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Analysis of proximal ALOX5 promoter binding proteins by quantitative proteomics
The FEBS Journal ( IF 5.5 ) Pub Date : 2020-02-24 , DOI: 10.1111/febs.15259 Katharina Schlag 1 , Dieter Steinhilber 1 , Michael Karas 1 , Bernd L. Sorg 1
The FEBS Journal ( IF 5.5 ) Pub Date : 2020-02-24 , DOI: 10.1111/febs.15259 Katharina Schlag 1 , Dieter Steinhilber 1 , Michael Karas 1 , Bernd L. Sorg 1
Affiliation
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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.
中文翻译：
定量蛋白质组学分析近端ALOX5启动子结合蛋白
5-Lipoxygenase（5-LO）是白三烯生物合成中的初始酶，白三烯是参与诸如哮喘和某些癌症类型等病理生理状况的介质。需要了解与5-LO途径调控有关的蛋白质（包括基因调控蛋白质）的知识，以评估针对这些疾病进行治疗性干预的所有选择。在这里，我们介绍了通过DNA下拉和无标记定量质谱技术获得的ALOX5启动子相互作用蛋白的质谱筛选。筛选了来自髓样和B淋巴细胞细胞系的蛋白质制备物中的启动子DNA相互作用子。通过统计分析，鉴定出66种蛋白为特异性ALOX5启动子结合蛋白。其中，最有可能在ALOX5基因调控中发挥重要作用的15个候选基因是已知的ALOX5相互作用子Sp1和Sp3，相关因子Sp2，两个Krüppel样因子（KLF13和KLF16）和其他六个锌指蛋白（MAZ，PRDM10） ，VEZF1，ZBTB7A，ZNF281和ZNF579）。有趣的是，我们还鉴定了两种解旋酶（BLM和DHX36）以及hnRNPD和hnRNPK蛋白，以及MAZ蛋白与DNA G-四链体结构相互作用。由于G四联体参与基因调控，因此使用了光谱学和基于抗体的方法来确认它们在ALOX5启动子的富含GC的序列中的存在。总之，我们已经系统地表征了ALOX5启动子，鉴定了几种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性作用。
更新日期：2020-02-24
中文翻译：
定量蛋白质组学分析近端ALOX5启动子结合蛋白
5-Lipoxygenase（5-LO）是白三烯生物合成中的初始酶，白三烯是参与诸如哮喘和某些癌症类型等病理生理状况的介质。需要了解与5-LO途径调控有关的蛋白质（包括基因调控蛋白质）的知识，以评估针对这些疾病进行治疗性干预的所有选择。在这里，我们介绍了通过DNA下拉和无标记定量质谱技术获得的ALOX5启动子相互作用蛋白的质谱筛选。筛选了来自髓样和B淋巴细胞细胞系的蛋白质制备物中的启动子DNA相互作用子。通过统计分析，鉴定出66种蛋白为特异性ALOX5启动子结合蛋白。其中，最有可能在ALOX5基因调控中发挥重要作用的15个候选基因是已知的ALOX5相互作用子Sp1和Sp3，相关因子Sp2，两个Krüppel样因子（KLF13和KLF16）和其他六个锌指蛋白（MAZ，PRDM10） ，VEZF1，ZBTB7A，ZNF281和ZNF579）。有趣的是，我们还鉴定了两种解旋酶（BLM和DHX36）以及hnRNPD和hnRNPK蛋白，以及MAZ蛋白与DNA G-四链体结构相互作用。由于G四联体参与基因调控，因此使用了光谱学和基于抗体的方法来确认它们在ALOX5启动子的富含GC的序列中的存在。总之，我们已经系统地表征了ALOX5启动子，鉴定了几种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性作用。
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