title: 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 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.
authors: Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors: Yu
authors: Y
authors: Wang
authors: John D
authors: JD
authors: Thompson
authors: William K
authors: WK
authors: Chan
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000595:Amino Acid Sequence; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D003035:Cobalt / Q000494:pharmacology; D015696:Gene Products, tat / Q000235:genetics; D006367:HeLa Cells; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D011485:Protein Binding; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D057846:Cell-Penetrating Peptides; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D003035:Cobalt; C018021:cobaltous chloride