Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins

Abstract

The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain-containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in "VNVN" and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function.

Keywords: JmjC demethylase; ankyrin; epigenetics; factor inhibiting HIF; hypoxia-inducible factor; iron and 2-oxoglutarate/alpha-ketoglutarate oxygenase; post-translational modification/hydroxylation.

Figure 1

Evidence for FIH-catalyzed double hydroxylation of ASPP2-V5 within the same ankyrin repeat in protein from U2OS cells but not in studies with peptide fragments.A, Western Blot analysis of V5-ASPP2 variants and HA-FIH using total cell lysates from ASPP2/FIH-double KO U2OS cells, transfected with an empty vector, or cotransfected with pcDNA3 vectors encoding for HA-tagged FIH and V5-tagged WT ASPP2 or “NVN” variants, immunoprecipitated (IP) with an anti-V5 antibody (Thermo Fisher Scientific). HA-tagged FIH, V5-ASPP2, and IgG light-chain (IgG_L) levels are indicated. B, LC–MS/MS analysis of V5-ASPP2 from U2OS FIH CRISPR KO cells cotransfected with vectors encoding for V5-ASPP2 and HA-FIH. The spectrum shows fragments from the elastase-catalyzed digestion of ASPP2 showing evidence for hydroxylation at Asp-984 and Asp-986 (indicated by lowercase n). Conditions: V5-ASPP2 in pcDNA3. The vectors encoding for ASPP2 and FIH variants were transfected and overexpressed for 24 h in U2OS FIH CRISPR KO cells. Immunoprecipitation was employed with an anti-V5 antibody. Proteins were separated by SDS-PAGE; the band corresponding to ASPP2 was excised and digested using elastase for 16 h at 37 °C. C, LC–MS-based hydroxylation assays of an ASPP1-derived peptide (residues 932–954), an ASPP2-derived peptide (residues 969–991), and an iASPP-derived peptide (residues 670–693). Conditions: 0.1 μM FIH, 100 μM sodium ascorbate, 10 μM 2OG disodium salt, 10 μM Fe(II), 50 mM Tris–HCl (pH 7.5), and 50 mM NaCl, at ambient temperature. 2OG, 2-oxoglutarate; ASPP, apoptosis stimulating of p53 protein; FIH, factor inhibiting hypoxia-inducible factor; HA, hemagglutinin; IgG, immunoglobulin G.

Figure 2

Views from crystal structures of FIH in complex with ASPP-derived peptides.A, left, sequence alignment of ASPPs with reported FIH substrates. Right, overlay of crystal structure–derived views of FIH in complex with ASPP2 and HIF-1α (Protein Data Bank code: 1H2K; 2.15 Å) showing the conserved nature of substrate binding. B, views from the dimeric structure of FIH in complex with an iASPP-derived peptide (residues 670–693), an ASPP1-derived peptide (residues 932–954), and an ASPP2-derived peptide (residues 969–991). C, close-up views from crystal structures of FIH in complex with ASPP-derived peptides, the Fo–Fc OMIT maps, shown in green mesh, are contoured to 3σ. ASPP, apoptosis stimulating of p53 protein; FIH, factor inhibiting hypoxia-inducible factor; HIF, hypoxia-inducible factor.

Figure 3

Evidence that FIH catalyzes two hydroxylations of ASB11 and p18.Top, domain structure of human “VNVN” motif–containing proteins. Middle, LC–MS/MS spectra implying a doubly hydroxylated ASB11 peptide at Asn-92 and Asn-90 (indicated by a lowercase n). Bottom, LC–MS/MS spectra for doubly hydroxylated peptides at Asn-32 and Asn-30 (indicated by a lowercase n). Reaction conditions: 1 μM ASB11, 0.25 μM FIH, 1 mM sodium ascorbate, 1 mM 2OG disodium salt, 200 μM Fe(II), and Tris–HCl (pH 7.5) incubated for 3 h at 37 °C. 2OG, 2-oxoglutarate; FIH, factor inhibiting hypoxia-inducible factor.