鸡chUSP1 His594Ala突变使其去泛素化酶活性丧失  

His 594-to-Ala mutation in chicken USP1 eliminated its deubiquitinating activity

作　　者：郑海南[1] 赵程程[1] 邓晨[1] 于佩峰 王梦云[1] 吴山力[2] 吕岩[1] 艾永兴[1] 

机构地区：[1]吉林大学动物科学学院,吉林长春130062 [2]吉林大学白求恩医学学院,吉林长春130021

出　　处：《中国兽医学报》2015年第5期745-750,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31272528);留学基金资助项目(教外司留2013-693号);跨世纪计划资助项目(NCET-12-0232)

摘　　要：泛素特异性蛋白酶1(ubiquitin-specific proteases 1,USP1)是一种去泛素化酶,它在DNA损伤修复应答过程中发挥重要功能。已证明USP1与UAF1(USP1-associated factor 1)形成复合物后,其酶活性会得到极大的提升。chUSP1为在鸡细胞中发现的一种去泛素化酶,生物信息学分析发现它与人USP1同源性高达72%。本研究以本室扩增的chUSP1基因和chUAF1基因,使用bac-to-bac系统表达了chUSP1GG680,681AA单体及chUSP1GG680,681AA/chUAF1复合体。并根据人USP1预测将chUSP1一个可能的活性中心位点第594位组氨酸突变为丙氨酸,并纯化了此突变型的单体和复合体。应用底物Di-Ub/Ub2 WT Chains(K63-linked)及Di-Ub/Ub2 WT Chains(K48-linked)对chUSP1GG680,681AA和突变型chUSP1HGG594,680,681AA进行酶活性分析。试验结果表明chUSP1具有切割Ub链底物的酶活性,且第594位组氨酸残基突变影响chUSP1的去泛素化酶活性。而chUAF1可以与chUSP1相互作用形成复合体,并且明显提高chUSP1酶活性。USP1(ubiquitin-specific protease 1)is a deubiquitinase in vertebrate cell and plays important roles in DNA damage repair,cell preliferation and genome stability etc.In human cells,UAF1(USP1associated factor 1)has been shown to interact directly with USP1 and enhances USP1 deubiquitinating activity.The gene encoding chicken USP1(chUSP1)is found in genomic analysis,and this deubiquitinase shares 72%amino acid sequence identity to the counterpart in human cells.In current study,we hypothesised that His-594 might be a potential activity site of chUSP1 and mutated His-594 to Ala according to human USP1 catalytic domain.Then,chUSP1GG680,681 AA, chUSP1GG680,681AA/chUAF1 complex, chUSP1HGG594,680,681 AAA and chUSP1HGG594,680,681AA/chUAF1 complex were expressed in Bac-to-Bac system and purified on NiNTA Agarose.The activity and cleavage specificity of these purified proteins were tested with two substrates,Di-Ub/Ub2 WT Chains(K63-linked)and Di-Ub/Ub2 WT Chains(K48-linked).The results showed that chUSP1 wild type and its complex,but not the mutants,were able to cleave two types of substrates without visable difference.Our results suggested that His-594 is an essential residue for USP1 deubiquitinating activity.Moreover,chUSP1 can interact with chUAF1 to form heterodimer in vivo during co-expression in insect cells.The interaction enhanced USP1 deubiquitinating activity.

关 键 词：chUSP1 杆状病毒 昆虫细胞 酶活性分析 

分 类 号：S831[农业科学—畜牧学]