Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

BACKGROUND: Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding

The wonders of flap endonucleases: structure, function, mechanism and regulation.

Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5' nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication

Recherches sur la microflore et les activites denitrifiantes dans les nappes

SIGLECNRS RP 185 (1737) / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Protein kinase D interacts with neuronal nitric oxide synthase and phosphorylates the activatory residue serine1412

Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser 916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser 1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.This work was supported by the Ministerio de Economía y Competitividad [SAF2011-26233 to T.I., BFU2009-10442 and BFU2012-37934 to I.R-C.]; Comunidad de Madrid [S2010/BMD-2331-Neurodegmodels-CM to T.I.]; and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas – CIBERNED, Instituto de Salud Carlos III, to T.I. Postdoctoral fellows L.S-R. and L.G-G. have been funded by research contracts from CIBERNED; Clara Aicart-Ramos is a recipient of a FPU predoctoral fellowship from Ministerio de Economía y Competitividad.Peer Reviewe

Respostas metabólicas à suplementação com frutose em exercício de força de membros inferiores Metabolic responses to fructose supplementation in strength exercise of lower limbs

A frutose, por seu metabolismo independente da insulina, realiza significativas alterações no metabolismo hepático, promovendo um entorno metabólico favorável ao metabolismo tanto da glicose como dos lipídios, durante o exercício. Essa condição tem sido bastante estudada em exercício de endurance; no entanto, nenhum estudo sobre a suplementação com frutose no exercício de força (EF) foi encontrado. O objetivo do presente estudo foi avaliar os efeitos agudos da adição de frutose a um suplemento de glicose sobre o metabolismo de lipídios em EF. Vinte homens treinados ingeriram suplemento de glicose (G) ou glicose mais frutose (G+F), 15 minutos antes de realizar exercício de força (10 séries de 10 repetições). Os sujeitos foram testados em ordem randômica em um desenho cruzado e com uma semana de intervalo em duas condições experimentais: EF+(G) e EF+(G+F). A análise dos resultados mostrou que os valores de triglicérides durante o exercício foram maiores (p < 0,05) quando os sujeitos foram suplementados com G+F do que quando suplementados apenas com G. Ao final do exercício, os valores de ácidos graxos livres foram maiores quando os sujeitos foram suplementados G+F (p < 0,05). A glicemia foi menor durante o exercício e maior na recuperação (p < 0,05) para essa condição. O comportamento da insulina não diferiu entre os experimentos durante o exercício de força (p > 0,05), mas foi maior em G+F que em G (p < 0,05) durante a recuperação. A percepção subjetiva de esforço (PSE) foi menor (p < 0,05) para a suplementação com G+F do que com G. Em conclusão, a suplementação com G+F afeta positivamente o metabolismo de lipídios durante o exercício de força e favorece seu metabolismo imediatamente após o esforço, proporcionando condição metabólica que reflete em uma condição que afeta favoravelmente a PSE.<br>Due to its insulin-independent metabolism, fructose promotes significant changes in liver metabolism, promoting a metabolic surrounding favorable to the glucose as well as lipids metabolism during the exercise. This condition has been widely studied in endurance exercises; however, none study about fructose supplementation in strength exercise (SE) was found. This study aimed to assess the acute effects of the fructose addition to a glucose supplement on lipid metabolism in strength exercise. Twenty trained male subjects ingested a glucose (G) or glucose plus fuctose (G+F) supplement, 15 minutes before practicing a strength exercise (10 sets of 10 repetitions). The subjects were tested randomly in a crossover design and with a week of pause in two experimental conditions: SE+(G) and SE+(G+F). The analysis of the results showed that values of triglycerides during the exercise were higher (p < 0.05) when the subjects were supplemented with G+F than when they were supplemented only with G. By the end of the exercise the values of free fatty acid were higher when in G+F (p < 0.05). Glycemia was lower during the exercise and higher in the recovery (p < 0.05) in this condition. Insulin values did not differ among the experiments during strength exercises (p > 0.05), but they were higher in G+F than in G (p < 0.05) during recovery. Perceived exertion (PE) was lower (p < 0.05) in G+F than in G. It can be concluded that the G+F supplementation positively affects the lipid metabolism during the strength exercise and favors its metabolism immediately after the effort, promoting a metabolic condition that reflects on a condition that favorably affects the PE