Reactive Oxygen Species (ROS) are required for cell signaling and host immune defense. ROS like superoxide (O2-) are generated by a family of enzymes called NADPH oxidases (Noxs). Nox enzymes have a variety of roles in cell and bodily homeostasis such as immune response, cellular signaling and cellular proliferation. While Nox enzymes share a core electron transfer mechanism to generate ROS, how each is activated varies from enzyme to enzyme. Nox5 is activated through Ca2+ flux which allows its EF-hand Domain (EFD) to bind to the Dehydrogenase Domain (DH). Previously two short sequences within the DH have been shown to interact with EFD in a Ca2+-dependent manner, these sequences are named Regulatory EF-hand Domain (REFBD) and Phosphorylatable region (PhosR). In these studies, only minimal to no characterization of the binding mechanism was performed, leaving many questions unanswered. It is suggested that both sequences play an important role in Nox5’s superoxide generating activity. Despite these findings, the activation mechanism for Nox5 is still unclear.
Here, EFD constructs and the synthetic peptides corresponding to the REFBD and PhosR sequences were used to study the interaction between EFD/REFBD and EFD/PhosR. Fluorescence measurements showed that REFBD binds to EFD in a Ca2+-dependent manner with a binding constant of 1.01×106 M-1. We determined that EFD’s C-terminal half domain binds to REFBD and forms a more compact structure that is resistant to tryptic digestion. The binding of EFD to REFBD was shown to be driven mainly through hydrophobic interactions. PhosR was observed to bind to EFD in a Ca2+-independent manner, with its binding to be 1.58×105 M-1. No specific binding region was observed through the use of our EFD constructs. We utilized Fluorescence Resonance Energy Transfer (FRET) to observe and measure the distances between the EFD to REFBD and REFBD to PhosR. Homology modeling and FRET experiments allowed us to extract potential orientation of REFBD upon binding to EFD.
|Commitee:||Lu, Yun, Rieth, Monica|
|School:||Southern Illinois University at Edwardsville|
|School Location:||United States -- Illinois|
|Source:||MAI 81/12(E), Masters Abstracts International|
|Keywords:||Calcium binding, Domain-domain interaction, EF-hand, NADPH oxidase 5|
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