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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays
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Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays

机译：使用细胞提取物和蛋白质微阵列大规模检测泛素化底物

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摘要

Identification of protein targets of post-translational modification is an important analytical problem in biology. Protein microarrays exposed to cellular extracts could offer a rapid and convenient means of identifying modified proteins, but this kind of biochemical assay, unlike DNA microarrays, depends on a faithful reconstruction of in vivo conditions. Over several years, concentrated cellular extracts have been developed, principally for cell cycle studies that reproduce very complex cellular states. We have used extracts that replicate the mitotic checkpoint and anaphase release to identify differentially regulated poyubiquitination. Protein microarrays were exposed to these complex extracts, and the polyubiquitinated products were detected by specific antibodies. We expected that among the substrates revealed by the microarray should be substrates of the anaphase promoting complex (APC). Among 8,000 proteins on the chip, 10% were polyubiquitinated. Among those, we found 11 known APC substrates (out of 16 present on the chip) to be polyubiquitinated. Interestingly, only 1.5% of the proteins were differentially ubiquitinated on exit from the checkpoint. When we arbitrarily chose 6 proteins thought to be involved in mitosis from the group of differentially modified proteins, all registered as putative substrates of the APC, and among 4 arbitrarily chosen non-mitotic proteins picked from the same list, 2 were ubiquitinated in an APC-dependent manner. The striking yield of potential APC substrates from a simple assay with concentrated cell extracts suggests that combining microarray analysis of the products of post-translational modifications with extracts that preserve the physiological state of the cell can yield information on protein modification under various in vivo conditions.
机译：鉴定翻译后修饰的蛋白质靶标是生物学中的重要分析问题。暴露于细胞提取物的蛋白质微阵列可以提供快速便捷的方法来鉴定修饰的蛋白质，但是与DNA微阵列不同，这种生化测定取决于对体内条件的忠实重建。几年来，已经开发出浓缩的细胞提取物，主要用于复制非常复杂的细胞状态的细胞周期研究。我们使用提取物复制有丝分裂检查点和后期释放来鉴定差异调节的泛素化。将蛋白质微阵列暴露于这些复杂的提取物，并通过特异性抗体检测多泛素化产物。我们期望在微阵列揭示的底物中，应该是后期促进复合物（APC）的底物。在芯片上的8,000种蛋白质中，有10％被泛素化。其中，我们发现11种已知的APC底物（芯片上存在的16种底物）被多泛素化。有趣的是，只有1.5％的蛋白质在从检查站退出时被差异泛素化。当我们从差异修饰的蛋白质组中任意选择6种被认为与有丝分裂有关的蛋白质时，所有蛋白质均被注册为APC的假定底物，并且在同一清单中任意选择的4种非有丝分裂蛋白质中，有2种在APC中被泛素化依赖的方式。从浓缩细胞提取物的简单测定中潜在的APC底物惊人的产率表明，将翻译后修饰产物与保留细胞生理状态的提取物进行微阵列分析相结合，可以在各种体内条件下产生有关蛋白质修饰的信息。

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