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.

Topical enzyme-replacement therapy restores transglutaminase 1 activity and corrects architecture of transglutaminase-1-deficient skin grafts.

Transglutaminase-1 (TG1)-deficient autosomal-recessive congenital ichthyosis (ARCI) is a rare and severe genetic skin disease caused by mutations in TGM1. It is characterized by collodion babies at birth, dramatically increased transepidermal water loss (TEWL), and lifelong pronounced scaling. The disease has a tremendous burden, including the problem of stigmatization. Currently, no therapy targeting the molecular cause is available, and the therapeutic situation is deplorable. In this study, we developed the basis for a causative therapy aiming at the delivery of the enzyme to the inner site of the keratinocytes' plasma membrane. We prepared sterically stabilized liposomes with encapsulated recombinant human TG1 (rhTG1) and equipped with a highly cationic lipopeptide vector to mediate cellular uptake. The liposomes overcame the problems of insufficient cutaneous delivery and membrane penetration and provided excellent availability and activity of rhTG1 in primary keratinocytes. To demonstrate the general feasibility of this therapeutic approach in a humanized context, we used a skin-humanized mouse model. Treatment with rhTG1 liposomes resulted in considerable improvement of the ichthyosis phenotype and in normalization of the regenerated ARCI skin: in situ monitoring showed a restoration of TG1 activity, and cholesterol clefts vanished ultrastructurally. Measurement of TEWL revealed a restoration of epidermal barrier function. We regard this aspect as a major advance over available nonspecific approaches making use of, for example, retinoid creams. We conclude that this topical approach is a promising strategy for restoring epidermal integrity and barrier function and provides a causal cure for individuals with TG1 deficiency.

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 Anti-Apoptotic Properties of APEX1 in the Endothelium Require the First 20 Amino Acids and Converge on Thioredoxin-1.

The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a disordered N-terminus, a redox, and a DNA repair domain. APEX1 has anti-apoptotic properties, which have been linked to both domains depending on cell type and experimental conditions. AIMS: As protection against apoptosis is a hallmark of vessel integrity, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and, if so, what the underlying mechanisms are. RESULTS: APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D (CatD) cleavage, potentially by binding to the unprocessed form. Diminished CatD activation results in increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently, apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1, because APEX1 (21-318) induces CatD activity, decreases Thioredoxin-1 protein levels, and, thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1 via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In an in vivo model of restenosis, which is characterized by oxidative stress, endothelial activation, and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the endothelium of the carotids. INNOVATION: APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends on the first 20 amino acids of APEX1. CONCLUSION: As proper function of the endothelium during life span is a hallmark for individual health span, a detailed characterization of the functions of the APEX1N-terminus is required to understand all its cellular properties. Antioxid. Redox Signal. 26, 616-629.

SPINK5 and Netherton syndrome: novel mutations, demonstration of missing LEKTI, and differential expression of transglutaminases.

Netherton syndrome (NTS) is an autosomal recessive congenital ichthyosis featuring chronic inflammation of the skin, hair anomalies, epidermal hyperplasia with an impaired epidermal barrier function, failure to thrive and atopic manifestations. The disease is caused by mutations in the SPINK5 gene encoding the serine proteinase inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI). Sequence analyses of SPINK5 in seven NTS patients from five different families allowed us to identify two known and three novel mutations all creating premature termination codons. We developed a monoclonal antibody giving a strong signal for LEKTI in the stratum granulosum of normal skin and demonstrated absence of the protein in NTS epidermis. Immunoblot analysis revealed presence of full length LEKTI and of LEKTI cleavage fragments in normal hair roots, whereas in NTS hair roots LEKTI and its cleavage products were completely missing. Transglutaminase1 activity was present throughout almost the entire suprabasal epidermis in NTS, whereas in normal skin it is restricted to the stratum granulosum. In contrast, immunostaining for transglutaminase3 was absent or faint. Moreover, comparable with the altered pattern in psoriatic skin the epidermis in NTS strongly expressed the serine proteinase inhibitor SKALP/elafin and the anti-microbial protein human beta-defensin 2. These studies demonstrate LEKTI deficiency in the epidermis and in hair roots at the protein level and an aberrant expression of other proteins, especially transglutaminase1 and 3, which may account for the impaired epidermal barrier in NTS.

Beta-actin is a target for transglutaminase activity at synaptic endings in chicken telencephalic cell cultures.

Transglutaminases are Ca(2+)-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 ß-actin was identified as a substrate. Furthermore, we were able to show that ß-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 ß-actin, thereby stabilising the actin cytoskeleton against depolymerising effects. In this way, transglutaminase 1 could modulate actin-dependent plasticity events at synaptic endings.

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

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.

The alpha and beta subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation.

The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation.

Bathing suit ichthyosis is caused by transglutaminase-1 deficiency: evidence for a temperature-sensitive phenotype.

Bathing suit ichthyosis (BSI) is a striking and unique clinical form of autosomal recessive congenital ichthyosis characterized by pronounced scaling on the bathing suit areas but sparing of the extremities and the central face. Here we report on a series of 10 BSI patients. Our genetic, ultrastructural and biochemical investigations show that BSI is caused by transglutaminase-1 (TGase-1) deficiency. Altogether, we identified 13 mutations in TGM1-among them seven novel missense mutations and one novel nonsense mutation. Structural modeling for the Tyr276Asn mutation reveals that the residue is buried in the hydrophobic interior of the enzyme and that the hydroxyl side chain of Tyr276 is exposed to solvent in a cavity of the enzyme. Cryosections of healthy skin areas demonstrated an almost normal TGase activity, in contrast to the affected BSI skin, which only showed a cytoplasmic and clearly reduced TGase-1 activity. The distribution of TGase-1 substrates in the epidermis of affected skin corresponded to the situation in TGase-1 deficiency. Interestingly, the expression of TGase-3 and cathepsin D was reduced. Digital thermography validated a striking correlation between warmer body areas and presence of scaling in patients suggesting a decisive influence of the skin temperature. In situ TGase testing in skin of BSI patients demonstrated a marked decrease of enzyme activity when the temperature was increased from 25 to 37 degrees C. We conclude that BSI is caused by TGase-1 deficiency and suggest that it is a temperature-sensitive phenotype.