The endosomal proteome of macrophage and dendritic cells
The endosomal proteome of macrophage and dendritic cells
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DOI：
10.1002/pmic.201000577
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发表时间：
2011-03-01
期刊：
影响因子：
3.4
通讯作者：
Desjardins, Michel
中科院分区：
文献类型：
--
作者：
Duclos, Sophie;Clavarino, Giovanna;Desjardins, Michel
液泡内系统在健康和疾病中的重要作用需要开发新的工具，以便更好地理解内吞过程中涉及的复杂分子机制。我们利用小乳胶珠 (sLB) 在不连续蔗糖梯度上的漂浮特性，在 J774 巨噬细胞和骨髓源性树突状细胞 (DC) 中内化小乳胶珠后，分离出高度纯化的核内体。我们特别关注巨噬细胞早期内涵体和晚期内涵体/溶酶体 (LE/LYS) 的分离，以及从未成熟和脂多糖激活（成熟）DC 中分离 LE/LYS。我们随后通过 MS 对它们各自的蛋白质含量进行了比较分析。正如预期的那样，已知定位于早期内涵体的蛋白质在 J774 内涵体的最早部分中富集，而已知在此过程后期积累的蛋白质（例如水解酶）在 LE/LYS 制剂中显着富集。接下来我们比较了未成熟 DC 和成熟 DC 的 LE/LYS 蛋白含量，已知它们会经历大规模重组，从而导致有效的免疫激活。通过关注先前描述不足的生化途径，强调了巨噬细胞和 DC 内吞细胞器的蛋白质含量之间的差异，这些途径可能在这些高度相关的免疫细胞类型的内体功能中发挥意想不到但重要的作用。
The essential roles of the endovacuolar system in health and disease call for the development of new tools allowing a better understanding of the complex molecular machinery involved in endocytic processes. We took advantage of the floating properties of small latex beads (sLB) on a discontinuous sucrose gradient to isolate highly purified endosomes following internalization of small latex beads in J774 macrophages and bone marrow-derived dendritic cells (DC). We particularly focused on the isolation of macrophages early endosomes and late endosomes/lysosomes (LE/LYS) as well as the isolation of LE/LYS from immature and lipopolysaccharide-activated (mature) DC. We subsequently performed a comparative analysis of their respective protein contents by MS. As expected, proteins already known to localize to the early endosomes were enriched in the earliest fraction of J774 endosomes, while proteins known to accumulate later in the process, such as hydrolases, were significantly enriched in the LE/LYS preparations. We next compared the LE/LYS protein contents of immature DC and mature DC, which are known to undergo massive reorganization leading to potent immune activation. The differences between the protein contents of endocytic organelles from macrophages and DC were underlined by focusing on previously poorly characterized biochemical pathways, which could have an unexpected but important role in the endosomal functions of these highly relevant immune cell types.