Medical School, University of Sheffield, Sheffield, UK.
Mol Cell Proteomics. 2011 Jun;10(6):M111.008193. doi: 10.1074/mcp.M111.008193. Epub 2011 Apr 7.
Macrophages are central effectors of innate immune responses to bacteria. We have investigated how activation of the abundant macrophage lysosomal protease, cathepsin D, regulates the macrophage proteome during killing of Streptococcus pneumoniae. Using the cathepsin D inhibitor pepstatin A, we demonstrate that cathepsin D differentially regulates multiple targets out of 679 proteins identified and quantified by eight-plex isobaric tag for relative and absolute quantitation. Our statistical analysis identified 18 differentially expressed proteins that passed all paired t-tests (α = 0.05). This dataset was enriched for proteins regulating the mitochondrial pathway of apoptosis or inhibiting competing death programs. Five proteins were selected for further analysis. Western blotting, followed by pharmacological inhibition or genetic manipulation of cathepsin D, verified cathepsin D-dependent regulation of these proteins, after exposure to S. pneumoniae. Superoxide dismutase-2 up-regulation was temporally related to increased reactive oxygen species generation. Gelsolin, a known regulator of mitochondrial outer membrane permeabilization, was down-regulated in association with cytochrome c release from mitochondria. Eukaryotic elongation factor (eEF2), a regulator of protein translation, was also down-regulated by cathepsin D. Using absence of the negative regulator of eEF2, eEF2 kinase, we confirm that eEF2 function is required to maintain expression of the anti-apoptotic protein Mcl-1, delaying macrophage apoptosis and confirm using a murine model that maintaining eEF2 function is associated with impaired macrophage apoptosis-associated killing of Streptococcus pneumoniae. These findings demonstrate that cathepsin D regulates multiple proteins controlling the mitochondrial pathway of macrophage apoptosis or competing death processes, facilitating intracellular bacterial killing.
巨噬细胞是固有免疫反应中细菌的主要效应细胞。我们研究了丰富的巨噬细胞溶酶体蛋白酶组织蛋白酶 D 的激活如何在肺炎链球菌杀伤过程中调节巨噬细胞蛋白质组。使用组织蛋白酶 D 抑制剂 Pepstatin A，我们证明组织蛋白酶 D 差异化调节了通过八重同位素标记相对和绝对定量法鉴定和定量的 679 种蛋白质中的多种靶标。我们的统计分析确定了 18 种差异表达的蛋白质，这些蛋白质通过所有配对 t 检验（α=0.05）。该数据集富含调节线粒体凋亡途径或抑制竞争死亡程序的蛋白质。选择了 5 种蛋白质进行进一步分析。Western blot 分析，随后用组织蛋白酶 D 的药理学抑制剂或基因操作，验证了肺炎链球菌暴露后这些蛋白质的组织蛋白酶 D 依赖性调节。超氧化物歧化酶 2 的上调与活性氧生成的增加呈时间相关。已知调节线粒体外膜通透性的凝胶蛋白，与细胞色素 c 从线粒体释放相关的下调。真核延伸因子（eEF2），一种蛋白质翻译的调节剂，也被组织蛋白酶 D 下调。使用 eEF2 的负调节剂 eEF2 激酶缺失，我们证实 eEF2 功能对于维持抗凋亡蛋白 Mcl-1 的表达是必需的，从而延迟巨噬细胞凋亡，并通过小鼠模型证实，维持 eEF2 功能与受损的巨噬细胞凋亡相关的肺炎链球菌杀伤有关。这些发现表明，组织蛋白酶 D 调节控制巨噬细胞凋亡或竞争死亡过程的线粒体途径的多种蛋白质，从而促进细胞内细菌杀伤。
