Molecular Mechanisms Underlying Mammalian NADPH Oxidase Activation and Regulation

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PROJECT SUMMARY Maintenance of the reactive oxygen species (ROS) homeostasis is essential to preserve cell integrity and vital for the survival and growth of almost all life. In multicellular organisms, ROS is actively generated outside of the cell or near the cell membrane to protect against invading pathogens as well as in normal physiological processes such as hormone biosynthesis. However, ROS are generally associated with causing damage to proteins and DNA within cells. Excessive ROS production leads to oxidative stress and contributes to the development of many chronic conditions such as aging, cancer, diabetes, cardiac disorders, and neurodegenerative diseases. The NADPH oxidases, a family of membrane enzymes whose primary function is to produce ROS, play an essential role in maintaining ROS homeostasis and thus serve as valid drug targets for combatting numerous diseases associated with oxidative stress. NADPH oxidases generate ROS by catalyzing cross-membrane electron transfer from cytosolic NADPH to extracellular oxygen. Mammals encode seven NADPH oxidases: DUOX1-2 and NOX1-5. To date, little is known about the molecular mechanism governing the activation and regulation of NADPH oxidase proteins, representing a critical knowledge gap. In this proposal, an interdisciplinary research program will be established to study the working mechanism of NADPH oxidases by combining cutting-edge structural biology techniques such as single-particle cryoEM with biochemical, biophysical, and cell biology approaches. We aim to address the two fundamental questions underlying the catalytic activity of NADPH oxidases: i) how do NADPH oxidases mediate cross-membrane electron transfer to catalyze the production of ROS? And ii) how is the catalytic function of NADPH oxidases activated and regulated at the molecular level? Using the DUOX1 as an example, we will establish a molecular paradigm for understanding the structure-function relationship of NADPH oxidases. The outcomes of our studies will advance our fundamental understanding of the NADPH oxidase biology and lay the foundation for novel drug development strategies to combat oxidative stress.