutaminase-1 and bathing suit ichthyosis: molecular analysis of gene/environment interactions

TO THE EDITOR Lamellar ichthyosis (LI) is a heterogeneous group of keratinization disorders characterized by scaling of the whole integument with differences in color and shape. Several LI cases (B40%) are due to mutations in the transglutaminase-1 (TGase-1) gene TGM1 on chromosome 14q11 (Huber et al., 1995; Russell et al., 1995). TGase-1, which catalyzes the calcium-dependent crosslinking of proteins through the formation of e-(g-glutamyl)lysine isopeptide bonds, is synthesized in the upper differentiated epidermal layers, where it facilitates the formation of the cornified cell envelope by cross-linking structural proteins, such as loricrin and involucrin. Moreover, TGase-1 catalyzes the linkage of specialized ceramides to cell envelope proteins (Nemes et al., 1999). Three clinical LI variants are related to mutations in TGM1. Patients with generalized LI are often born as collodion babies and develop a scaling on the entire body. A minority, referred as ‘‘self-healing collodion baby,’’ display a mild phenotype or heal spontaneously within the first few weeks of life (Reed et al., 1972) and have particular TGM1 mutations leading to impaired TGase-1 activity at higher intrauterine water pressure (Raghunath et al., 2003). Patients with bathing suit ichthyosis (BSI) develop a scaling pattern only affecting the trunk (Jacyk, 2005; Oji et al., 2006; Arita et al., 2007). We showed that the BSI phenotype is due to mutations in TGM1 and hypothesized that the scaling pattern correlates with the local body temperature (Oji et al., 2006). We show here that BSI mutations, when compared with wild type, (a) have a decreased enzyme activity (Figure 1a–b) and (b) exhibit a marked shift in temperature optimum from 37 to 31 1C (Figure 1c–e). Deficient activity of BSI mutants can be reconstituted by decreasing the temperature to below 33 1C (Table 1). These data explain the clinical phenotype with healthy areas on arms and legs having a lower body temperature. Mutations of 10 patients (Oji et al., 2006) have been studied regarding their impact on the enzymatic activity of TGase-1. Eleven mutations (Table 1) were expressed in HEK 293 cells, maintained at 32 1C. The TGase-1 cDNA was subcloned into the mammalian expression vector pcDNA3.1(þ ) (Invitrogen, Karlsruhe, Germany) and was used as a template for site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands) following the manufacturer’s instructions (http://www.stratagene. com). Eight of these mutations, namely Tyr276Asn, Arg126Cys, Arg264Trp, Arg307Gly, Arg264Gln, Arg687His, Arg315Cys, and Arg315His, are associated with BSI and three mutations (Arg142His, Arg389Pro, and Ser358fsX26) are further described in patients with classical LI. All constructs were confirmed by complete sequencing (Seqlab, Göttingen, Germany). At 96 hours after transfection, HEK293 cells were homogenized for 30 minutes on ice in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 104 mM AEBSF, 0.08 mM aprotinin, 2 mM leupeptin, 4 mM bestatin, 1.5 mM pepstatin A, 1.4 mM E-64, pH 7.5). Cell lysates were assayed for protein using the BCA Protein Assay Kit (Pierce, Rockford, IL). Western blot analysis using a specific anti-TGase-1 antibody confirmed high expression levels of the wild type, and all but one of the mutants (Figure 1a). Several mutations — despite similar transfection efficiencies — showed reduced expression compared with the wild-type gene (Figure 1b). No expression was detectable in cell lysates that were repeatedly transfected with the mutant Ser358fsX26, indicating that this mutation causes protein instability. No TGase-1 expression was detected in HEK cells transfected with the pcDNA3.1 lacZ vector used as negative control and to determine transfection efficiency to show that different expression levels of the mutants are not because of different transfection efficiencies. We analyzed the recombinantly expressed enzymes for activity between 25 and 45 1C by florescence spectrometry (LS55, Perkin Elmer, Rodgau, Germany; excitation: 332 nm, emission: 500 nm, slit: 5.0 nm). Each protein sample of 5mg was incubated in prewarmed assay buffer (50 mM Tris-HCl, 10 mM CaCl2, 10 mM reduced glutathione, 2.5% glycerol, 2.5% DMSO, 25 mM biotinylated cadaverine, and 20 mM N,N-dimethylcasein) and the activity was measured for 15 minutes. The incorporation of cadaverine into casein results in an increment of fluorimetric intensity and a shift in emission wavelength. The determined slope of an arithmetic mean of five measurements (n1⁄45) served as a measure for TGase activity. Lysates of untransfected cells served as a negative control. The wild-type TGase-1 showed high activity over a wide temperature LETTERS TO THE EDITOR

