Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

<p>Abstract</p> <p>Background</p> <p>Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1^stand 2^ndEF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.</p> <p>Results</p> <p>The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca²⁺binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10⁵and 5.0 × 10²M^-1with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1^stnon-canonical EF-hand is largely enhanced by the binding of Ca²⁺to the 2^ndcanonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca²⁺binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg²⁺-binding in NCaBD.</p> <p>Conclusions</p> <p>We demonstrated that the 1^stnon-canonical EF-hand of NOX5 has very weak Ca²⁺binding affinity compared with the 2^ndcanonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca²⁺binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca²⁺specific.</p> <p>Graphical abstract</p> <p><display-formula><graphic file="1752-153X-6-29-i1.gif"/></display-formula></p

Expression of NADPH Oxidase (NOX) 5 in Rabbit Corneal Stromal Cells

To determine whether NOX 5 is expressed in rabbit corneal stromal cells (RCSC). NADPH oxidases (NOXes) are enzymes that preferentially use NADPH as a substrate and generate superoxide. Several isoforms of NOXes function as multi-protein complexes while NOX5 and DUOXs do not require the accessory proteins for their activity and possess calcium binding EF hands.Human NOX5 primers were used to amplify the rabbit NOX5 by RT-PCR. Amplified product was sequenced to confirm its identity. The protein encoded by the NOX5 was identified by western blot analysis. NOX5 siRNA was used to reduce transcript, protein, and calcium stimulated activity. In silico analyses were performed to establish the putative structure, functions, and evolution of rabbit NOX5.NOX activity was measured in RCSC with NADPH rather than NADH as a substrate. RT-PCR with NOX5 primers amplified 288 bp product using RCSC cDNA, which, when sequenced, confirmed its identity to human NOX5 mRNA. This sequence was used to predict the rabbit (Oryctolagus cuniculus) NOX5 gene. NOX5 siRNA reduced amounts of NOX5 mRNA in RCSC and reduced ionomycin stimulated superoxide production. A protein of about 65 to 70 kDa encoded by the NOX5 was detected by western blot analysis. In silico analysis predicted a putative rabbit NOX5 protein containing 801 amino acids. Motif searches predicted the presence of at least 3 putative EF-hands in N-terminus and a NOX domain in C terminal region.The data document that the NOX5 gene was expressed in cells of lagomorphs unlike rodents, making the rabbit an interesting model to study NOX5 functions. The activity of the rabbit NOX5 was calcium stimulated, a trait of NOX5 in general. NOX5 may also prove to be a useful genetic marker for studying the taxonomic position of lagomorphs and the Glires classification

NOX5 cell-free assay for the high-throughput screening of small molecules

NADPH oxidases (NOX) are a family of transmembrane enzymes, which catalyze the formation of O2˙- and H2O2. Membrane fractions of leukocytes are highly enriched in the phagocyte NOX isoform (NOX2). This feat has allowed the development of a complex NOX2 cell-free assay, which has been a key tool for the understanding of the mode of action of NOX2, its biochemistry, pharmacology, and identification of NOX2-specific inhibitors. In addition to NOX2, there are six other NOX isoforms in humans, but cell-free assays of non-phagocytic oxidases are infrequently used, and their specificity has recently been debated. Here we describe a NOX5 cell-free assay. We present a method to purify the membranous component of cells stably transduced with NOX5 and to measure O2˙- in a high-throughput format (96-w or 384-w plates). The experimental description allows high-throughput screening of small molecules with limited cost