A cell-penetrating peptide suppresses the hypoxia inducible factor-1 function by binding to the helix-loop-helix domain of the aryl hydrocarbon receptor nuclear translocator.
A cell-penetrating peptide suppresses the hypoxia inducible factor-1 function by binding to the helix-loop-helix domain of the aryl hydrocarbon receptor nuclear translocator.
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DOI：
10.1016/j.cbi.2013.02.003
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发表时间：
2013-04-25
影响因子：
5.1
通讯作者：
Chan, William K.
中科院分区：
文献类型：
--
作者：
Wang, Yu;Thompson, John D.;Chan, William K.
异二聚体低氧诱导因子-1 （HIF-1）复合物由低氧诱导因子-1α (HIF-1α)和芳烃受体核转运子（ARNT）组成。HIF-1功能的激活对肿瘤的生长和转移至关重要。我们之前的研究表明，转染含有arnt相互作用肽（Ainp1） cDNA的质粒可抑制Hep3B细胞中的HIF-1信号传导。在这里，我们生成了Ainp1肽（6His-TAT-Ainp1）的TAT融合，以确定Ainp1肽是否以及如何抑制HIF-1功能。细菌表达的6His-TAT-Ainp1在变性条件下纯化，然后通过有限的透析重新折叠。重新折叠的6His-TAT-Ainp1以与原生6His-Ainp1相似的方式与ARNT的螺旋-环-螺旋（HLH）结构域相互作用。his - tat - ainp1在HeLa和Hep3B细胞蛋白转导后与ARNT共定位。转导的蛋白在2小时内达到细胞内的最高水平，而在HeLa细胞中可检测到长达96小时。在2µM浓度下，6his - tata - ainp1在HeLa细胞中没有细胞毒性，但以剂量依赖的方式抑制氯化钴激活的、缺氧反应增强剂驱动的荧光素酶的表达。此外，它还降低了HIF-1靶基因在信息（血管内皮生长因子和醛缩酶C）和蛋白质（碳酸酐酶IX和葡萄糖转运蛋白1）水平上的氯化钴依赖性诱导。HIF-1α和ARNT的蛋白水平在6His-TAT-Ainp1的存在下没有改变。综上所述，我们提供的证据支持Ainp1肽通过与ARNT HLH结构域相互作用直接抑制HIF-1功能，进而干扰HIF-1α和ARNT的异源二聚化。
The heterodimeric hypoxia inducible factor-1 (HIF-1) complex is composed of the hypoxia inducible factor-1 alpha (HIF-1α) and the aryl hydrocarbon receptor nuclear translocator (ARNT). Activation of the HIF-1 function is essential for tumor growth and metastasis. We previously showed that transfection of a plasmid containing an ARNT-interacting peptide (Ainp1) cDNA suppresses the HIF-1 signaling in Hep3B cells. Here we generated TAT fusion of the Ainp1 peptide (6His-TAT-Ainp1) to determine whether and how the Ainp1 peptide suppresses the HIF-1 function. The bacterially expressed 6His-TAT-Ainp1 was purified under denatured condition and then refolded by limited dialysis. The refolded 6His-TAT-Ainp1 interacts with the helix-loop-helix (HLH) domain of ARNT in a similar fashion as the native 6His-Ainp1. 6His-TAT-Ainp1 colocalizes with ARNT in the nucleus of HeLa and Hep3B cells after protein transduction. The transduced protein reaches the maximum intracellular levels within 2 h while remains detectable up to 96 h in HeLa cells. At 2 µM concentration, 6His-TAT-Ainp1 is not cytotoxic in HeLa cells but suppresses the cobalt chloride-activated, hypoxia responsive enhancer-driven luciferase expression in a dose-dependent manner. In addition, it decreases the cobalt chloride-dependent induction of the HIF-1 target genes at both the message (vascular endothelial growth factor and aldolase C) and protein (carbonic anhydrase IX and glucose transporter 1) levels. The protein levels of HIF-1α and ARNT are not altered in the presence of 6His-TAT-Ainp1. In summary, we provided evidence to support that the Ainp1 peptide directly suppresses the HIF-1 function by interacting with the ARNT HLH domain, and in turn interfering with the heterodimerization of HIF-1α and ARNT.
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