Proteomics-based Identification of Novel Factor Inhibiting Hypoxia-inducible Factor (FIH) Substrates Indicates Widespread Asparaginyl Hydroxylation of Ankyrin Repeat Domain-containing Proteins

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 IB 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 ϳ300 ARD-containing species in the human proteome. Following the identification of the HIF hydroxylases, searches for alternative (non-HIF) substrates of these enzymes have identified certain IB and Notch family members and ASB4 (ankyrin repeat and SOCS box protein 4) as substrates of FIH (3-6). These intracellular proteins all contain ARDs, and in each case the target asparagine residues lie within the ARD. The ARD is one of the most common amino acid motifs in nature; it is present in over 300 proteins in the human genome (SMART (simple modular architecture research tool) database (7)) and conserved in all kingdoms of life (for a review, see Ref. 8). ARDs are composed of a variable number of 33-residue repeats that individually fold into paired antiparallel ␣-helices linked by a ␤-hairpin type turn. The hydroxylated asparagine residue is positioned within the hairpin loop that links individual repeats.These findings suggest that asparaginyl hydroxylation might be much more prevalent in intracellular proteins than has been appreciated previously, particularly among ARDFrom the §Central Proteomics Facility,

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

“…The false positive rate (FPR) of the algorithm for identification was set to 4% with a randomized database, appended to the original one. Only those proteins with 50% or more probability to be present in the mixture and detected with a score above 20, as calculated by the software, were selected for proteomic analysis (29). Data sets were normalized using the "autonormalization" function of PLGS, and label-free quantitative analysis was performed by comparing the normalized peak area/intensity of the peptides identified.…”

microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation

“…Significantly, proteins containing the ankyrin repeat domain (ARD) such as Notch are other substrates for FIH-1 (8). Moreover, the binding affinity of FIH-1 for Notch 1 is appreciably greater than for HIF-1α (8,10). With respect to Notch 1, FIH-1 hydroxylates the Notch ICD at two asparagine residues (N 1945 and N 2012 ) (11).…”

“…The asparaginyl hydroxylase factor inhibiting HIF-1α (FIH-1) can negatively regulate Notch signaling (8). FIH-1 was originally identified as a protein that interacts with and inhibits the activity of HIF-1α in the C-terminal transactivation domain (CAD) (9) by coupling the oxidative decarboxylation of 2-oxoglutarate to the hydroxylation of HIF-1α (8).…”

“…FIH-1 was originally identified as a protein that interacts with and inhibits the activity of HIF-1α in the C-terminal transactivation domain (CAD) (9) by coupling the oxidative decarboxylation of 2-oxoglutarate to the hydroxylation of HIF-1α (8). Significantly, proteins containing the ankyrin repeat domain (ARD) such as Notch are other substrates for FIH-1 (8). Moreover, the binding affinity of FIH-1 for Notch 1 is appreciably greater than for HIF-1α (8,10).…”

HIF Prolyl-4-hydroxylase Interacting Proteins: Consequences for Drug Targeting

“…While prolyl hydroxylation data are missing in this study, other factors of the NF-B signalling pathway (I B , I B and p105) have been shown to be efficiently hydroxylated by FIH [94,95]. However, no functional alterations in the NF-B pathway could be identified following asparaginyl hydroxylation.…”