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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.

PubMed | Institute Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2010

Insulin-induced gene 2 (INSIG2) and its homolog INSIG1 encode closely related endoplasmic reticulum proteins that regulate the proteolytic activation of sterol regulatory element-binding proteins, transcription factors that activate the synthesis of cholesterol and fatty acids in animal cells. Several studies have been carried out to identify INSIG2 genetic variants associated with metabolic diseases. However, few data have been published regarding the regulation of INSIG2 gene expression. Two Insig2 transcripts have been described in rodents through the use of different promoters that produce different noncoding first exons that splice into a common second exon. Herein we report the cloning and characterization of the human INSIG2 promoter and the detection of an INSIG2-specific transcript homologous to the Insig2b mouse variant in human liver. Deletion analyses on 3 kb of 5-flanking DNA of the human INSIG2 gene revealed the functional importance of a 350-bp region upstream of the transcription start site. Mutated analyses, chromatin immunoprecipitation assays, and RNA interference analyses unveiled the significance of an Ets-consensus motif in the proximal region and the interaction of the Ets family member SAP1a (serum response factor (SRF) accessory protein-1a) with this region of the human INSIG2 promoter. Moreover, our findings suggest that insulin activated the human INSIG2 promoter in a process mediated by phosphorylated SAP1a. Overall, these results map the functional elements in the human INSIG2 promoter sequence and suggest an unexpected regulation of INSIG2 gene expression in human liver.

PubMed | Institute Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas
Type: Journal Article | Journal: Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

PII, an ancient and widespread signaling protein, transduces nitrogen/carbon/energy abundance signals through interactions with target proteins. We clarify structurally how PII regulates gene expression mediated by the transcription factor NtcA, the global nitrogen regulator of cyanobacteria, shedding light on NtcA structure and function and on how NtcA is activated by 2-oxoglutarate (2OG) and coactivated by the nonenzymatic PII target, protein PipX. We determine for the cyanobacteria Synechococcus elongatus the crystal structures of the PII-PipX and PipX-NtcA complexes and of NtcA in active and inactive conformations (respective resolutions, 3.2, 2.25, 2.3, and 3.05 A). The structures and the conclusions derived from them are consistent with the results of present and prior site-directed mutagenesis and functional studies. A tudor-like domain (TLD) makes up most of the PipX structure and mediates virtually all the contacts of PipX with PII and NtcA. In the PII-PipX complex, one PII trimer sequesters the TLDs of three PipX molecules between its body and its extended T loops, preventing PipX activation of NtcA. Changes in T loop conformation triggered by 2OG explain PII-PipX dissociation when 2OG is bound. The structure of active dimeric NtcA closely resembles that of the active cAMP receptor protein (CRP). This strongly suggests that with these proteins DNA binding, transcription activation, and allosteric regulation occur by common mechanisms, although the effectors are different. The PipX-NtcA complex consists of one active NtcA dimer and two PipX monomers. PipX coactivates NtcA by stabilizing its active conformation and by possibly helping recruit RNA polymerase but not by providing extra DNA contacts.

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