Deciphering the dichotomy of oxygen metabolism in renal cancer using a novel genetically encoded oxygen biosensor

Abstract Molecular oxygen (O2) plays a crucial role in shaping Earth's biosphere. It is generally accepted that the rise of oxygen in the atmosphere led living systems to evolve a sophisticated respiratory chain, which allowed organisms to harvest energy by oxidative phosphorylation. Oxygen is an indispensable aspect of the eukaryotic life and is a reactant in many enzymatic reactions beyond oxidative phosphorylation. However, it has frequently been an overlooked variable in biochemical and cellular studies. Recently, there is increasing evidence that oxygen tension is a crucial parameter of tissue, cellular and even subcellular metabolism. Oxygen (as O2) concentrations directly influence physiology, redox signaling, the manifestation of a wide range of pathologies, as well as developmental and differentiation programs in eukaryotic cells and tissues. A significant obstacle to a comprehensive understanding of how different O2 tensions control and modulate these phenotypes is the absence of versatile and reliable technology to monitor oxygen gradients in living cells. This proposal aims to address this technological gap by developing a protein-based fluorescence biosensor to monitor O2 levels in mammalian cells. Based on currently available biochemical and structural information, we will engineer conjugates between naturally occurring heme-based protein scaffolds that bind oxygen reversibly (sensing unit) and a fluorescent protein UnaG (reporter unit). The sensor constructs will be designed so that the UnaG-based reporting unit relays and visualizes conformational and absorption changes occurring upon O2 binding within the heme-based sensing unit. Our approach takes advantage of the fact that maturation of the chromophore of UnaG is not oxygen dependent and therefore UnaG can be used as a reporter unit at various oxygen tensions. Biosensor candidates will be rigorously characterized using biochemical, spectroscopic, and imaging approaches, both as purified recombinant proteins as well as in mammalian cell culture systems. Finally, we will apply our genetically encoded biosensor to elucidate intracellular O2 concentration dependent metabolic remodeling in renal cancer cell lines with either intact or deficient von Hippel–Lindau tumor suppressor (VHL) gene. Overall, the technology we are developing will provide transformative opportunities for understanding the role of O2 metabolism in cancer and beyond by directly visualizing oxygen gradients at sub-organellar resolution within living cells.