Mechanism of extracellular ubiquitination in the mammalian epididymis
Mechanism of extracellular ubiquitination in the mammalian epididymis
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DOI：
10.1002/jcp.21349
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发表时间：
2008-06-01
影响因子：
5.6
通讯作者：
Sutovsky, Peter
中科院分区：
文献类型：
--
作者：
Baska, Kathleen M.;Manandhar, Gaurishankar;Sutovsky, Peter
通过泛素化的翻译后修饰标记了缺陷的或寿命过长的细胞内蛋白质，用于26 S蛋白酶体的蛋白水解降解。底物蛋白上的多聚泛素链的ATP依赖性共价连接和形成需要一组泛素活化和缀合酶的存在和活性。虽然蛋白质泛素化通常发生在细胞质溶胶或细胞核中，但有缺陷的哺乳动物精子在睾丸后精子在附睾中成熟期间在其表面上变得泛素化，这表明精子质量控制的积极分子机制。因此，我们假设泛素-蛋白酶体途径的生物活性成分在哺乳动物附睾液（EF）中分泌，并能够泛素化外源性底物。蛋白质印迹法确实检测到存在的泛素活化酶E I和推定的E1-泛素巯基-酯中间体，泛素载体酶E2和推定的E2-泛素巯基-酯中间体和泛素C-末端水解酶PGP 9.5/UCHL 1在分离的牛EF。巯基酯测定利用重组泛素活化和泛素缀合酶，生物素化底物，和分离的牛EF证实了EF内的泛素活化和缀合酶的活性。泛素化的蛋白质被发现是丰富的缺陷公牛精子部分和适当的蛋白酶体去泛素化和蛋白水解活性进行了测量，在分离的EF由特定的荧光底物。在泛素-C启动子指导下表达增强型绿色荧光蛋白（eGFP）的转基因小鼠和大鼠中，胞浆蛋白质的顶浆分泌是可视化的。虽然存在区域特异性差异，但在大鼠和小鼠附睾上皮的顶浆分泌部位--顶囊泡中检测到eGFP、泛素和蛋白酶体的积累。在体外培养的大鼠附睾上皮细胞的长期培养过程中，eGFP和蛋白酶体的分泌持续。这项研究提供的证据表明，泛蛋白系统的活动并不局限于细胞内环境，有助于更好地了解精子在附睾通过过程中的成熟过程。
Posttranslational modification by ubiquitination marks defective or outlived intracellular proteins for proteolytic degradation by the 26S proteasome. The ATP-dependent, covalent ligation and formation of polyubiquitin chains on substrate proteins requires the presence and activity of a set of ubiquitin activating and conjugating enzymes. While protein ubiquitination typically occurs in the cell cytosol or nucleus, defective mammalian spermatozoa become ubiquitinated on their surface during post-testicular sperm maturation in the epididymis, suggesting an active molecular mechanism for sperm quality control. Consequently, we hypothesized that the bioactive constituents of ubiquitin-proteasome pathway were secreted in the mammalian epididymal fluid (EF) and capable of ubiquitinating extrinsic substrates. Western blotting indeed detected the presence of the ubiquitin-activating enzyme E I and presumed E1-ubiquitin thiol-ester intermediates, ubiquitin-carrier enzyme E2 and presumed E2-ubiquitin thiol-ester intermediates and the ubiquitin C-terminal hydrolase PGP 9.5/UCHL1 in the isolated bovine EF. Thiol-ester assays utilizing recombinant ubiquitin-activating and ubiquitin-conjugating enzymes, biotinylated substrates, and isolated bovine EF confirmed the activity of the ubiquitin activating and conjugating enzymes within EF. Ubiquitinated proteins were found to be enriched in the defective bull sperm fraction and appropriate proteasomal deubiquitinating and proteolytic activities were measured in the isolated EF by specific fluorescent substrates. The apocrine secretion of cytosolic proteins was visualized in transgenic mice and rats expressing the enhanced green fluorescent protein (eGFP) under the direction of ubiquitin-C promoter. Accumulation of eGFP, ubiquitin and proteasomes was detected in the apical blebs, the apocrine secretion sites of the caput epididymal epithelia of both the rat and mouse epididymal epithelium, although region-specific differences exist. Secretion of eGFP and proteasomes continued during the prolonged culture of the isolated rat epididymal epithelial cells in vitro. This study provides evidence that the activity of the ubiquitin system is not limited to the intracellular environment, contributing to a greater understanding of the sperm maturation process during epididymal passage.