Remodeling Ca2+ dynamics by targeting a promising E-box containing G-quadruplex at ORAI1 promoter in triple-negative breast cancer

IF 4.3 2区 生物学 Q2 CELL BIOLOGY
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Abstract

ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca2+ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca2+ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca2+ imaging.

Abstract Image

针对三阴性乳腺癌 ORAI1 启动子中前景看好的含 E-box 的 G- 四叉链,重塑 Ca2+ 动力学
ORAI1 是贮存操作钙离子通道(SOCE)的内在组成部分,严格调节大多数非兴奋细胞中的 Ca2+ 流入。ORAI1 在多种癌症中过表达,其信号转导与化疗耐药性有关。ORAI1 与其他通道和效应器存在广泛的蛋白质组相互作用,从而导致各种表型的改变。然而,人们对 ORAI1 的转录调控还不甚了解。我们在该基因的上游启动子区域发现了一个具有调控功能的推定 G-四叠体(G4)基团 ORAI1-Pu。高分辨率三维核磁共振结构分析表明,ORAI1-Pu 是一个稳定的平行链 G4,它有一个长 8-nt 的环,在不影响结构稳定性的情况下赋予其动态性。突出的环还包含一个 E-box 基序,为 Zeb1 等转录因子提供了一个对接位点。在过表达 ORAI1 的 MDA-MB-231 细胞系中,使用抗 G4 抗体(BG4)进行染色质免疫沉淀(ChIP),也能观察到 G4 结构。配体介导的稳定化表明,稳定化的 G4 可抑制癌细胞株 MDA-MB-231 中的转录。通过活细胞 Fura-2 Ca2+ 成像观察到,转录的下调进一步导致通过 SOCE 途径进入的 Ca2+ 减少。
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来源期刊
Cell calcium
Cell calcium 生物-细胞生物学
CiteScore
8.70
自引率
5.00%
发文量
115
审稿时长
35 days
期刊介绍: Cell Calcium covers the field of calcium metabolism and signalling in living systems, from aspects including inorganic chemistry, physiology, molecular biology and pathology. Topic themes include: Roles of calcium in regulating cellular events such as apoptosis, necrosis and organelle remodelling Influence of calcium regulation in affecting health and disease outcomes
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