Liu Zhiqian, Oughtred Rose, Wing Simon S
Polypeptide Hormone Laboratory, McGill University, Strathcona Anatomy and Dentistry Building, 3640 University St., Room W315, Montreal, Quebec, Canada, H3A 2B2.
Mol Cell Biol. 2005 Apr;25(7):2819-31. doi: 10.1128/MCB.25.7.2819-2831.2005.
During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone, which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone-dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domain-containing protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation.
在精子发生过程中，随着精子细胞向细长的成熟形式演化，大部分细胞蛋白质会被降解。特别是，组蛋白必须在早期伸长的精子细胞中被降解，以允许染色质浓缩。我们实验室先前证明了精子发生过程中泛素缀合的激活。这种激活依赖于泛素缀合酶（E2）UBC4，并且当组蛋白被降解时，一种睾丸特异性同工型UBC4-睾丸会被诱导产生。因此，我们测试了是否存在能使组蛋白泛素化的UBC4依赖性泛素蛋白连接酶（E3）。确实，发现了一种新酶E3组蛋白，它在体外可将泛素缀合到组蛋白H1、H2A、H2B、H3和H4上。只有E2的UBC4/UBC5家族支持E3组蛋白依赖性的组蛋白H2A泛素化，在这个家族中，UBC4-1和UBC4-睾丸是首选的E2。我们将这种连接酶活性纯化了3600倍，达到近乎同质的状态。对最终产物进行质谱分析，发现存在一种含有482-kDa HECT结构域的蛋白质，该蛋白质先前被命名为LASU1。抗LASU1抗体免疫去除了E3组蛋白活性。质谱分析以及通过凝胶过滤和甘油梯度离心进行的大小分析表明，E3组蛋白是LASU1的单体。我们的测定还表明，这种酶是主要的UBC4-1依赖性组蛋白泛素化E3。因此，E3组蛋白是一种HECT结构域E3，可能在精子细胞成熟过程中发生的染色质浓缩中起重要作用。
