Deciphering the reductive pathways in phagosomes and lysosomes.

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Phagosomes are organelles that are formed after the engulfment of material by phagocytes such as macrophages and dendritic cells. The lumen of phagosome is a dynamic and complex microenvironment. It is the site of microbial killing, precise proteolytic processing of antigens and the degradation and recycling of macromolecules during tissue remodeling, growth and homeostasis. Despite the importance of the phagosome, there are critical gaps in our understanding of its function and dysfunction. One particular gap is the impact of redox chemistries on phagosomal function.*** Over the past seven years, my group has investigated the redox control of critical chemistries within phagosomes and lysosomes. We discovered that NOX2 oxidizes the normally reductive lumen of the phagosome and inhibits both oxidation-sensitive proteolysis and disulfide reductiontwo key chemistries for antigen processing and protein turn-over. However, the other side to this story was yet to be told: what re-reduces these oxidized proteases and how is the reductive capacity of phagosomes and lysosomes maintained? We recently addressed this by conducting a screen that identified a cytosolic pathway that utilizes NADPH and a selenoprotein reductase to maintain the reductive environment within the phagosome (30). This study was highlighted as “Leading Edge Research” in the July 2016 issue of the Journal of Leukocyte Biology with a dedicated editorial. The authors of the editorial stated: that “[while the study] represents an excellent starting platform to discern how the reductive capacity of the phagosome is maintained”“[it] is not the final conclusion to the story” (29). We agree. *** This proposal aims to continue the interrogation of the fundamental biochemical processes that promote essential reductive chemistries in phagosomes. This is critically needed in the field of phagocyte biology, but will also likely uncover key pathways that are necessary to maintain the redox balance in endosomes and lysosomescommon to all eukaryotic cells. Additionally, my research program will be broadened beyond the phagosome, as we propose to adapt our technologies to explore the control of reductive chemistries in a new cellular locationthe osteoclast lacunae. The three aims propose a breadth of unique and cutting edge technologies to test specific hypotheses and facilitate discovery-based knowledge creation. ****** Aim 1: Specific identification of components of the newly identified cytosolic pathway that provides reductive equivalents to the phagosomal lumen. *** Aim 2: Identification of upstream and downstream interacting partners of GILT.*** Aim 3: Exploration of the role of GILT and reductive pathways in the lacunae of the osteoclast.****** Funding of this proposal will allow my group to build upon our strong track record of pioneering new research areas in phagocyte biology and creating an engaging, rigorous and unique training environment.