Deciphering the reductive pathways in phagosomes and lysosomes.

破译吞噬体和溶酶体中的还原途径。

• 批准号: RGPIN-2017-04147
• 负责人: Yates, Robin
• 金额: $ 2.91万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687620
• 关键词: Deciphering; reductive; pathways; phagosomes; lysosomes.

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.

吞噬体是吞噬细胞如巨噬细胞和树突状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。在组织重塑、生长和体内平衡过程中，它是微生物杀灭、抗原的精确蛋白水解加工和大分子降解和再循环的场所。尽管吞噬体很重要，但我们对其功能和功能障碍的理解仍存在重大差距。一个特别的空白是氧化还原化学对吞噬体功能的影响。***在过去的七年中，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NOX2氧化吞噬体的正常还原性管腔，并抑制氧化敏感的蛋白质水解和二硫还原，这是抗原加工和蛋白质周转的两种关键化学反应。然而，这个故事的另一面还没有被告知：是什么重新还原了这些氧化蛋白酶，吞噬体和溶酶体的还原能力是如何维持的？我们最近通过筛选确定了利用NADPH和硒蛋白还原酶维持吞噬体内还原环境的细胞质途径来解决这个问题（30）。该研究在2016年7月出版的《白细胞生物学杂志》（Journal of Leukocyte Biology）上以专门社论的形式被评为“前沿研究”。这篇社论的作者说：“[虽然这项研究]代表了一个很好的开始平台，可以看出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。***本研究旨在继续探索吞噬体中促进基本还原化学的基本生化过程。这在吞噬细胞生物学领域是非常需要的，但也可能揭示维持所有真核细胞共有的核内体和溶酶体氧化还原平衡所必需的关键途径。此外，我的研究计划将扩展到吞噬体之外，因为我们建议调整我们的技术来探索在一个新的细胞位置-破骨细胞腔隙中的还原性化学控制。这三个目标提出了一系列独特的前沿技术，以测试特定的假设，并促进基于发现的知识创造。******目标1：特异性鉴定新鉴定的提供吞噬体管腔还原等效物的细胞质途径的组分。***目的2：确定GILT上下游相互作用伙伴。***目的3：探讨GILT和还原通路在破骨细胞腔隙中的作用。******这项提案的资金将使我的团队能够建立在我们在吞噬细胞生物学领域开拓新研究领域的良好记录上，并创造一个引人入胜、严谨而独特的培训环境。