Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/47857
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Type: Journal article
Title: Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor
Author: Karttunen, S.
Hampton-Smith, R.
Peet, D.
Citation: Methods in Enzymology, 2007; 435:61-85
Publisher: Elsevier Academic Press Inc
Issue Date: 2007
ISSN: 0076-6879
1557-7988
Editor: Sies, H.
Brune, B.
Organisation: Centre for the Molecular Genetics of Development
Statement of
Responsibility: 
Sarah Linke, Rachel J. Hampton‐Smith and Daniel J. Peet
Abstract: The hypoxia-inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. The HIFs are regulated by four oxygen-sensitive hydroxylases-three prolyl hydroxylases and one asparaginyl hydroxylase. These hydroxylases are all members of the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily and convey changes in cellular oxygen concentration to the HIF-alpha (alpha) subunit, leading to potent accumulation and activity in hypoxia versus degradation and repression in normoxia. HIF-alpha asparaginyl hydroxylation is catalyzed by factor-inhibiting HIF-1 (FIH-1) and directly regulates the transcription activity of the HIF-alpha proteins. Recent work has demonstrated that, in addition to hydroxylating HIF-alpha, FIH-1 can also hydroxylate the ankyrin domains of a wide range of proteins. This paper presents in vitro and cell-based techniques for the preliminary characterization of ankyrin domain-containing proteins as FIH-1 substrates and interacting proteins. Strategies are presented for the expression and purification of FIH-1 from mammalian or bacterial cells. Similar to the HIF-alpha proteins, the ankyrin-containing substrates are examined as purified proteins expressed in bacteria and overexpressed in mammalian cells or in the form of synthetic peptides. Specific conditions for the efficient expression of ankyrin-containing proteins compared with the HIF-alpha substrates in Escherichia coli are detailed. Hydroxylation is rapidly inferred, utilizing the described in vitro CO(2) capture assay. Finally, substrate and non-substrate interactions are examined using in vitro affinity pull-down assays and mammalian cell-based co-immunoprecipitation assays. Together, these methods are rapid and well suited to the preliminary characterization of potential substrates of the therapeutically relevant oxygen-sensing enzyme FIH-1.
Keywords: Animals
Humans
Escherichia coli
Carbon Dioxide
Mixed Function Oxygenases
Proteins
Recombinant Proteins
Transcription Factors
Repressor Proteins
Immunoprecipitation
Ankyrin Repeat
Substrate Specificity
Description: Available online 12 November 2007.
Rights: Copyright © 2007 Elsevier Inc. All rights reserved.
DOI: 10.1016/S0076-6879(07)35004-0
Description (link): http://www.sciencedirect.com/science/bookseries/00766879
Published version: http://dx.doi.org/10.1016/s0076-6879(07)35004-0
Appears in Collections:Aurora harvest 6
Centre for the Molecular Genetics of Development publications

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