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首页> 外文期刊>Cell and Tissue Research >Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa
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Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa

机译：哺乳动物精子中RING手指泛素连接酶UBR7的鉴定与表征

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摘要

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB+1, to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.
机译：泛素-蛋白酶体系统（UPS）通过E3型泛素连接酶以底物特异性方式控制细胞内蛋白更新。 UPS会控制哺乳动物的受精，尤其是精子通过卵母细胞卵黄质外壳（透明带）的渗透。我们使用蛋白酶体依赖性泛素融合降解途径的外在底物，突变体泛素UBB + 1，以提供证据表明精子顶体部分存在E3型连接酶活性。这种从头进行的泛素化实验的蛋白质电泳凝胶包含一个独特的蛋白质带，该蛋白质带经串联质谱鉴定，与泛素连接酶UBR7（替代名称：C14ORF130）相似。从公猪睾丸中扩增出相应的mRNA，并通过Western印迹在公猪，小鼠和人精子提取物中检测到UBR7蛋白的几种变体。基因组分析表明，UBR7基因具有高度的进化保守性，非常恒定的纯化选择和保守的睾丸表达。通过免疫荧光，UBR7定位于精子顶体帽和精子顶体，以及精子中蛋白酶体活性的热点，例如胞质叶，尾巴手足，核和中心体。受精过程中，顶体胞吐作用后，UBR7与ZP结合的顶体护罩保持在一起。因此，UBR7存在于圆形精子的顶体帽和成熟公猪精子的顶体基质中。这些数据提供了哺乳动物精子中泛素连接酶活性的第一个证据，并表明UBR7参与精子发生。

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