Structural and functional analysis of single‐nucleotide polymorphic variants of purine‐rich element‐binding protein B

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.

YAP and TAZ Regulate Cc2d1b and Purβ in Schwann Cells

“…PURβ belongs to the purine-rich element binding (PUR) protein family, which includes of PURα, PURβ and PURγ. There is substantial evidence for PUR role in DNA binding (Rumora et al, 2013; Ferris and Kelm, 2019). Among them, PURα was studied the most, for its implication in fragile × syndrome and PURA syndrome, a disorder characterized by intellectual disability and delayed development of speech and motor skills, such as walking (Johnson et al, 2013; Hunt et al, 2014; Lalani et al, 2014).…”

Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B

“…A previously described P223L variant, which demonstrates reduced interaction with the 210−229 antibody, was included as an internal control. 37 These findings indicate that the amino acid substitutions do not affect the recognition of epitopes that are remote from the site of mutation and further highlight the overall structural similarity of the point mutants relative to Purβ WT. Assessment of the ssDNA-Binding Activity of Purβ Y/ F Mutants.…”

“…Thermal Shift Assay. The thermostability of purified proteins was assessed by differential scanning fluorimetry as described previously 37,40 Celsius increase in temperature with a total run time of approximately 25 min. Fluorescence intensities (F) were averaged to yield a single value per one-degree Celsius increment.…”

“…Briefly, the interaction of Purβ with various biotinylated ssDNA probes (Table S3) immobilized on streptavidin-coated wells was assessed by an enzyme-linked immunosorbent assay (ELISA). 37,40 The interaction of Purβ with purified mouse Ybx1 45 immobilized on microplate wells was similarly evaluated by ELISA. 37,40 The relative reactivity of several different Purβ antibodies for WT or mutated Purβ adsorbed to microplate wells was also assessed by ELISA.…”

“…37,40 The interaction of Purβ with purified mouse Ybx1 45 immobilized on microplate wells was similarly evaluated by ELISA. 37,40 The relative reactivity of several different Purβ antibodies for WT or mutated Purβ adsorbed to microplate wells was also assessed by ELISA. 37,40 The primary rabbit polyclonal antibodies employed in the various immunoassays recognize distinct epitopes spanning residues 210−229 or 302−324 of mouse Purβ.…”