Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Biochemistry. 2013 Jul 2;52(26):4439-50. doi: 10.1021/bi400283r. Epub 2013 Jun 20.
A hallmark of dysfunctional fibroblast to myofibroblast differentiation associated with fibrotic disorders is persistent expression of ACTA2, the gene encoding the cyto-contractile protein smooth muscle α-actin. In this study, a PURB-specific gene knockdown approach was used in conjunction with biochemical analyses of protein subdomain structure and function to reveal the mechanism by which purine-rich element binding protein B (Purβ) restricts ACTA2 expression in mouse embryo fibroblasts (MEFs). Consistent with the hypothesized role of Purβ as a suppressor of myofibroblast differentiation, stable short hairpin RNA-mediated knockdown of Purβ in cultured MEFs promoted changes in cell morphology, actin isoform expression, and cell migration indicative of conversion to a myofibroblast-like phenotype. Promoter-reporter assays in transfected Purβ knockdown MEFs confirmed that these changes were attributable, in part, to derepression of ACTA2 transcription. To map the domains in Purβ responsible for ACTA2 repression, several recombinant truncation mutants were generated and analyzed based on hypothetical, computationally derived models of the tertiary and quaternary structure of Purβ. Discrete subdomains mediating sequence- and strand-specific cis-element binding, protein-protein interaction, and inhibition of a composite ACTA2 enhancer were identified using a combination of biochemical, biophysical, and cell-based assays. Our results indicate that the Purβ homodimer possesses three separate but unequal single-stranded DNA-binding modules formed by subdomain-specific inter- and intramolecular interactions. This structural arrangement suggests that the cooperative assembly of the dimeric Purβ repressor on the sense strand of the ACTA2 enhancer is dictated by the association of each subdomain with distinct purine-rich binding sites within the enhancer.
与纤维化疾病相关的功能失调的成纤维细胞向肌成纤维细胞分化的一个标志是 ACTA2 的持续表达，ACTA2 基因编码细胞收缩蛋白平滑肌α-肌动蛋白。在这项研究中，使用 PURB 特异性基因敲低方法结合生化分析蛋白质亚结构域的结构和功能，揭示嘌呤丰富元件结合蛋白 B (Purβ) 限制小鼠胚胎成纤维细胞 (MEFs) 中 ACTA2 表达的机制。与 Purβ 作为肌成纤维细胞分化抑制因子的假设作用一致，稳定的短发夹 RNA 介导的 Purβ 在培养的 MEFs 中的敲低促进了细胞形态、肌动蛋白同工型表达和细胞迁移的变化，表明向肌成纤维细胞样表型的转化。转染 Purβ 敲低 MEFs 的启动子报告基因分析证实，这些变化部分归因于 ACTA2 转录的去抑制。为了绘制 Purβ 中负责 ACTA2 抑制的结构域，根据 Purβ 三级和四级结构的假设计算模型生成并分析了几个重组截断突变体。使用生化、生物物理和基于细胞的测定的组合，鉴定了介导序列和链特异性顺式元件结合、蛋白质-蛋白质相互作用以及抑制复合 ACTA2 增强子的离散亚域。我们的结果表明，Purβ 同源二聚体具有三个单独但不等同的单链 DNA 结合模块，由亚域特异性的分子间和分子内相互作用形成。这种结构排列表明，二聚体 Purβ 抑制剂在 ACTA2 增强子的有义链上的协同组装是由每个亚域与增强子内独特的嘌呤丰富结合位点的关联决定的。
