ABSTRACT Red Blood Cells (RBCs) represent ~83% of the total human cells in the body. RBC hemoglobin (Hb), which is critical for their function to carry and deliver oxygen to peripheral tissues, constitutes ~90% of the total protein content of a mature RBCs. During their lifespan of 120 days in the bloodstream, RBCs are constantly exposed to oxidant stress, which mostly arises from Fenton and Haber-Weiss reactions triggered by Hb-Iron in the presence of oxygen. However, RBCs lack nuclei and organelle and, as such, they cannot synthesize new proteins to replace oxidatively damaged components. Therefore, in order to cope with oxidant stress, RBCs have evolved unique mechanisms that rely on signaling axes and their capacity to trigger metabolic reprogramming to favor antioxidant defenses (the Pentose Phosphate Pathway – PPP) over energy metabolism (glycolysis). One such mechanism relies on the two most abundant proteins in RBC cytosols and membranes: Hb and anion exchanger 1 (AE1), respectively. Owing to its capacity to “sense” oxygen, in response to hypoxia, deoxygenated Hb migrates to the membrane, where it binds the N-terminus of AE1. This mechanism releases a series of glycolytic enzymes, which are inhibited under high-oxygen tensions owing to their binding to the same region of AE1 with high affinity for deoxygenated Hb. This phenomenon favors energy metabolism under low oxygen tensions (e.g., high-altitude hypoxia), while it creates a metabolic bottleneck in energy metabolism to promote a critical antioxidant pathway when oxidant stress is high: the Pentose Phosphate Pathway (PPP). This mechanism is referred to as the AE1-Hb switch in this proposal. Of note, glucose 6-phosphate dehydrogenase (G6PD) is not only the rate-limiting enzyme of the PPP, but also the target of the most common enzymopathy in humans, G6PD deficiency, which affects ~400 million people. While RBCs from G6PD-deficient subjects are perfectly healthy in the absence of oxidant stress, RBCs from these individuals are characterized by a shorter lifespan and susceptibility to lysis following oxidative insults. Oxidative stress to RBCs is not only relevant within the context of RBC senescence. Significant oxidant stress arises during RBC storage under blood bank conditions for blood transfusion purposes, a common in hospital medical procedure and a life-saving intervention for ~3-4 million Americans every year. However, little is known about the impact of the AE1-Hb switch and G6PD deficiency in the context of RBC aging in vitro (blood bank). In parallel, when studying human acclimatization to high-altitude hypoxia, we discovered a novel axis, the ADORA2b/Sphk1 axis, that interplays with the AE1-Hb switch to favor oxygen off-loading in healthy individuals as they climb to high-altitude, where oxygen is limited in comparison to sea level. By leveraging a combination of state-of-the art omics technologies (fluxomics and Xlinking proteomics) and a mix of well-established and novel an...