Antizyme family and their functions

从斑马鱼cDNA文库中克隆到抗酶（antizymes，AZ）家族的两个成员（AZS和AZL），它们与ODC和AZ抑制剂一起在斑马鱼胚胎至成鱼的组织中差异表达。两种AZ均能抑制ODC，AZ抑制剂可逆转这种抑制作用。AZS而非AZL促进ODC的体外降解。在斑马鱼细胞系中，亚精胺的加入导致ODC以蛋白质合成依赖的方式快速降解，表明存在多胺/AZ介导的ODC调节系统。在粟酒裂殖酵母中，ODC被证明是快速阻遏响应添加亚精胺通过AZ依赖ODC降解的蛋白酶体催化。哺乳动物AZ 2不促进ODC的体外降解，但在HTC细胞中表达AZ 2可缩短ODC的半衰期，抑制多胺的摄取，AZ 2本身可促进ODC的降解，而AZ 2释放的AZ不促进ODC的降解。在AZ 2基因敲除小鼠细胞中，亚精胺以蛋白质合成依赖的方式引起ODC的快速降解，并在细胞提取物中检测到AZ 2。AZi或2的过量表达引起HTC细胞的生长抑制。从这些结果我们得出结论，AZ 2负调节哺乳动物细胞中的多胺代谢.检测AZ的细胞定位和降解。与ODC复合的AZ 1和AZ 2均定位于胞浆，而游离的AZs（未与ODC结合）主要定位于细胞核。用抑制剂的实验结果表明，AZ 1和AZ 2都在胞质溶胶中被蛋白酶体降解。体外降解实验表明，两种AZ均通过泛素途径降解。我们还表明，在体外ODC降解的抑制抗酶3，小鼠肾脏ODC的组织特异性调节和缺乏AZi纳入26 S蛋白酶体在ODC降解。

Two members of antizymes (AZ) family were cloned from a zebrafish cDNA library together with ODC and AZ inhibitor.The the two AZs (AZS and AZL) were differentially expressed in the tissues from embryos to adult fish. Both AZs inhibited ODC and the inhibition was reversed by AZ inhibitor. AZS but not AZL accelerated ODC degradation in vitro. In a zebrafish cell line, the addition of spermidine caused a rapid degradation of ODC in a protein synthesis-dependent manner, suggesting the presence of the polyamine/AZ-mediated ODC regulatory system. In Schizosaccharomyces pombe, ODC was shown to be rapidly repressed in response to addition of spermidine through AZ-dependent ODC degradation catalyzed by the proteasome. Mammalian AZ 2 did not accelerate ODC degradation in vitro, although AZ2 expression in HTC cells decreased the ODC half-life and inhibited polyamine uptake.The ODC degradation was accelerated by AZ2 itself but not by released AZi by AZ2.In AZiknockout cells derived from knockout mice, spermidine caused rapid ODC degradation in a protein synthesis-dependent manner and AZ 2 was detected in the cell extract. Expression of AZi or 2 in excess caused the growth inhibition in HTC cells. From these results we concluded that AZ 2 negatively regulates polyamine metabolism in mammalian cells. Cellular localization and degradation of AZs were examined. Both AZ1 and AZ2 complexed with ODC were localized in cytosol, whereas free AZs (unbound with ODC) were mainly localized in nucleus. Experimental results with inhibitors suggested that both AZi and AZ2 were degraded in cytosol by the proteasome. Furthermore, in vitro degradation study suggested that both AZs were degraded through the ubiquitin pathway. We also showed the inhibition of ODC degradation in vitro by antizyme 3, tissue specific regulation of mouse kidney ODC and the lack of incorporation of AZi into the 26S proteasome during ODC degradation.