Jules Stein Eye Institute and Department of Ophthalmology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
PLoS One. 2012;7(5):e35317. doi: 10.1371/journal.pone.0035317. Epub 2012 May 31.
Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4's secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts.
Zbed4 是锌指蛋白 BED 亚家族的成员，在人视网膜的视锥细胞和神经胶质 Müller 细胞中表达，而在鼠视网膜中仅存在于 Müller 细胞中。为了表征 Zbed4 的结构和功能特性，需要足够量的纯化蛋白。因此，重组 Zbed4 在大肠杆菌中表达，并优化其复性条件，以生产同质且具有功能活性的蛋白。通过圆二色性光谱学确定的 Zbed4 的二级结构表明，该蛋白包含 32%的α-螺旋、18%的β-折叠、20%的转角和 30%的无规卷曲结构。CASTing 用于鉴定 Zbed4 在 DNA 中的靶位。获得的大多数 DNA 片段含有聚-Gs，其中一些片段除了具有 GC 盒的核心特征外，还有一些仅含有 GC 盒。通过电泳迁移率变动分析，我们证明 Zbed4 不仅与 DNA 而且与 RNA 寡核苷酸具有非常高的亲和力结合，与具有至少 5 个 Gs 的聚-G 区相互作用；其与 GC 盒的结合取决于 GC 盒侧翼核苷酸。我们还发现 Zbed4 在 Y79 视网膜母细胞瘤细胞中与核蛋白和细胞质蛋白 Scaffold Attachment Factor B1（SAFB1）、雌激素受体 alpha（ERα）和细胞肌球蛋白 9（MYH9）相互作用，如免疫沉淀和质谱研究以及凝胶覆盖分析所示。此外，免疫染色证实了 Zbed4 与这些蛋白的共定位。最重要的是，使用包含指导荧光素酶基因表达的启动子的构建体进行的体外实验表明，Zbed4 可转录激活具有聚-G 区的这些启动子的转录。
