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.

Abstract: 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 2h while remains detectable up to 96h in HeLa cells. At 2μM 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. [Copyright &y& Elsevier]