Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
PLoS One. 2012;7(1):e30588. doi: 10.1371/journal.pone.0030588. Epub 2012 Jan 24.
ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1-8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes.
Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB(-) mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment.
Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis.
ALKBH 蛋白是大肠杆菌 AlkB 双氧酶的同源物，构成了一个直接的、单一蛋白修复系统，保护细胞 DNA 和 RNA 免受烷化剂的细胞毒性和诱变活性的影响，烷化剂是导致肿瘤形成的重要化学物质，并用于癌症治疗。计算机分析和体内研究表明，几乎所有生物体中都存在 AlkB 同源物。人类已鉴定出 9 种 AlkB 同源物（ALKBH1-8 和 FTO）。研究发现高 ALKBH 水平会促进肿瘤的发展，这对烷化剂在化疗中的应用提出了质疑。本工作的目的是通过研究其在原核生物核酸修复中的活性及其在真核生物中的亚细胞定位，赋予多种 AlkB 同源物生物学意义。
通过对蛋白质序列数据库的生物信息学分析，鉴定出 1943 个 AlkB 序列，其中包含 8 个新的 AlkB 亚家族。由于蓝藻和拟南芥含有多个 AlkB 同源物，因此它们被选为体内研究的模式生物。我们使用大肠杆菌 alkB(-)突变体和表达蓝藻 AlkB 的质粒，研究了甲基甲磺酸（MMS）和氯乙醛（CAA）诱导的 ssDNA、ssRNA 和基因组 DNA 损伤的修复。基于 GFP 融合，我们研究了 ALKBHs 在拟南芥中的亚细胞定位，并确定其主要核质分布。发现一些 ALKBH 蛋白在 MMS 处理后发生了定位改变。
我们的体内研究表明，蓝藻 AlkB 蛋白对损伤和核酸类型具有高度特异性的活性。ALKBHs 在拟南芥中的亚细胞定位和易位表明，这些蛋白可能参与了烷化损伤的修复。我们假设 ALKBH 蛋白的多样性是由于它们参与核蛋白复合物的代谢；我们发现 ALKBH 蛋白对这些损伤的修复是一种经济有效的替代降解和从头合成的方法。
