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Sola-Carvajal A.,University of Murcia | Gil-Ortiz F.,Institute Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Gil-Ortiz F.,ALBA Synchrotron Light Facility | Garcia-Carmona F.,University of Murcia | And 2 more authors.
Biochemical Journal | Year: 2014

NAMDH (N-acetyl-D-mannosamine dehydrogenase), from the soil bacteroidete Flavobacterium sp. 141-8, catalyses a rare NAD+ -dependent oxidation of ManNAc (N-acetyl-Dmannosamine) into N-acetylmannosamino-lactone, which spontaneously hydrolyses into N-acetylmannosaminic acid. NAMDH belongs to the SDR (short-chain dehydrogenase/reductase) superfamily and is the only NAMDH characterized to date. Thorough functional, stability, site-directed mutagenesis and crystallographic studies have been carried out to understand better the structural and biochemical aspects of this unique enzyme. NAMDH exhibited a remarkable alkaline pH optimum (pH 9.4) with a high thermal stability in glycine buffer (Tm =64°C) and a strict selectivity towards ManNAc and NAD+ . Crystal structures of ligand-free and ManNAc- and NAD + -bound enzyme forms revealed a compact homotetramer having point 222 symmetry, formed by subunits presenting the characteristic SDR α3β7α3 sandwich fold. A highly developed C-terminal tail used as a latch connecting nearby subunits stabilizes the tetramer. A dense network of polar interactions with the substrate including the encasement of its acetamido group in a specific binding pocket and the hydrogen binding of the sugar 4OH atom ensure specificity for ManNAc. The NAMDH-substrate complexes and site-directed mutagenesis studies identify the catalytic tetrad and provide useful traits for identifying new NAMDH sequences. © 2014 Biochemical Society.

Latorre V.,Institute Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Sevilla L.M.,Institute Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Sanchis A.,Institute Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Perez P.,Institute Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas
Journal of Investigative Dermatology | Year: 2013

We recently demonstrated that mice lacking the epidermal glucocorticoid (GC) receptor (GR) (GR epidermal knockout (GR EKO) mice) have developmental defects and sensitivity to epidermal challenge in adulthood. We examined the susceptibility of GR EKO mice to skin chemical carcinogenesis. GR EKO mice treated with a low dose of 12-dimethylbenz(a) anthracene (DMBA) followed by phorbol 12-myristate 13-acetate (PMA) promotion exhibited earlier papilloma formation with higher incidence and multiplicity relative to control littermates (CO). Augmented proliferation and inflammation and defective differentiation of GR EKO keratinocytes contributed to the phenotype, likely through increased AKT and STAT3 (signal transducer and activator of transcription 3) activities. GR EKO tumors exhibited signs of early malignization, including delocalized expression of laminin A, dermal invasion of keratin 5 (K5)-positive cells, K13 expression, and focal loss of E-cadherin. Cultured GR EKO keratinocytes were spindle like, with loss of E-cadherin and upregulation of smooth muscle actin (SMA) and Snail, suggesting partial epithelial-mesenchymal transition. A high DMBA dose followed by PMA promotion generated sebaceous adenomas and melanocytic foci in GR EKO and CO. Importantly, the number, growth kinetics, and extent of both tumor types increased in GR EKO mice, suggesting that in addition to regulating tumorigenesis from epidermal lineages, GR in keratinocytes is important for cross-talk with other skin cells. Altogether, our data reinforce the importance of GR in the pathogenesis of skin cancer. © 2013 The Society for Investigative Dermatology.

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