Abstract
Purine-rich element-binding protein B (Purβ) inhibits myofibroblast differentiation by repressing the expression of the smooth muscle α-actin gene (Acta2). Several reports have identified the structural domains in Purβ that enable its characteristic interaction with purine-rich single-stranded DNA (ssDNA) sequences in the Acta2 promoter. However, little is known about the physical and functional effects of single-nucleotide polymorphisms that alter individual amino acid residues in Purβ. This study evaluated seven rare single amino acid variants of human PURB engineered into the homologous mouse Purβ protein. Mapping the location of variant residues on a homology model of the Purβ homodimer suggested that most of the altered residues are remote from the predicted ssDNA-binding regions of the protein. The repressor activity of each Purβ variant was assessed in transfected fibroblasts and smooth muscle cells via Acta2 promoter-reporter assays. A Q64* nonsense variant was completely inactive while missense variants exhibited repressor activity that ranged from ~1.5-fold greater to ~2-fold less than wild-type Purβ. Lower activity variants P223L and R297Q were expressed in bacteria and purified to homogeneity. Each variant was physically indistinguishable from wild-type Purβ in terms of quaternary structure and thermostability. Results of DNA and protein-binding assays indicated that the P223L and R297Q variants retained high affinity and specificity for purine-rich ssDNA sequences but differed in their interaction with other Acta2 regulatory proteins. These findings suggest that the presence of certain variant residues affects the Acta2 repressor activity of Purβ by altering its interaction with other transcription factors but not with ssDNA.