Reactive oxygen species (ROS) are a class of oxygen compounds that are highly reactive due to their partially reduced oxygen atom, with examples including superoxide (O2-), hydrogen peroxide (H2O2), and peroxide (O2-2). ROS play a critical role in the body with their involvement in cell signaling and host cell defense when proper levels are maintained. The production of the ROS superoxide in particular is largely facilitated by the enzyme family of NADPH oxidases (Noxs).
Production of the superoxide via the Nox enzyme family has been shown to play a role in the regulation of homeostasis conditions in the body. However, overproduction or underproduction can lead to disease development or trouble combating pathogens, respectively. Two homologs of the Nox family, NADPH Oxidase 5 (Nox5) and Dual oxidase (Duox), contain an additional EF-Hand Domain (EFD) that requires Ca2+ coordination to its EF-Hand motifs to stimulate superoxide production. Binding of Ca2+ to the EFD stimulates a conformational change that involves the exposure of hydrophobic amino acid residues. Previous studies have suggested that the hydrophobic residue exposure prompts the interaction of the EFD with the dehydrogenase domain (DH) within the same enzyme. However, the exact structure of the EFD as well as the conformational change remain unknown.
To elucidate the structure of Nox5-EFD and the conformational change that occurs upon Ca2+ coordination, recombinant protein constructs for the Nox5 EFD were expressed and purified. The Ca2+ binding assessed by isothermal titration calorimetry (ITC) revealed that the binding constant of Ca2+ is much higher in the third EF of Nox5 (108 M-1) than in the other EF motifs. Assessment of cooperativity showed that the C-terminal of Nox5-EFD (C-EFD) exhibits cooperativity to the order of 14000, where N-EFD has a lower cooperativity of 1500, likely attributed to the high affinity of EF3 and the low affinity of EF1. Assessment of hydrophobicity by the ANS interaction with Nox5-EFD suggested that the most significant hydrophobic exposure occurs in a portion of the C-terminal linker region and a portion of the C-terminal lobe, complementary to the findings from the determination of heat capacity (ΔCp) for Nox5-EFD. The limited trypsin digestion revealed burial of the polar Lys and Arg residues in the C-terminus, leading to digestion resistance in the 10 kDa digestion fragment. The potential use of CaM as a model protein for Nox5-EFD is discussed.
|Commitee:||Dixon, Robert, Shaw, Michael|
|School:||Southern Illinois University at Edwardsville|
|School Location:||United States -- Illinois|
|Source:||MAI 58/06M(E), Masters Abstracts International|
|Keywords:||Calcium binding, Ef-hand, Fluorescence, Isothermal titration calorimetry, Nadph oxidase 5, Nox5|
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