Affinity purification allied to mass spectrometry is a powerful method that has been successfully applied to the characterization of discrete protein-protein interactions and large interaction networks (20
). Despite the high sensitivity of MS, a limitation of affinity purification in protein discovery is in the identification of transient interactions exemplified by those between an enzyme and substrate. In the present study we describe a proteomics approach that has led to the identification of several novel substrates of the asparaginyl hydroxylase FIH. A key component in the success of our strategy was the use of DMOG as a substrate-trapping agent.
Combining the substrate trapping methodology with SILAC and in-solution digestion provided the most efficient method for identifying FIH substrates. From a single AP experiment we were able to identify 10 ARD substrates that were all binding in a DMOG-inducible manner. Although this approach was more successful than gel-based methods in terms of identifying substrates from a single experiment, a further refinement could be beneficial. Because we digested the immunopurified material in solution, the most abundant peptides were derived from the FIH “bait.” In fact over 160 FIH tryptic peptides were identified by MS/MS. It is conceivable that a large excess of bait peptides could have masked less abundant peptides derived from additional substrates. Prefractionation strategies may thus enhance the detection of substrate-derived peptides. The utility of combining SILAC with substrate trapping by DMOG is not restricted to FIH because the agent inhibits all 2OG oxygenases for which it has been tested (21
), and in other experiments we have found that DMOG greatly stabilizes interactions between HIF-α subunits and the HIF prolyl hydroxylase enzymes.2
Thus the methodology we describe might be used to reveal substrates for other enzymes among more than 60 known and predicted 2OG-dependent oxygenases encoded in the human genome (22
At present the mechanism by which DMOG promotes interaction between FIH and protein substrates is not entirely clear. It is possible that the effect is kinetic, i.e.
inhibition of catalysis prolongs otherwise transient interactions between enzyme and substrate. Alternatively because the affinity of FIH for hydroxylated ARD proteins is much lower than that for unhydroxylated ARD proteins it is possible that increased interaction reflects the accumulation of unhydroxylated species that bind FIH more tightly (4
). It is also interesting that our data and those of others reveal variation in enhancement of FIH-substrate interactions by DMOG (compare Tankyrase and RNase L; and Ref. 10
) raising the possibility that there are additional FIH substrates that do not behave in this way and might be detected in the absence of DMOG pretreatment.
Another experimental approach that facilitated the discovery of novel asparaginyl hydroxylation sites in FIH substrates was the use of a recently developed MS data acquisition method (MSE
allows the collection of 5–10 times more precursor ions and fragmentation data when compared with data-directed acquisition modes because of a sequential low and high collision energy data acquisition cycle, resulting in significantly higher protein sequence coverage (). Data collection in low collision energy mode can be used for the quantification of peak ion intensities, and data collected in high collision mode provide fragmentation information that can be used for protein identification, both of which can be obtained from a single chromatography run. Allied to the data-directed MS/MS analysis, which generates optimized MS/MS spectra for precise assignment of post-translational modifications, the two MS/MS modes have provided complementary approaches toward identifying and characterizing novel FIH substrates.
Prior to this study a limited repertoire of ARD-containing proteins had been assigned as in vivo
substrates of FIH, namely p105 and IκBα (3
), Notch1 (4
), and ASB4 (5
). This work has identified Rabankyrin-5, RNase L, and Tankyrase-2 as novel ARD substrates and a further eight proteins as presumed substrates. Together with the definition of an FIH recognition consensus that conforms to that of the ankyrin repeat (23
), these data strongly suggest that FIH-catalyzed intracellular asparaginyl hydroxylation is a common post-translational modification that likely extends to many of the ~300 ARD-containing proteins encoded by the human genome (8
). FIH-dependent hydroxylation may also extend to other proteins. Notably we have identified several non-ARD-containing proteins as binding to FIH in a DMOG-inducible manner,2
but as yet it is unclear whether these species bind FIH directly or as part of a ternary complex with ARD-containing proteins.
Alignment of the 13 newly assigned asparagine residues hydroxylated by FIH with established sites of hydroxylation reveals a largely degenerate FIH consensus motif with only the target asparagine showing absolute conservation (). Notably the acidic residue (glutamate/aspartate) at the −2 position, which was conserved in previously assigned substrates, is not present in three of the four sites that were readily hydroxylated in Tankyrase-2 and is, therefore, clearly not an absolute requirement for FIH activity. However, significant conservation was observed at distinct positions, namely −8 (leucine), −3 (alanine), and −1 (valine) positions (see for logo representation).
Fig. 6. Distribution of amino acids surrounding all known sites of FIH-dependent asparaginyl hydroxylation. A, ClustalW non-gapped multiple sequence alignment of all known FIH-mediated hydroxylation sites. Novel sites identified in Tankyrase-2, RNase L, and Rabankyrin-5 (more ...)
