Disturbances of the redox environment in various cellular compartments are linked to many pathologies, including neurodegenerative diseases, cancer, cardiovascular disease and aging. Since the ratio of oxidized to reduced nicotinamide adenine dinucleotides is a major contributor to the cellular redox environment, engineering tools to perturb this ratio would enable the study the role of redox imbalances in driving these pathologies. In this work we examine the fundamental mechanisms used by some microorganisms to control their optimal redox environment, as well as the possible applications of these mechanisms in mammalian cells. A number of microaerophilic bacteria and protozoa lack a conventional multi-complex respiratory chain, and instead rely on enzymes which catalyze a H2O-forming NADH oxidase reaction to recycle NAD+ and to eliminate toxic oxygen from the environment. Since the product of this reaction is benign water and not hydrogen peroxide (H2O2), these enzymes represent attractive reagents to perturb metabolism in mammalian cells. In this work we propose to engineer a bacterial H2O-forming NADH oxidase towards NADPH specificity. This NADPH oxidase will then be expressed in different compartments of mammalian cells, and its effects on cell viability and metabolism will be systematically evaluated. Since no tool has previously been reported to safely increase the NADP+/NADPH ratio in cells in a compartment specific manner, our work will provide fundamental insights into NADPH metabolism and its regulation, as well as into the sizes of NAD(P)H pools in different cellular compartments. Our work also explores the mechanisms of how microaerophilic human protozoan parasites like Giardia intestinalis, Trichomonas vaginalis and Entamoeba histolytica, which lack conventional respiratory chains, control their redox environments in order to support energy metabolism. We have identified in T.vaginalis a natural protein which represents a fusion between a flavodiiron core protein with its redox partners: rubredoxin and rubredoxin oxidoreductase. This fusion protein catalyzes a four-electron reduction of oxygen to water using reducing equivalents of NAD(P)H. Our studies of the structure and mechanism of this fusion protein will provide insights into how these human parasites are able to maintain their optimal redox environment. These mechanisms can be attractive targets for therapeutic intervention, allowing us to combat diseases caused by human protozoan parasites.
Regulation of the redox environment is essential to life. We propose to develop novel tools to study important contributors to the redox environment and its regulation in mammalian cells. We additionally propose to study mechanisms of how human protozoan parasites, including those causing the infectious diseases giardiasis and trichomoniasis, maintain their optimal redox environment, since understanding these mechanisms can provide attractive therapeutic strategies.
| Cracan, Valentin; Titov, Denis V; Shen, Hongying et al. (2017) A genetically encoded tool for manipulation of NADP+/NADPH in living cells. Nat Chem Biol 13:1088-1095 |
