Insights into human cardiovascular zinc finger proteins (ZFPs): Profiling, identification and functional characterization of a novel RING ZFP (SMRZ)

The zinc finger proteins (ZFPs) are a superfamily of proteins containing at least one of several conserved zinc-binding domains. ZFPs have a wide range of biological functions and evidence is increasing that these proteins are involved in the pathogenesis of cardiac disease. Elucidating the roles of ZFPs in the molecular regulatory dynamics of the cardiovascular system is thus becoming essential to molecular cardiology. This research project aims to profile unique ZFPs and to identify novel ZFPs in the cardiovascular system. Using an EST sequence similarity search, we established a profile of cardiovascular-based ZFPs (cvbZFPs). Structural classification divided cvbZFPs into seven types: C₂H₂-, C₂C₂-, C₂HC-, C₂HC₄C(HD)-, C₃H-, C₃HC₄ - and combination-types, with the C₂H₂-type predominating. To expand the cvbZFP profile, three domain sequences (KRAB, BTB/POZ and RING) were also used as probes to screen our EST database in silico. An analysis of genomic assignments of cvbZFPs suggests that chromosome 19 appears to be enriched in C₂H₂-type ZFP genes. Over 90% of cvbZFPs demonstrated widespread tissue distribution as shown by in silico Northern blot. Three developmentally regulated genes (HFHZ, BPOZ and SMRZ) were identified and characterized. Of these, SMRZ, containing a RING domain (a protein-protein interaction interface), was selected for further functional investigation in searching for its interaction partner. Using a yeast two hybrid system, SMT3b, a human homolog of yeast mutation suppressor of the essential centromere protein, MIF2, was identified as an interaction partner of SMRZ, and this was mediated by the RING domain. This interaction was further confirmed by in vitro interaction assay using both wild-type and RING-domain mutated SMRZ. Our observation of SMRZ-SMT3b interaction taken together with evidence that SMRZ is: (1) restrictedly expressed in heart and skeletal muscle, (2) developmentally regulated in heart tissue, (3) localized in nucleus, and observation that overexpression of SMT3b inhibited C2C12 myoblast growth, lead us to conclude that SMRZ must play an important role in developmental regulation, perhaps cell cycle regulation, of striated muscle cells. The present cvbZFP study combines EST in silico and conventional molecular biology approaches to provide an important first step toward understanding the ZFP regulatory network in the cardiovascular system.