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Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.
- Source :
-
Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2009 Mar; Vol. 8 (3), pp. 535-46. Date of Electronic Publication: 2008 Oct 20. - Publication Year :
- 2009
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Abstract
- Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IkappaB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological "substrate trapping" together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the approximately 300 ARD-containing species in the human proteome.
- Subjects :
- Amino Acid Sequence
Amino Acids, Dicarboxylic pharmacology
Cell Line, Tumor
Endoribonucleases chemistry
Endoribonucleases metabolism
Humans
Hydroxylation drug effects
Immunoblotting
Mass Spectrometry
Mixed Function Oxygenases
Molecular Sequence Data
Protein Binding drug effects
Repressor Proteins chemistry
Reproducibility of Results
Substrate Specificity drug effects
Tankyrases chemistry
Tankyrases metabolism
Ankyrin Repeat
Asparagine metabolism
Proteomics methods
Repressor Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1535-9484
- Volume :
- 8
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Molecular & cellular proteomics : MCP
- Publication Type :
- Academic Journal
- Accession number :
- 18936059
- Full Text :
- https://doi.org/10.1074/mcp.M800340-MCP200