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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Activity-based probes for the proteomic profiling of metalloproteases.
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Activity-based probes for the proteomic profiling of metalloproteases.

机译：基于活性的金属蛋白酶蛋白质组分析探针。

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Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.
机译：金属蛋白酶（MPs）是一类广泛多样的酶，与许多生理和病理过程有关，包括组织重塑，肽激素加工和癌症。 MPs在体内受到多种翻译后机制的严格调控，阻碍了其通过常规基因组和蛋白质组学方法进行功能分析。在这里，我们描述了通过将锌螯合异羟肟酸酯偶联至二苯甲酮光交联剂来创建MP的基于活性的蛋白质组探针的一般策略，这分别促进了MP活性位点的选择性结合和修饰。这些探针标记了活性MP，但未标记其酶原或抑制剂结合的对应物，用于鉴定在浸润性癌细胞中上调的该酶类别的成员，并评估整个蛋白质组中MP抑制剂的选择性。有趣的是，发现基质金属蛋白酶抑制剂GM6001（伊洛马司他）目前正在临床上开发，它也靶向MPs的脑啡肽酶，氨基肽酶和二肽基肽酶家族。这些结果表明，尽管缺乏序列同源性，MP仍可显示出重叠的抑制剂敏感性，并强调需要广泛评估该酶类别的MP抑制剂以开发具有合适靶标选择性的体内试剂。基于活动的分析提供了进行此类筛选的有力手段，因为这种方法可以直接在整个蛋白质组中进行，从而有助于与疾病相关的MPs的发现以及选择性靶向这些蛋白的抑制剂。

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