Deciphering the reductive pathways in phagosomes and lysosomes.

吞噬体是在吞噬细胞如巨噬细胞和树突细胞吞噬物质后形成的细胞器。吞噬体内腔是一个动态的、复杂的微环境。它是微生物杀灭、抗原的精确蛋白水解加工以及组织重塑、生长和体内平衡期间大分子的降解和再循环的位点。尽管吞噬体的重要性，但我们对其功能和功能障碍的理解存在重大差距。一个特殊的差距是氧化还原化学对吞噬体功能的影响。 在过去的七年里，我的团队研究了吞噬体和溶酶体中关键化学物质的氧化还原控制。我们发现，NOX 2氧化吞噬体的正常还原腔，并抑制氧化敏感性蛋白水解和二硫键还原-抗原加工和蛋白质翻转的两个关键化学反应。然而，这个故事的另一面还没有被告知：是什么重新还原这些氧化的蛋白酶，以及吞噬体和溶酶体的还原能力是如何维持的？我们最近通过进行筛选来解决这个问题，该筛选鉴定了利用NADPH和硒蛋白还原酶来维持吞噬体内还原环境的胞质途径（30）。这项研究在2016年7月出版的《白细胞生物学杂志》上被列为“前沿研究”，并有专门的社论。社论的作者说：“[虽然这项研究]代表了一个很好的起点平台，可以辨别吞噬体的还原能力是如何维持的”“[它]不是故事的最终结论”（29）。 我们同意。*** 这项建议的目的是继续审问的基本生化过程，促进必要的还原化学吞噬体。这在吞噬细胞生物学领域是迫切需要的，但也可能揭示维持所有真核细胞共同的内体和溶酶体中氧化还原平衡所必需的关键途径。此外，我的研究计划将扩大到吞噬体之外，因为我们建议调整我们的技术，以探索在一个新的细胞位置破骨细胞腔隙中控制还原化学。这三个目标提出了广泛的独特和尖端技术，以测试特定的假设，并促进基于发现的知识创造。****** 目的1：特异性鉴定新鉴定的胞质途径的组分，该途径为吞噬体腔提供还原等价物。*** 目标2：确定GILT的上游和下游相互作用伙伴。 目的3：探索GILT和还原途径在破骨细胞腔隙中的作用。** 这项提案的资助将使我的团队能够建立在我们在吞噬细胞生物学领域开创新研究领域的良好记录之上，并创造一个引人入胜，严谨和独特的培训环境。

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.