Interaction between 52 kDa SSA/Ro and deubiquitinating enzyme UnpEL: a clue to function
Interaction between 52 kDa SSA/Ro and deubiquitinating enzyme UnpEL: a clue to function
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DOI：
10.1016/s1357-2725(01)00055-3
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发表时间：
2001-09-01
影响因子：
4
通讯作者：
Buyon, JP
中科院分区：
文献类型：
--
作者：
Di Donato, F;Chan, EKL;Buyon, JP
在子宫内检测到孤立的心脏传导阻滞强烈预测与52 kDa反应的母体自身抗体的存在。SSA/Ro.通过定义靶抗原的功能，可以阐明这一观察结果的机制。最初的方法是利用酵母双杂交系统中的转录活性来鉴定与52 Ro相互作用的蛋白质。使用从人胎儿心脏（12-23周）分离的RNA构建cDNA文库，并将其克隆到编码GAL 4（AD）激活结构域的HybriZAP载体中作为靶标。将约7 × 106个cDNA与诱饵共转化到YRG-2中。对来自五个相互作用菌落的质粒进行测序，其中三个被鉴定为特异性人类去泛素化酶UnpEL。UnpEL不与编码52 β的诱饵质粒相互作用，52 β是52 kDa SSA/Ro的替代亮氨酸拉链-减形式，其在胎儿生命中最大程度地表达。哺乳动物双杂交试验证实了全长52 Ro和UnpEL之间的相互作用。生物学相互作用的进一步支持是在将两种蛋白质共转染到培养的人心脏细胞、人肾癌细胞（293细胞）和猴肾成纤维细胞（COS-I）中后，UnpEL的细胞定位发生了显著的再分布。总之，全长52 Ro和UnpEL的相互作用意味着前者也可能参与泛素途径，特别感兴趣的观察，因为52 Ro包含一个环指结构域，一个共同的基序，最近报道的几个蛋白质参与调节泛素化。缺乏与52 β的相互作用引起了这样的考虑，即蛋白质泛素化的调节可能在胎儿生活中有所不同。(C)2001爱思唯尔科技有限公司版权所有。
The detection of isolated heart block in utero strongly predicts the presence of maternal autoantibodies reactive with 52 kDa. SSA/Ro. The mechanisms that underlie this observation may be elucidated by defining the function of the target antigen. The initial approach was to identify proteins interactive with 52Ro using transcriptional activity in the yeast 2-hybrid system. A cDNA library was constructed using RNA isolated from human fetal hearts (12-23 weeks) and cloned into the HybriZAP vector encoding the activation domain of GAL4(AD) as target. Approximately 7 x 10(6) cDNAs were cotransformed with the bait into YRG-2. Plasmids from five interactive colonies were sequenced and three identified as the specific human deubiquitinating enzyme, UnpEL. UnpEL did not interact with bait plasmid encoding 52 beta, an alternative leucine zipper-minus form of 52 kDa SSA/Ro which is maximally expressed in fetal life. The mammalian 2-hybrid assay confirmed the interaction between full-length 52Ro and UnpEL. Further support for a biologic interaction was the marked redistribution in cellular localization of UnpEL following cotransfection of the two proteins into cultured human cardiocytes, human renal carcinoma cells (293 cells), and monkey kidney fibroblasts (COS-I). In conclusion, the interaction of full-length 52Ro and UnpEL implies that the former may also be involved in the ubiquitin pathway, an observation of particular interest since 52Ro contains a RING finger domain, a motif common to several recently reported proteins involved in modulating ubiquitination. The absence of an interaction with 52 beta raises the consideration that regulation of protein ubiquitination might differ in fetal life. (C) 2001 Elsevier Science Ltd. All rights reserved.