Dissertation/Thesis Abstract

Expression and Purification of the Nox5 Dehydrogenase Domain and Characterization of Its Interaction with Calmodulin
by Lloyd, Laura, M.S., Southern Illinois University at Edwardsville, 2020, 106; 27956895
Abstract (Summary)

Reactive oxygen species (ROS) are highly chemically reactive and form from molecular oxygen. They include molecules such as hydrogen peroxide and superoxide anion. ROS play an important role in the human body, where it must be very tightly regulated. If they are under-regulated, formation of granulomas can occur. If ROS are overproduced, on the other hand, it causes oxidative stress and has been thought to contribute to the development of different diseases such as heart disease, prostate cancer, and Alzheimer’s disease. ROS are produced both exogenously and endogenously, which involves electron transfers within different enzymes to produce these species, such as the NADPH oxidase (Nox) family.

The Nox superfamily regulates superoxide production through an internal electron transfer eventually transferring to molecular oxygen outside the cell. The molecular mechanism for many Nox enzymes in regulating superoxide production is already well understood; the same cannot be said about Ca2+-dependent Nox5. Nox5 is an essential enzyme expressed in human cells that produces superoxide. Superoxide activity is regulated by Ca2+ through its EF-Hand domain (EFD). It is believed that the mechanism of superoxide production involves Ca2+ binding to the EFD, leading to a conformational change allowing the interaction with its own dehydrogenase domain (DH), which in turn produces superoxide. Recently, it has been shown that Nox5’s DH domain contains a putative Calmoduin (CaM) binding site. Therefore, it is suggested that CaM increases the Ca2+ sensitivity of Nox5, allowing it to easily bind to Ca2+ in the EFD, thus inducing the conformational change within the Nox5 protein, allowing for the production of superoxide.

To reveal the contribution of CaM binding to the DH domain of Nox5, we expressed the recombinant partial DH protein constructs for the characterization. The isothermal titration calorimetry (ITC) measurement indicated a binding stoichiometry of 2 CMBD per 1 CaM. The binding affinity between CaM and CMBD was determined by fluorescence spectroscopy, including steady-state and anisotropy. These characterizations yields the binding is a biphasic process with the strong binding in the range of 107 M-1 and the weak binding near 105 M-1. Our results concluded that CaM may induce the dimerization of Nox5, which plays a role the electron transfer.

Indexing (document details)
Advisor: Wei, Chin-Chuan
Commitee: Tucker, Kevin, Voss, Eric
School: Southern Illinois University at Edwardsville
Department: Chemistry
School Location: United States -- Illinois
Source: MAI 81/12(E), Masters Abstracts International
Source Type: DISSERTATION
Subjects: Biochemistry
Keywords: Calmodulin, Nox5, Protein-protein interaction
Publication Number: 27956895
ISBN: 9798645477226
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