Acral self-healing collodion baby: report of a new clinical phenotype caused by a novel TGM1 mutation

A minority of collodion babies, called ‘self‐healing collodion babies’, heal spontaneously. We describe a novel clinical phenotype of acral self‐healing collodion baby caused by a new TGM1 mutation. The proband, born to healthy parents, presented at birth as a collodion baby strictly localized to the extremities. The skin condition returned to normal at the age of 3 weeks. The older sister was born as a generalized collodion baby; the condition then developed into lamellar ichthyosis. Molecular analysis of TGM1 revealed three novel mutations in the family. The proband was compound heterozygous for the p.Val359Met and p.Arg396His mutations, whereas the older sister was compound heterozygous for p.Arg396His and a deletion mutation c.1922_1926+2delGGCCTGT. Structural modelling of the p.Val359Met mutation suggested a minor disruption of the protein structure, whereas a modification of protein–protein interaction was predicted for p.Arg396His. These predictions corroborated the analysis of recombinant transglutaminase (TGase)‐1 proteins carrying the p.Val359Met and p.Arg396His mutations. Both showed decreased levels of protein expression: p.Val359Met displayed residual activity (12·8%), while p.Arg396His caused a dramatic loss of activity (3·3%). These observations demonstrate for the first time that TGM1 mutations can be associated with acral self‐healing collodion baby, and expand the clinical spectrum of TGase‐1 deficiency.

Complete filaggrin deficiency in ichthyosis vulgaris is associated with only moderate changes in epidermal permeability barrier function profile

Ichthyosis vulgaris (IV) is caused by loss‐of‐function mutations in the profilaggrin (FLG) gene. Filaggrin drives complex interrelated functions, with strategic roles in establishing structural and chemical barrier function, hydration of the skin and maintaining epidermal homeostasis. Data on the effect of FLG mutations on epidermal barrier function in IV are very scarce.

Ichthyosis vulgaris: novel FLG mutations in the German population and high presence of CD1a+ cells in the epidermis of the atopic subgroup

Background  Ichthyosis vulgaris (IV) is a genetic disorder with a prevalence of 1 : 250–1000 caused by filaggrin (FLG) mutations, which also predispose to atopic diseases.

Long-Term Faithful Recapitulation of Transglutaminase 1–Deficient Lamellar Ichthyosis in a Skin-Humanized Mouse Model, and Insights from Proteomic Studies

Abbreviations: CE, cornified envelope; DEJ, dermoepidermal junction; KPRP, keratinocyte proline-rich protein; LI, lamellar ichthyosis; NMF, natural moisturizing factor; TEWL, transepidermal water loss; TG1, transglutaminase 1

Beta-Actin is a Target for Transglutaminase Activity at Synaptic Endings in Chicken Telencephalic Cell Cultures

Transglutaminases are Ca2+-dependent enzymes that catalyse the covalent cross-linking of protein-bound glutamine and lysine residues, which can stabilise proteins or protein aggregates. In the brain, elevated expression levels and activity of transglutaminases are known to be linked with several neurodegenerative diseases. However, little is known about the physiological functions of transglutaminases in the central nervous system. In this study, we examined the expression and activity of transglutaminase 1 in chicken telencephalic cell cultures. We observed a cytosolic expression of transglutaminase 1 in telencephalic neurons. However, transglutaminase 1 activity was restricted to synaptic endings. Transglutaminase targets in the cultured cells were characterised via a biotinylation assay and β-actin was identified as a substrate. Furthermore, we were able to show that β-actin is a target for the activity of recombinant human transglutaminase 1 in vitro. We propose a mechanism where neuronal transglutaminase 1 is activated by synaptic activity-dependent influx of calcium ions and thereupon catalyse the formation of an intramolecular cross-link in β-actin, thereby stabilising the actin cytoskeleton against depolymerising effects. In this way, transglutaminase 1 could modulate actin-dependent plasticity events at synaptic endings.

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

S1 guidelines for the diagnosis and treatment of ichthyoses – update

Ichthyoses are a group of rare genetic skin disorders that pose numerous clinical challenges, in particular with respect to the correct diagnosis and appropriate management. The present update of the German ichthyosis guidelines addresses recent diagnostic advances that have resulted in the Sorèze consensus classification. In this context, we provide an updated diagnostic algorithm, taking into account clinical features as well as the molecular genetic basis of these disorders. Moreover, we highlight current therapeutic approaches such as psychosocial support, balneotherapy, mechanical scale removal, topical therapy, and systemic retinoid therapy. General aspects such as the indication for physical therapy, ergotherapy, or genetic counseling are also discussed. The present update was consented by an interdisciplinary consensus conference that included dermatologists, pediatricians, human geneticists, and natural scientists as well as representatives of the German patient support organization Selbsthilfe Ichthyose e. V.