中文摘要：

目的探讨类泛素蛋白FAT10的C末端双甘氨酸基团及双泛素结构域结合修饰底物蛋白质的活性。方法分别将野生型全长FATIO，C末端双甘氨酸的缺失突变体FAT10△SGG，分别包含N端、C端泛素结构域的截断突变体FAT10-N和FAT10—C构建到真核表达载体中，转染HEK293T细胞，并使用26S蛋白酶体抑制剂MG132处理细胞。利用Western blot方法检测野生型FAT10及其突变体结合底物蛋白质的活性。结果与野生型FAT10的蛋白质底物结合能力相比，FAT10△GG、FAT10-N、FAT10-C与底物蛋白质结合能力均显著下降。蛋白酶体抑制剂MG132能够促进FAT10及其突变体的底物结合活性。结论FAT10的C末端双甘氨酸基团及双泛素结构域都是FAT10与底物共价结合所需的活性基团，被FAT10标记后的底物蛋白质通过26S蛋白酶体途径降解。

英文摘要：

Objective To investigate the substrate-modifying ability of FAT10~s carboxyl-terminal diglycine motif and its two ubiquitin-like domains. Methods The coding sequences of wild-type FAT10, carboxyl-terminal diglycine deficient mutant FAT10AGC,, and two separate ubiquitin domains FAT10-N, FAT10-C were cloned into pCMV-HA expression vector respectively. After transfected with these FAT10 constructs, HEK293T cells were treated with 26S proteasome inhibitor MG132 before harvest. The whole cell lysates were then subjected to Western blot analysis. Results Wild FAT10 was able to covalently conjugate to unidentified target proteins, while the covalent binding abilities of its diglycine deficient mutant and two ubiquitin domains to substrates were reduced significantly. Treatment of MG132 resulted in an increase in the protein-binding abilities of FAT 10 and its mutants. Conclusions Both carboxyl-terminal diglycine motif and two ubiquitin-like domains are crucial to the substrate-binding ability of FAT10. FAT 10- conjugated proteins undergo degradation in a 26S proteasome-dependent manner.