Novel mechanisms of purine regulation and their roles in human pathophysiology - PROJECT SUMMARY Mitochondria regulate essential biological activities through their effects on cellular metabolism, particularly via oxidative phosphorylation (OXPHOS). Mitochondrial dysfunction leads to numerous human pathologies, including neurological disorders, autoimmune diseases, and cancer. However, while permanent impairment of the electron transport chain (ETC) in human tissues is detrimental, reversible suppression of OXPHOS is integral to some physiological processes, such as the activation of inflammatory responses in macrophages. Metabolic dysregulation in macrophages can either weaken their anti-microbial strength or drive inflammation to pathological levels. Therefore, defining the context-specific metabolic consequences of OXPHOS suppression is crucial for identifying the mechanistic basis of mitochondrial diseases and pathological inflammation. Previous studies, including mine, have shown that under conditions of suppressed OXPHOS, both ETC-deficient cancer cells and proinflammatory bone marrow-derived macrophages (BMDMs) exhibit suppressed de novo purine biosynthesis and increased purine salvage. Although enhanced purine salvage maintains nucleotide pools in response to ETC deficiency-induced DNPB suppression in vitro, it is unclear if this mechanism operates under in vivo conditions with more relevance to human health, including tumors and splenic macrophages. My recent work demonstrated that ETC deficiency potentiates purine salvage through increasing the levels of HPRT1 substrate, phosphoribosyl pyrophosphate (PRPP) via the pentose phosphate pathway (PPP) in cultured cancer cells. However, the molecular mechanisms promoting purine salvage and their impact on inflammatory signaling in macrophages are unknown. The overarching objective of this proposal is to elucidate molecular mechanisms underlying purine salvage regulation in response to suppressed OXPHOS under both pathological (e.g., ETC- deficient tumors) and physiological (e.g., inflammatory macrophages) conditions. Building upon my previous study, I will test the central hypothesis that ETC deficiency enhances purine salvage in tumors and inflammatory macrophages to support cell growth and function. In Aim1, I will test whether ETC-deficient tumors enhance purine salvage and utilize environmental nucleobases to maintain cellular purine nucleotide pools in vivo. In Aim2, I will characterize the role of purine salvage in inflammatory macrophages. Specifically, I will test if purine salvage is enhanced via the increased PPP to promote innate immune responses in BMDMs upon inflammatory stimulation. I will also characterize the role of purine salvage in splenic macrophages in vivo during systemic inflammation. Taken together, the proposed study will advance our understanding of purine nucleotide regulation in vivo under conditions of suppressed OXPHOS and facilitate the development of novel therapeutic strategies to treat patients with mitochondrial defects, purine abnormalities, or pathological inflammation.
