Baska Kathleen M, Manandhar Gaurishankar, Feng Dawn, Agca Yuksel, Tengowski Mark W, Sutovsky Miriam, Yi Young-Joo, Sutovsky Peter
Division of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri, USA.
J Cell Physiol. 2008 Jun;215(3):684-96. doi: 10.1002/jcp.21349.
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 E1 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.
泛素化介导的翻译后修饰会标记有缺陷或寿命已尽的细胞内蛋白质，以便26S蛋白酶体进行蛋白水解降解。在底物蛋白上进行依赖ATP的多聚泛素链的共价连接和形成，需要一组泛素激活酶和缀合酶的存在及活性。虽然蛋白质泛素化通常发生在细胞质或细胞核中，但有缺陷的哺乳动物精子在附睾中睾丸后精子成熟过程中，其表面会发生泛素化，这表明存在一种活跃的精子质量控制分子机制。因此，我们推测泛素-蛋白酶体途径的生物活性成分会分泌到哺乳动物附睾液（EF）中，并能够使外源底物泛素化。蛋白质印迹法确实在分离出的牛附睾液中检测到了泛素激活酶E1以及推测的E1-泛素硫酯中间体、泛素载体酶E2以及推测的E2-泛素硫酯中间体，还有泛素C末端水解酶PGP 9.5/UCHL1。利用重组泛素激活酶和泛素缀合酶、生物素化底物以及分离出的牛附睾液进行的硫酯测定，证实了附睾液中泛素激活酶和缀合酶的活性。在有缺陷的公牛精子部分中发现泛素化蛋白富集，并且通过特定荧光底物在分离出的附睾液中检测到了适当的蛋白酶体去泛素化和蛋白水解活性。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，观察到了细胞质蛋白的顶浆分泌。在大鼠和小鼠附睾上皮的附睾头顶端小泡（顶浆分泌位点）中检测到了eGFP、泛素和蛋白酶体的积累，尽管存在区域特异性差异。在体外对分离出的大鼠附睾上皮细胞进行长时间培养期间，eGFP和蛋白酶体持续分泌。这项研究提供了证据，表明泛素系统的活性不限于细胞内环境，这有助于更深入地了解精子在附睾中通过时的成熟过程。
