Bacterially produced human HIF-1alpha is competent for heterodimerization and specific DNA-binding.
Hypoxia-inducible factor 1alpha (HIF-1alpha) is the regulatory subunit of HIF-1, the transcriptional activator and key mediator of the cellular response to hypoxia. Regulation of HIF-1alpha occurs at multiple levels and involves several different post-translational modifications. In order to examine the importance of these modifications for the basic function of HIF-1alpha, we have produced in bacteria recombinant full-length human HIF-1alpha using different expression systems. We show that this unmodified form of HIF-1alpha is able to form a stable heterodimer with the second subunit of HIF-1 (HIF-1beta or ARNT) when both proteins are co-expressed in Escherichia coli. Furthermore, this bacterially reconstituted heterodimer exhibits specific DNA-binding activity. These data indicate that post-translational modification of HIF-1alpha is not essential for its interaction with ARNT and DNA, and provide an in vitro system for the characterization of the molecular properties of HIF-1alpha.
Chachami
Georgia
G
Laboratory of Biochemistry, School of Medicine, University of Thessaly, Papakyriazi 22, 41222 Larissa, Greece.
Paraskeva
Efrosyni
E
Georgatsou
Eleni
E
Bonanou
Sofia
S
Simos
George
G
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C497396:ARNT protein, human; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004247:DNA