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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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