Interaction between 52 kDa SSA/Ro and deubiquitinating enzyme UnpEL: a clue to function

The detection of isolated heart block in utero strongly predicts the presen ce of maternal autoantibodies reactive with 52 kDa. SSA/Ro. The mechanisms that underlie this observation may be elucidated by defining the function o f the target antigen. The initial approach was to identify proteins interac tive 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 d omain of GAL4(AD) as target. Approximately 7 x 10(6) cDNAs were cotransform ed with the bait into YRG-2. Plasmids from five interactive colonies were s equenced and three identified as the specific human deubiquitinating enzyme , UnpEL. UnpEL did not interact with bait plasmid encoding 52 beta, an alte rnative leucine zipper-minus form of 52 kDa SSA/Ro which is maximally expre ssed in fetal life. The mammalian 2-hybrid assay confirmed the interaction between full-length 52Ro and UnpEL. Further support for a biologic interact ion was the marked redistribution in cellular localization of UnpEL followi ng cotransfection of the two proteins into cultured human cardiocytes, huma n 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 comm on to several recently reported proteins involved in modulating ubiquitinat ion. The absence of an interaction with 52 beta raises the consideration th at regulation of protein ubiquitination might differ in fetal life. (C) 200 1 Elsevier Science Ltd. All rights reserved.