Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain; Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain.
J Proteomics. 2013 Oct 8;91:106-35. doi: 10.1016/j.jprot.2013.06.026. Epub 2013 Jul 5.
Macrophages play a pivotal role in the prevention of Candida albicans infections. Yeast recognition and phagocytosis by macrophages is mediated by Pattern Recognition Receptors (PRRs) that initiate downstream signal transduction cascades by protein phosphorylation and dephosphorylation. We exposed RAW 264.7 macrophages to C. albicans for 3h and used SILAC to quantify macrophage proteins and phosphoproteins by mass spectrometry to study the effects of infection. We identified 53 macrophage up-regulated proteins and 15 less abundant in the presence of C. albicans out of a total of 2071 identified proteins. 922 unique protein phosphorylation sites were identified by phosphopeptide enrichment and mass spectrometry, including 327 previously unidentified mouse protein phosphorylation sites. 126 peptides showed an increase and 70 a decrease in their phosphorylation level. The majority of the differentially expressed and phosphorylated proteins are receptors, mitochondrial ribosomal proteins, cytoskeletal proteins, and transcription factor activators involved in inflammatory and oxidative responses. In addition, we identified 22 proteins and phosphoproteins related to apoptosis. The analysis of apoptotic markers revealed that anti-apoptotic signals prevailed during the interaction of the yeast. Our proteomics study suggests that besides inflammation, apoptosis is a central pathway in the immune defense against C. albicans infection.
This work uses SILAC and SIMAC methodology combined with CPP (+ TiO2) to study protein and phosphopeptide changes in RAW 264.7 macrophages in response to coincubation with Candida albicans for 3h. We show that the presence of C. albicans induces inflammatory responses and inhibits apoptosis in the macrophages. Our phosphoproteomic analysis identified 327 new mouse protein phosphorylation sites.
巨噬细胞在预防白色念珠菌感染中起着关键作用。巨噬细胞对酵母的识别和吞噬作用是由模式识别受体（PRRs）介导的，这些受体通过蛋白质磷酸化和去磷酸化启动下游信号转导级联反应。我们将 RAW 264.7 巨噬细胞暴露于白色念珠菌中 3 小时，并用 SILAC 通过质谱法定量巨噬细胞蛋白和磷酸化蛋白，以研究感染的影响。我们总共鉴定出 2071 种蛋白质，发现了 53 种巨噬细胞上调的蛋白质和 15 种在存在白色念珠菌时含量较低的蛋白质。通过磷酸肽富集和质谱法鉴定了 922 个独特的蛋白质磷酸化位点，包括 327 个以前未鉴定的小鼠蛋白质磷酸化位点。126 个肽段的磷酸化水平增加，70 个肽段的磷酸化水平降低。差异表达和磷酸化的蛋白质大多数是受体、线粒体核糖体蛋白、细胞骨架蛋白和转录因子激活剂，涉及炎症和氧化反应。此外，我们还鉴定了 22 种与细胞凋亡相关的蛋白质和磷酸化蛋白。凋亡标志物的分析表明，在酵母相互作用过程中，抗凋亡信号占主导地位。我们的蛋白质组学研究表明，除了炎症外，细胞凋亡是免疫防御白色念珠菌感染的中心途径。
这项工作使用 SILAC 和 SIMAC 方法结合 CPP（+TiO2）研究了 RAW 264.7 巨噬细胞在与白色念珠菌共孵育 3 小时后对蛋白和磷酸肽变化的反应。我们表明，白色念珠菌的存在诱导了巨噬细胞中的炎症反应并抑制了细胞凋亡。我们的磷酸蛋白质组学分析鉴定了 327 个新的小鼠蛋白质磷酸化位点。
