Antizyme family and their functions

从斑马鱼cDNA文库中克隆了两个抗酶（AZ）家族成员以及ODC和AZ抑制剂。两种AZs （AZs和AZL）在胚胎到成鱼的组织中差异表达。两种AZ均对ODC有抑制作用，且这种抑制作用被AZ抑制剂逆转。AZS能加速ODC的体外降解，而AZL不能。在斑马鱼细胞系中，添加亚精胺导致ODC以蛋白质合成依赖的方式快速降解，这表明存在多胺/ az介导的ODC调控系统。在Schizosaccharomyces pombe中，ODC通过蛋白酶体催化az依赖性ODC降解而迅速受到亚精胺的抑制。尽管AZ2在HTC细胞中的表达降低了ODC的半衰期并抑制了多胺的摄取，但AZ2在哺乳动物体内并没有加速ODC的体外降解。AZ2本身加速ODC降解，而AZ2释放的AZi不加速ODC降解。在来自敲除小鼠的AZiknockout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，并且在细胞提取物中检测到az2。过量表达AZi或2导致HTC细胞生长受到抑制。由此得出az2负调控哺乳动物细胞多胺代谢的结论。检测了AZs的细胞定位和降解。与ODC络合的AZ1和AZ2均定位于细胞质中，而未与ODC结合的游离AZ1主要定位于细胞核中。用抑制剂进行的实验结果表明，AZi和AZ2在细胞质中均被蛋白酶体降解。此外，体外降解研究表明，这两种az均通过泛素途径降解。我们还发现了抗酶3对ODC体外降解的抑制作用，对小鼠肾脏ODC的组织特异性调节，以及ODC降解过程中AZi不掺入26S蛋白酶体。

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.