RESUMO
Impaired redox metabolism is a key contributor to the etiology of many diseases, including primary mitochondrial disorders, cancer, neurodegeneration and aging. However, mechanistic studies of redox imbalance remain challenging due to limited strategies that can perturb redox metabolism in various cellular or organismal backgrounds. Most studies involving impaired redox metabolism have focused on oxidative stress; consequently, less is known about the settings where there is an overabundance of NADH reducing equivalents, termed reductive stress. Here we introduce a soluble transhydrogenase from Escherichia coli (EcSTH) as a novel genetically encoded tool to promote reductive stress in living cells. When expressed in mammalian cells, EcSTH, and a mitochondrially targeted version (mitoEcSTH), robustly elevated the NADH/NAD+ ratio in a compartment-specific manner. Using this tool, we determined that metabolic and transcriptomic signatures of the NADH reductive stress are cellular background specific. Collectively, our novel genetically encoded tool represents an orthogonal strategy to promote reductive stress.
RESUMO
Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H2O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H2O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX-like enzyme and instead harbors three paralogous genes (FDPF1-3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a "stand-alone" FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.
