Institute of Pharmaceutical Chemistry/ZAFES, Goethe-University, Frankfurt am Main, Germany.
FEBS J. 2020 Oct;287(20):4481-4499. doi: 10.1111/febs.15259. Epub 2020 Mar 13.
5-Lipoxygenase (5-LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5-LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter-interacting proteins, obtained by DNA pulldown and label-free quantitative mass spectrometry. Protein preparations from myeloid and B-lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel-like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G-quadruplex structures. As G-quadruplexes are implicated in gene regulation, spectroscopic and antibody-based methods were used to confirm their presence within the GC-rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G-quadruplex structures, which may play a functional role in ALOX5 gene regulation.
5-脂氧合酶（5-LO）是白三烯生物合成中的初始酶，白三烯是参与哮喘和某些癌症类型等病理生理状况的介质。为了评估这些疾病中治疗干预的所有选择，需要了解参与 5-LO 途径调节的蛋白质，包括基因调节蛋白。在这里，我们通过 DNA 下拉和无标记定量质谱法进行了 5-LO 启动子相互作用蛋白的质谱筛选。从髓样和 B 淋巴细胞系的蛋白质制剂中筛选启动子 DNA 相互作用蛋白。通过统计分析，鉴定出 66 种特定 ALOX5 启动子结合蛋白作为 ALOX5 启动子结合蛋白。其中，已知的 ALOX5 相互作用蛋白 Sp1 和 Sp3、相关因子 Sp2、两个 Krüppel 样因子（KLF13 和 KLF16）和另外六个锌指蛋白（MAZ、PRDM10、VEZF1、ZBTB7A、ZNF281 和 ZNF579）是最有可能在 ALOX5 基因调控中发挥重要作用的 15 个候选蛋白。有趣的是，我们还鉴定出两种解旋酶（BLM 和 DHX36）以及 hnRNPD 和 hnRNPK 蛋白，它们与蛋白 MAZ 一起，已知与 DNA G-四链体结构相互作用。由于 G-四链体结构参与基因调控，因此使用光谱和抗体方法来确认它们在 ALOX5 启动子的 GC 丰富序列内的存在。总之，我们系统地描述了 ALOX5 启动子的相互作用组，鉴定出几个锌指蛋白作为新型潜在的 ALOX5 基因调节剂。此外，我们已经表明，ALOX5 启动子可以形成 DNA G-四链体结构，这可能在 ALOX5 基因调控中发挥功能作用。
