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rdf:type |
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lifeskim:mentions |
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pubmed:dateCreated |
2007-11-13
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pubmed:abstractText |
The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:issn |
0076-6879
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
435
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
43-60
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pubmed:meshHeading |
pubmed-meshheading:17998048-Animals,
pubmed-meshheading:17998048-Aryl Hydrocarbon Receptor Nuclear Translocator,
pubmed-meshheading:17998048-Chromatography, Thin Layer,
pubmed-meshheading:17998048-Decarboxylation,
pubmed-meshheading:17998048-Glutarates,
pubmed-meshheading:17998048-Humans,
pubmed-meshheading:17998048-Hypoxia-Inducible Factor 1, alpha Subunit,
pubmed-meshheading:17998048-Oxidation-Reduction,
pubmed-meshheading:17998048-Oxygen,
pubmed-meshheading:17998048-Peptides,
pubmed-meshheading:17998048-Procollagen-Proline Dioxygenase,
pubmed-meshheading:17998048-Protein Structure, Tertiary,
pubmed-meshheading:17998048-Recombinant Proteins,
pubmed-meshheading:17998048-Tissue Extracts
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pubmed:year |
2007
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pubmed:articleTitle |
Determination and modulation of prolyl-4-hydroxylase domain oxygen sensor activity.
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pubmed:affiliation |
Institute of Physiology and Zürich Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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