proteomic approach for the elucidation of stimulus- and substrate-specificity of ectodomain shedding.
proteomic approach for the elucidation of stimulus- and substrate-specificity of ectodomain shedding.
复制标题
用于阐明胞外域脱落的刺激和底物特异性的蛋白质组学方法。
DOI：
10.1016/j.jprot.2014.01.012
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发表时间：
2013
期刊：
影响因子：
3.3
通讯作者：
Hattori S.
中科院分区：
文献类型：
--
作者：
Shirakabe K.;Shibagaki Y.;Yoshimura A.;Koyasu S.;Hattori S.
胞外结构域脱落（shedding）是一种翻译后修饰机制，通过膜加工释放膜蛋白的胞外结构域。由于脱落以快速和不可逆转的方式改变细胞特性，因此必须对其进行严格调控。然而，调节机制的脱落，以应对环境的变化仍然模糊不清。为了评估内源性脱落的调控机制，我们先前开发了一种蛋白质组学筛选系统来鉴定脱落靶点。该系统揭示了在限定条件下脱落的膜蛋白的全面图片。在这项研究中，我们改进了筛选系统，比较了两种不同的脱落诱导剂，脂多糖（LPS）和12-O-十四烷酰佛波醇13-乙酸酯（TPA）处理的小鼠巨噬细胞系的脱落模式。我们在这里表明，LPS同时激活多种膜蛋白的脱落。我们进一步表明，TPA特异性激活αM/β2整联蛋白（Mac-1）的脱落，而巨噬细胞在LPS刺激后不会脱落。这些结果清楚地表明，内源性膜蛋白脱落的调节是刺激和底物特异性的。生物学意义迄今报道的脱落靶点在各种生物学现象中起着关键作用，包括免疫反应，细胞生长，细胞粘附和细胞运动。此外，一些疾病相关的膜蛋白正在脱落的目标。因此，了解脱落的调节对于阐明发病机制和发展治疗策略是重要的。我们认为，内源性脱落的全面表征是理解脱落的调控机制所不可或缺的，因此开发了一种蛋白质组学筛选系统来识别脱落靶点。在这项研究中，使用我们的筛选系统，我们证明了不同的细胞外刺激激活不同类型的脱落，即使在单个细胞中。我们的研究结果证明，这种蛋白质组学的方法是非常有效的脱落的调节机制的阐明。
Ectodomain shedding (shedding) is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing. Because shedding alters cell characteristics in a rapid and irreversible manner, it must be strictly regulated. However, the regulatory mechanisms of shedding in response to environmental changes remain obscure. To evaluate the regulatory mechanisms of endogenous shedding, we previously developed a proteomic screening system to identify shedding targets. This system revealed a comprehensive picture of membrane proteins shed under defined conditions. In this study, we have improved the screening system to compare the shedding patterns in a mouse macrophage cell line treated with two different shedding inducers, lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol 13-acetate (TPA). We show here that LPS simultaneously activates the shedding of multiple membrane proteins. We further show that TPA specifically activates the shedding of αM/β2 integrin (Mac-1), which was not shed upon LPS-stimulation of macrophages. These results clearly demonstrate that the regulation of endogenous membrane protein shedding is both stimulus- and substrate-specific.Biological significanceThe shedding targets reported to date play pivotal roles in a variety of biological phenomena, including the immunological response, cell growth, cell adhesion and cell movement. In addition, several disease-related membrane proteins are shedding targets. Thus, understanding the regulation of shedding is important for the elucidation of pathogenesis and the development of therapeutic strategies. We submit that a comprehensive characterization of endogenous shedding is indispensable for understanding the regulatory mechanisms of shedding, and thus have developed a proteomic screening system to identify shedding targets. In this study, using our screening system, we demonstrate that different extracellular stimuli activate different types of shedding, even in a single cell. Our results prove that this proteomic approach is quite effective for the elucidation of the regulatory mechanisms of shedding.