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首页> 外文期刊>Molecular and Cellular Biology >Pharmacoproteomics of a Metalloproteinase Hydroxamate Inhibitor in Breast Cancer Cells: Dynamics of Membrane Type 1 Matrix Metalloproteinase-Mediated Membrane Protein Shedding
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Pharmacoproteomics of a Metalloproteinase Hydroxamate Inhibitor in Breast Cancer Cells: Dynamics of Membrane Type 1 Matrix Metalloproteinase-Mediated Membrane Protein Shedding

机译：乳腺癌细胞中金属蛋白酶异羟肟酸酯抑制剂的药代动力学：膜类型1基质金属蛋白酶介导的膜蛋白脱落的动力学。

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Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90α, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment.
机译：广谱基质金属蛋白酶（MMP）抑制剂（MMPI）在癌症临床试验中不成功，部分原因是由于对MMP底物的全部组成（称为底物降解组）的了解有限而产生的副作用，以及MMP的体内功能。为了进一步了解MMP-14（膜类型1 MMP）的降解，使用了MMPI抑瘤剂（药物代码AG3340）来减少MMP-MB-231转染的人MDA-MB-231乳腺癌细胞的蛋白水解过程和胞外域脱落。 14。我们报告了在基于细胞的系统中抑制剂的目标和作用的定量蛋白质组学评估。通过同位素编码的亲和标签（ICAT）标记和串联质谱法鉴定细胞条件培养基（分泌组）中的蛋白质和具有MMPI调节水平的膜级分。 MMP-14的表达与载体对照的表达比较导致条件培养基中许多蛋白质的MMP-14 /载体ICAT比增加，表明MMP-14介导的胞外域脱落。 MMPI处理后，MMPI /车辆ICAT比例逆转，表明抑制剂阻止了MMP-14介导的这些蛋白质的脱落。 MMPI /车辆ICAT比例高表明，在MMPI存在下，脱落或底物从细胞周围位点释放的减少通常伴随着蛋白质在质膜中的积累。综合考虑，这是在细胞环境中裂解的生物学相关底物的强力预测因子，这些底物导致鉴定了许多未描述的MMP-14底物，我们对其中20种底物进行了生化验证，包括DJ-1，galectin-1，Hsp90α，pentraxin 3 ，颗粒蛋白原，Cyr61，肽基脯氨酰顺-反异构酶A和dickkopf-1。其他水平发生变化的蛋白质，例如Kunitz型蛋白酶抑制剂1和β-2-微球蛋白，在生化分析中不是底物，表明MMPI的间接影响，这可能对作为生物标记物的药物开发或在临床前阶段很重要，以预测全身性药物作用和不良副作用。因此，这种方法描述了细胞膜胞外域脱落的动态模式及其对金属蛋白酶药物治疗的干扰。

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