蟾蜍泛素羧基末端水解酶(tUCH)以不依赖其UCH活性的方式参与卵母细胞成熟调控

本实验室已报道中华大蟾蜍卵母细胞的p28蛋白具泛素羧基末端水解酶活性,称作tUCH,它和哺乳类中发现的UCH L1的氨基酸序列具高度同源性,二级结构同源性比较发现,二者可能具类似的功能。本文实验表明:未成熟卵母细胞和成熟卵母细胞的可溶性蛋白中均含有tUCH,约占提取物中蛋白质总量的2%。根据测定所得到的GST-tUCH和GST-UCH L1对底物Ub-AMC的酶动力学参数,说明卵母细胞中tUCH可能与小鼠UCH L1有类似的生物学功能;anti-tUCH单抗可以与原核细胞表达的tUCH和显性失活突变类型tUCH C(90)S特异结合,但不识别小鼠的UCH L1。Anti-tUCH单抗能够和tUCH结合但不能封闭它的UCH活性。当anti-tUCH单抗注入卵母细胞内,则孕酮诱导的生发泡破裂(germinal vesicle breakdown,GVBD)过程受到抑制,足见tUCH参与GVHD调节并不依赖其UCH活性。

THE FUNCTIONS IN THE PROGESTERONE-INDUCED OOCYTE MATURATION OF TOAD UBIQUITIN CARBOXYL-TERMINAL HYDROLASE(tUCH) IS INDEPENDENT OF ITS UCH ACTIVITY

p28, a protein derived from toad ( Bufo bufo gargarizans) oocytes, has a high level of sequence homology to mouse UCH L1. We report here that it is a toad ubiquitin carboxyl-terminal hydrolase (UCH), termed tUCH for its ability to hydrolyze the UCH substrate ubiquitin ethyl ester (UboEt). The similarities of secondary structures between tUCH and UCH L1 highlight that they might have common functions. The total extracted proteins both from immature and matured oocytes contain 2% tUCH. The enzyme kinetic constants (Km and Kc) both of the tUCH and UCH L1 reveal that they possess similar catalytic properties on their common substrate Ub-AMC. Anti-tUCH monoclonal antibody (tUCH mAb) can recognize tUCH and dominant-negative tUCH (tUCH C(90)S), but not mouse UCH L1, suggesting that it does not target on the conservative UCH active sites. Furthermore, anti-tUCH mAb when injected into oocytes blocked the progesterone-induced GVBD but anti-tUCH mAb could not inhibit the tUCH catalytic hydrolysis of Ub-AMC, revealing that the implication of tUCH in the oocyte maturation regulation is not dependent on its UCH activity.