Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, Shijiazhuang, China.
PLoS One. 2011;6(10):e26297. doi: 10.1371/journal.pone.0026297. Epub 2011 Oct 17.
Deubiquitinating enzymes (DUBs) regulate diverse cellular functions by their activity of cleaving ubiquitin from specific protein substrates. Ubiquitin-Specific Protease 46 (USP46) has recently been identified as a quantitative trait gene responsible for immobility in the tail suspension test and forced swimming test in mice. Mice with a lysine codon (Lys 92) deletion in USP46 exhibited loss of 'behavioral despair' under inescapable stresses in addition to abnormalities in circadian behavioral rhythms and the GABAergic system. However, whether this deletion affects enzyme activity is unknown. Here we show that USP46 has deubiquitinating enzyme activity detected by USP cleavage assay using GST-Ub52 as a model substrate. Interestingly, compared to wild type, the Lys 92 deletion mutant resulted in a decreased deubiquitinating enzyme activity of 27.04%. We also determined the relative expression levels of Usp46 in rat tissues using real-time RT-PCR. Usp46 mRNA was expressed in various tissues examined including brain, with the highest expression in spleen. In addition, like rat USP46, both human and mouse USP46 are active toward to the model substrate, indicating the USP cleavage assay is a simple method for testing the deubiquitinating enzyme activity of USP46. These results suggest that the Lys 92 deletion of USP46 could influence enzyme activity and thereby provide a molecular clue how the enzyme regulating the pathogenesis of mental illnesses.
去泛素化酶（DUBs）通过其从特定蛋白质底物上切割泛素的活性来调节多种细胞功能。最近发现泛素特异性蛋白酶 46（USP46）是负责小鼠尾部悬垂试验和强迫游泳试验中不动性的数量性状基因。USP46 中的赖氨酸密码子（Lys 92）缺失的小鼠除了昼夜节律行为和 GABA 能系统异常外，在不可逃避的应激下还表现出“行为绝望”的丧失。然而，这种缺失是否会影响酶活性尚不清楚。在这里，我们使用 GST-Ub52 作为模型底物的 USP 切割测定法显示 USP46 具有去泛素化酶活性。有趣的是，与野生型相比，Lys 92 缺失突变体导致去泛素化酶活性降低了 27.04%。我们还使用实时 RT-PCR 确定了大鼠组织中 Usp46 的相对表达水平。在检查的各种组织中都表达了 Usp46 mRNA，包括大脑，脾脏中的表达最高。此外，与大鼠 USP46 一样，人 USP46 和小鼠 USP46 均对模型底物具有活性，表明 USP 切割测定法是测试 USP46 去泛素化酶活性的简单方法。这些结果表明，USP46 的 Lys 92 缺失可能会影响酶活性，从而为该酶如何调节精神疾病的发病机制提供了分子线索。
