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 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-1). 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.
Di Donato
F
F
Department of Rheumatology, Hospital for Joint Diseases, New York University School of Medicine, Room 1608, 301 East 17th Street, New York, NY 10003, USA.
Chan
E K
EK
Askanase
A D
AD
Miranda-Carus
M
M
Buyon
J P
JP
D000818:Animals; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019556:COS Cells; D002292:Carcinoma, Renal Cell; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005260:Female; D005318:Fetal Heart / Q000502:physiology* / Q000503:physiopathology; D015723:Gene Library; D005865:Gestational Age; D006327:Heart Block / Q000175:diagnosis / Q000196:embryology; D006801:Humans; D007680:Kidney Neoplasms; D011247:Pregnancy; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D000099207:SS-A Antigen
D001323:Autoantibodies; D001324:Autoantigens; D010450:Endopeptidases; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human; C438343:UnpEL enzyme