Zhang Nannan, Guan Fangxia, Zhang Yanting, Li Qinghua, Xu Yao, Yang Lu, Zhu Xiangzhan, Zhang Mingzhi
Laboratory for Cell Biology, The First Affiliated Hospital, Zhengzhou University, 40 Daxue Road, Zhengzhou, 450052, China.
School of Life Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, 450001, China.
Folia Microbiol (Praha). 2017 Jan;62(1):57-62. doi: 10.1007/s12223-016-0472-9. Epub 2016 Sep 22.
Ring box protein-1 (RBX1), also called Regulator of Cullins-1 (ROC1), is a key component of SCF (Skp-1, cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting protein substrates for degradation. Although RBX1 plays an important role in ubiquitination machinery of both prokaryotes and eukaryotes, studies on the RBX1 have not been involved in the unicellular green alga Dunaliella salina. In this study, a full-length RBX1 cDNA fragment of 817 bp was cloned using rapid amplification of cDNA end (RACE) technique. The full-length sequence contained an open reading frame of 411 bp encoding 136 amino acids. The predicted protein had a molecular molar mass of 14.8 kDa and pI of 5.9 with a high degree of homology to RBX1 from Chlamydomonas reinhardtii (92 %). Recombinant RBX1 was expressed in Escherichia coli BL21 and was purified and characterized. The apparent molecular mass of the recombinant protein was approximately 17 kDa, and the optimal induction time and concentration were 3 h and 0.1 mmol/L IPTG, respectively. The predicted 3D structures of RBX1 proteins contained RING-H2 finger domain including "Cys59-X2-Cys62-X30-Cys93-X1-His95-X2-His98-X2-Cys101-X10-Cys112-X2-Cys115." The expression of RBX1 protein was increased by 132 % during flagellar disassembly and decreased by 76 % during flagellar assembly of D. salina. The expression of RBX1 mRNA had a similar tendency with the expression of RBX1 protein. The results indicated that RBX1 responded to flagellar disassembly of D. salina.
环形盒蛋白1（RBX1），也称为Cullin-1调节因子（ROC1），是SCF（Skp-1、Cullin、F-box蛋白）E3泛素连接酶的关键组成部分，该酶通过靶向蛋白质底物进行降解来调节多种细胞过程。尽管RBX1在原核生物和真核生物的泛素化机制中都发挥着重要作用，但关于RBX1的研究尚未涉及单细胞绿藻盐生杜氏藻。在本研究中，使用cDNA末端快速扩增（RACE）技术克隆了一个817 bp的全长RBX1 cDNA片段。全长序列包含一个411 bp的开放阅读框，编码136个氨基酸。预测的蛋白质分子量为14.8 kDa，pI为5.9，与莱茵衣藻的RBX1具有高度同源性（92%）。重组RBX1在大肠杆菌BL21中表达，并进行了纯化和表征。重组蛋白的表观分子量约为17 kDa，最佳诱导时间和浓度分别为3 h和0.1 mmol/L IPTG。RBX1蛋白的预测三维结构包含RING-H2指结构域，包括“Cys59-X2-Cys62-X30-Cys93-X1-His95-X2-His98-X2-Cys101-X10-Cys112-X2-Cys115”。在盐生杜氏藻鞭毛拆卸过程中，RBX1蛋白的表达增加了132%，而在鞭毛组装过程中减少了76%。RBX1 mRNA的表达与RBX1蛋白的表达具有相似的趋势。结果表明，RBX1对盐生杜氏藻的鞭毛拆卸有反应。