FIH has been crystallized with Notch1 peptide substrates, providing an insight into how AR substrates bind FIH (4
). Very few side-chain interactions were observed between Notch and FIH, compatible with the degenerate FIH consensus. However, a distinct interaction was observed with the leucine −8 residue of Notch1 that was buried in a hydrophobic pocket on the surface of FIH. The importance of this interaction is supported by the current data with leucine at −8 being the most conserved residue outside the target asparagine. Hydroxylation was observed at sites bearing non-conservative substitutions at the −8 position when FIH was overexpressed; for example Asn-518 of Tankyrase-2 contains a lysine residue at the −8 position (supplemental Fig. S3D). This was not the case when ARD-containing proteins were expressed in cells without exogenous FIH, indicating that although the leucine residue is not absolutely required for FIH-dependent catalysis it is likely to be important under physiological conditions.
At present, the precise role of FIH-dependent hydroxylation of ARD-containing proteins is unclear. FIH has been shown to hydroxylate the ARD within the intracellular domain of the Notch receptor (4
) and, in certain circumstances, to antagonize Notch signaling (6
). Notch hydroxylation sites lie within protein domains that are involved in the formation of higher order complexes at paired DNA binding sites (24
), and it has been proposed that hydroxylation might affect assembly of these complexes (6
). FIH has also been shown to hydroxylate the ARD of ASB4; wild type ASB4, but not a hydroxylation site mutant, is able to regulate vascular differentiation (5
). This has led to the proposal that oxygen-regulated vascular differentiation, promoted by ASB4, is regulated by ARD hydroxylation (5
). Nevertheless the complexity of these pathways means that the role of FIH-dependent hydroxylation events has not yet been defined with complete clarity. Given the functional diversity among the FIH-target ARD proteins that our present study has identified, including characterized or predicted roles in endocytosis/macropinocytosis (Rabankyrin-5 (13
)), antiviral immunity (RNase L (25
)), and vesicle trafficking/telomere regulation (Tankyrase-2 (26
)), we believe that a generic signaling role for ARD hydroxylation is unlikely. However, it is possible that the modification is used in signaling by specific ARD proteins. Interestingly hypoxia has been implicated in telomere regulation (27
), and it is possible that hydroxylation of Tankyrase-2 could contribute to this phenomenon.
The best characterized signaling role for FIH-dependent hydroxylation is in regulation of the association between the C terminus of HIF-α subunits and p300/CREB-binding protein co-activators. Previous work in cells has defined cross-competition between HIF-α and Notch receptor ARDs for FIH-mediated Asn hydroxylation, which modulates the HIF transcriptional response (4
). Taken together with the current work, which indicates that cells contain numerous FIH-dependent hydroxylation sites, the data suggest that it is likely to be the hydroxylation status of the ARD pool rather than any one individual ARD protein that provides the effective competition.
In recent work, we have demonstrated that hydroxylation can enhance the stability of certain ARDs, including both natural3
and synthetic ARDs (28
), which are designed around the consensus repeat. Although the biological function of these changes in thermodynamic stability is unclear, it is of interest that mutational studies of the ARD-containing protein IκBα have indicated that precisely tuned stability is important for the proper function of the ARD as a protein-protein interaction domain (29
). It is therefore possible that FIH-dependent hydroxylation serves to fine-tune the stability of the ARD interaction domain.
Our studies demonstrate that FIH-mediated hydroxylation is saturated by enhanced ARD protein expression in transfected cells, leading to partial hydroxylation at several sites. Incomplete hydroxylation has also been demonstrated on the ARDs of endogenous proteins including IκBα and Notch receptors (3
). At present it is unclear whether incomplete hydroxylation of these proteins represents a steady-state level common to individual protein molecules or whether it represents progressive accumulation of hydroxylation at these sites during the lifetime of the protein species. Further work will be required to resolve these possibilities and to address the potential structural and/or signaling roles of FIH-mediated hydroxylation. The clearest and arguably the only biological function of FIH-mediated hydroxylation defined to date is in the regulation of HIF.
Nevertheless the current analyses together with recently published work on endogenous ARD proteins indicate that many or even most ARD proteins are likely to be hydroxylated by FIH in vivo
). Given the abundance of ARD proteins in the proteome, the findings raise a question of why asparaginyl hydroxylation has not been recognized previously in proteomics surveys that must have included ARD-containing species. It seems possible that allowance for artifactual protein oxidation in computerized database searching may have confounded some analyses. In the current work we utilized data-directed MS/MS and MSE
to combine the rigorous assignment of sites of hydroxylation with quantitation, thus enabling the assay of sequence-specific Asn hydroxylation that is responsive to genetic suppression of FIH in cells. Interrogation of peptide sequences by MS should in the future consider the possibility of FIH-catalyzed hydroxylation on asparaginyl residues particularly at appropriately sited residues within ARD-containing proteins.