Screening And Identification Of Proteins Interacting With Zinc Finger Transcription Factor ZNF580

Background:Human ZNF580 gene (Gene ID:AF184939) was cloned by stimulating vascular endothelial cells with atherogenic factor low-density lipoprotein (LDL) and screening human aorta cDNA library. Bioinformatics analysis reveals that human ZNF580 gene encodes a Cys2-His2 (C2H2) zinc finger protein containing 172 amino acids. The protein has highly conserved carboxy terminus containing three tandem repeated C2H2 zinc finger domains, and the amino terminus is proline-riched domain. The protein structure of ZNF580 is very similar to the Spl-like or Kruppel-like transcriptional factors (KLFs) which are characterized by the three tandem repeated C2H2 zinc fingers in C-terminus. These findings suggest that ZNF580 may be a new member of the Sp1-like/KLFs superfamily. Recent studies have shown that members of the Spl-like/KLFs family are important components of the eukaryotic cellular transcriptional machinery in regulating many of the cellular functions.Objective:To screen and identify the interactive proteins with zinc finger transcription factor ZNF580 and reveal the possible signaling pathway in which ZNF580 being involved.Methods:Human ZNF580 ORF (open reading frame, ORF) was amplified by polymerase chain reaction and ligated into pGB to construct recombinant plasmid pGB-ZNF580. After eliminating bait proteins self-autoactivation and its toxicity to Y190 yeast strain, human fetal brain cDNA library was transformed into the bait yeast strain and screened with yeast two-hybrid (Y2H) system. The co-transformants were plated onto a SD/-Trp/-Leu/-His selective medium. The firm colonies were then performedβ-galactosidase colony-lift filter assay by detecting the initiation of LacZ reporter gene transcription. Finally, the blue colonies were selected as positive clones. Positive colonies plasmids were extracted. The inserted cDNAs cloned into library plasmids pACT2 were amplified by PCR (polymerase chain reaction, PCR). The PCR sequences were checked for homology by using the BLAST program against the GenBank/EMBL/DDBJ non-redundant sequence database. SMAD2, a component of the transforming growth factor-B (TGF-B) signaling cascade, was focused on for further analyses. The interaction between ZNF580 and SMAD2 was detected by co-immunoprecipitation in HEK293 cells, and the co-localization between endogenous ZNF580 and SMAD2 was observed in EA.hy926 endothelial cells with immunofluorescence and confocal microscopy.Results:Fourteen proteins were identified to interact with ZNF580. These proteins are as follows:Activity-dependent neuroprotective protein (ADNP), Smad family member 2 (SMAD2), Glyoxalase domain containing 4 (GLOD4), Kinesin family member 3A (KIF3A), Kinesin-associated protein 3 (KIFAP3), Phospholipase A2-activating protein (PLAA), Phospholipase C gamma 1 (PLCG1), XPA binding protein 2 (XAB2), Tripartite motif-containing 32 (TRIM32), Suppressor of Ty 16 homolog (SUPT16H), Wilms tumor 1 interacting protein (WTIP), DIP2 disco-interacting protein 2 homolog C (DIP2C), SH3-domain GRB2-like 3 (SH3GL3), D4, zinc and double PHD fingers family 1 (DPF1). The interaction between ZNF580 and SMAD2 was confirmed by co-immunoprecipitation in HEK293 cells. The co-localization between endogenous ZNF580 and SMAD2 was mainly found in nuclei of EA.hy926 cells.Conclusions:1. Fourteen potential proteins interacting with ZNF580 were obtained through the yeast two-hybrid screen.2. The interaction between ZNF580 and SMAD2 was confirmed by co-immunoprecipitation and immunofluorescence assay in eukaryotic cells. The co-localizaion of endogenous ZNF580 and SMAD2 was identified in nuclei.3. Our results suggest that ZNF580 is a binding partner of SMAD2 and involved in transforming growth factor-βsignaling pathway. The expression of downstream target genes may depend on the interaction between ZNF580 and SMAD2.4. Our studies provide basis for additional research to further investigate the role of ZNF580 playing in atherosclerotic diseases.