Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae.
Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae.
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DOI：
10.1074/mcp.m111.008193
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发表时间：
2011-06
期刊：
影响因子：
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通讯作者：
Dockrell DH
中科院分区：
文献类型：
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作者：
Bewley MA;Pham TK;Marriott HM;Noirel J;Chu HP;Ow SY;Ryazanov AG;Read RC;Whyte MK;Chain B;Wright PC;Dockrell DH
巨噬细胞是对细菌的先天免疫反应的中心效应物。我们已经研究了如何激活丰富的巨噬细胞溶酶体蛋白酶，组织蛋白酶D，调节巨噬细胞蛋白质组在杀死肺炎链球菌。使用组织蛋白酶D抑制剂胃蛋白酶抑制剂A，我们表明，组织蛋白酶D差异调节多个目标的679个蛋白质的鉴定和定量的八重同量异位素标签的相对和绝对定量。我们的统计分析鉴定了18个差异表达的蛋白，通过所有配对t检验（α = 0.05）。该数据集富集了调节细胞凋亡的线粒体途径或抑制竞争性死亡程序的蛋白质。选择五种蛋白质进行进一步分析。蛋白质印迹法，随后通过药理学抑制或组织蛋白酶D的遗传操作，证实了这些蛋白质在暴露于S.肺炎。超氧化物歧化酶-2的上调与活性氧产生的增加在时间上相关。凝溶胶蛋白是一种已知的线粒体外膜透化的调节剂，其下调与线粒体释放细胞色素c有关。真核细胞延伸因子（eEF 2），一种蛋白质翻译的调节因子，也被组织蛋白酶D下调。使用eEF 2的负调节因子eEF 2激酶的缺失，我们证实eEF 2功能是维持抗凋亡蛋白Mcl-1表达所必需的，延迟巨噬细胞凋亡，并使用小鼠模型证实维持eEF 2功能与受损的巨噬细胞凋亡相关的肺炎链球菌杀伤相关。这些发现表明，组织蛋白酶D调节多种蛋白质，控制巨噬细胞凋亡或竞争性死亡过程的线粒体途径，促进细胞内细菌杀伤。
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.