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https://hdl.handle.net/2440/47857
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dc.contributor.author | Karttunen, S. | - |
dc.contributor.author | Hampton-Smith, R. | - |
dc.contributor.author | Peet, D. | - |
dc.contributor.editor | Sies, H. | - |
dc.contributor.editor | Brune, B. | - |
dc.date.issued | 2007 | - |
dc.identifier.citation | Methods in Enzymology, 2007; 435:61-85 | - |
dc.identifier.issn | 0076-6879 | - |
dc.identifier.issn | 1557-7988 | - |
dc.identifier.uri | http://hdl.handle.net/2440/47857 | - |
dc.description | Available online 12 November 2007. | - |
dc.description.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. | - |
dc.description.statementofresponsibility | Sarah Linke, Rachel J. Hampton‐Smith and Daniel J. Peet | - |
dc.description.uri | http://www.sciencedirect.com/science/bookseries/00766879 | - |
dc.language.iso | en | - |
dc.publisher | Elsevier Academic Press Inc | - |
dc.rights | Copyright © 2007 Elsevier Inc. All rights reserved. | - |
dc.source.uri | http://dx.doi.org/10.1016/s0076-6879(07)35004-0 | - |
dc.subject | Animals | - |
dc.subject | Humans | - |
dc.subject | Escherichia coli | - |
dc.subject | Carbon Dioxide | - |
dc.subject | Mixed Function Oxygenases | - |
dc.subject | Proteins | - |
dc.subject | Recombinant Proteins | - |
dc.subject | Transcription Factors | - |
dc.subject | Repressor Proteins | - |
dc.subject | Immunoprecipitation | - |
dc.subject | Ankyrin Repeat | - |
dc.subject | Substrate Specificity | - |
dc.title | Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor | - |
dc.type | Journal article | - |
dc.contributor.organisation | Centre for the Molecular Genetics of Development | - |
dc.identifier.doi | 10.1016/S0076-6879(07)35004-0 | - |
pubs.publication-status | Published | - |
dc.identifier.orcid | Peet, D. [0000-0002-6085-8936] | - |
Appears in Collections: | Aurora harvest 6 Centre for the Molecular Genetics of Development publications |
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