Mapping and structural dissection of human 20 S proteasome using proteomic approaches
Mapping and structural dissection of human 20 S proteasome using proteomic approaches
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DOI：
10.1074/mcp.m200030-mcp200
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发表时间：
2002-08-01
影响因子：
7
通讯作者：
Monsarrat, B
中科院分区：
文献类型：
--
作者：
Claverol, S;Burlet-Schiltz, O;Monsarrat, B
蛋白酶体是存在于所有真核细胞中的蛋白水解复合物，是ATP依赖性泛素/蛋白酶体途径的一部分。它在许多生理过程的调节中起着关键作用。20 S蛋白酶体是26 S蛋白酶体的催化核心，由四个堆叠的环组成，每个环有七个亚基（α 7 β 7 β 7 α 7）。本文采用蛋白质组学方法对人20 S蛋白酶体进行了研究.这导致了精细亚基参考图谱的建立和翻译后修饰的鉴定。我们发现，从红细胞中纯化的人20 S蛋白酶体，表现出高度的结构异质性，其特征在于存在多个亚型的大部分的α和13亚基，包括催化的，导致在考马斯蓝染色后，总共至少32个可见的斑点。给定亚基的不同亚型显示出pi值的变化，这表明它们可能是由翻译后修饰引起的。然后，我们利用互补质谱方法的效率，在结构水平上进一步研究这些蛋白质修饰。特别是，我们把我们的努力集中在α 7亚基，并表征其N-乙酰化和定位于Ser（250）上的磷酸化位点。
The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the a and 13 subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pi values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250).