Oxygen-dependent asparagine hydroxylation of the ubiquitin-associated (UBA) domain in Cezanne regulates ubiquitin binding

Abstract

Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain-containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn³⁵ within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBA^Cez), which lacks conserved UBA domain properties. We show that UBA^Cez binds Lys¹¹-, Lys⁴⁸-, Lys⁶³-, and Met¹-linked ubiquitin chains in vitro, establishing UBA^Cez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBA^Cez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn³⁵ inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBA^Cez Our results indicate that UBA^Cez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.

Keywords: Cezanne; FIH1; OTU domain-containing protein 7B (OTUD7B); UBA domain; deubiquitinase (DUB); deubiquitylation (deubiquitination); hydroxylation; nuclear magnetic resonance (NMR); posttranslational modification (PTM); protein-protein interaction; ubiquitin.

Figure 1.

Cezanne contains a putative consensus site for FIH1-dependent hydroxylation. A, overview of the domain architecture of Cezanne and sequence alignment showing an FIH1 consensus sequence in Cezanne (where Φ indicates aliphatic amino acids and * represents the modified asparagine residue). B, co-IP of GFP-Cezanne and FIH1 from HEK293 cells. C, co-IP of GFP-Cezanne and catalytic inactive FIH1 (H199A) and dimerization-deficient FIH1 (L340R) mutants. D, co-IP of GFP-Cezanne truncated versions and FIH1 to narrow down the region in Cezanne that interacts with FIH1. E, co-IP of GFP-Cezanne and FIH1, assessing the effect of hypoxia on the interaction of the two proteins.

Figure 2.

Cezanne is hydroxylated within its UBA-like domain. A, FIH1 was efficiently depleted by CRISPR/Cas9-mediated knockout in HEK293 cells. B, cartoon depicting design of SILAC-based MS experiment for the detection of asparagine hydroxylation in Cezanne. C, annotated MS2 spectrum showing hydroxylated Asn³⁵ of Cezanne in HEK293 cells. D, FIH1- and oxygen-dependent enrichment of Asn³⁵ hydroxylation. Data are represented as mean of SILAC ratios plus S.E. (error bars). *, p < 0.1; **, p < 0.01 by one-sample t test. The experiment was performed with three biological replicates.

Figure 3.

Cezanne's UBA domain binds polyubiquitin of different linkage types. A, sequence alignment of various UBA domains revealed that UBA^Cez lacks both the MGF motif and the dileucine motif. B, Western blotting showing in vitro GST pulldown assay. GST-UBA^Cez co-precipitated Lys¹¹-, Lys⁴⁸-, and Lys⁶³-linked tetraubiquitin. C, in vitro pulldown of WT and mutant UBA^Cez and Halo-tagged tetraubiquitin (Met¹-linked). Mutation of Asn³⁵ in UBA^Cez greatly reduced ubiquitin binding. D, semi-in cellulo pulldown of recombinant WT or mutant UBA^Cez and ubiquitinated proteins from HEK293 cells treated with the proteasome inhibitor MG132.

Figure 4.

Hydroxylation of Asn³⁵ modulates UBA-ubiquitin interaction. A, cartoon depicting experimental strategy for assessing the effect of asparagine hydroxylation on ubiquitin binding. B, verification of FIH1-dependent enrichment of Asn³⁵ hydroxylation of recombinant UBA^Cez by MS analysis. Data are represented as mean of log₂ -fold change intensity plus S.E. (error bars). **, p < 0.01; ***, p < 0.001 by one-sample t test. The experiment was performed with three biological replicates. C, in vitro GST pulldown assay comparing interaction of linear tetraubiquitin with unmodified and hydroxylated UBA^Cez, respectively.

Figure 5.

Binding between linear diubiquitin and the UBA domain of Cezanne. Shown are the results for NMR titrations of ¹⁵N-labeled linear diubiquitin with nonlabeled UBA^Cez (A–D) and NMR titrations of ¹⁵N-labeled UBA^Cez with nonlabeled linear diubiquitin (E–H). A, representative area of ¹⁵N,¹H BEST-TROSY HSQC spectra for linear diubiquitin, to which UBA^Cez was added in a specified molar ratio (1:8, 1:4, 1:2, 1, 2, 4, and 8), is shown. The rainbow color code indicates increased molar ratios upon titration from free linear diubiquitin (red) to full saturation upon 8-fold excess of UBA^Cez (dark gray). B, CSP mapping on the ubiquitin sequence. Yellow and red lines indicate the 1× and 2× S.D. (δ) calculated from CSP values of all residues, respectively. Residues of linear diubiquitin, which showed double peaks (either in the free or UBA^Cez-bound form) and significant line broadening, are colored in blue. C, CSP mapping on the linear diubiquitin structure (PDB code 2L2D). The diubiquitin is presented as a ribbon diagram; P and D symbols indicate proximal and distal ubiquitin moieties. Residues with intermediately (δ ≤ CSP ≤ 2×δ) and strongly (CSP ≥ 2×δ) perturbed backbone HN resonances are marked in yellow and red, respectively. Residues with anomalous exchange behavior (described above) are marked in blue. Side chains of residues from the ubiquitin hydrophobic patch (Leu⁸-Ile⁴⁴-Val⁷⁰) are shown as spherical atom models. D, K_D calculated for the selected linear diubiquitin residues upon titration with UBA^Cez. Normalized CSP values of residues I3p (where p represents proximal ubiquitin), Leu⁸, Ile⁴⁴, Lys⁶³p, Val⁷⁰, and Leu⁷¹, showing significant CSP in the fast exchange mode, were used in the global fit. E, representative area of ¹⁵N,¹H BEST-TROSY HSQC spectra for UBA^Cez, to which linear diubiquitin was added in the specified molar ratio (1:8, 1:4, 1:2, 1, 2, 4, and 8), is shown. The rainbow color code indicates increased molar ratios upon titration from the free UBA^Cez (red) to full saturation upon 8-fold excess of linear diubiquitin (dark gray). F, CSP mapping on the UBA^Cez sequence. Yellow and red lines, 1× and 2× S.D. (δ) calculated from CSP values of all residues, respectively. Residues of UBA^Cez, which showed intermediate exchange and significant line broadening, are colored in blue. *, CSP for the side chain of Gln⁴³, which shows intermediate and big CSP upon binding of linear diubiquitin. G, CSP mapping on the modeled UBA^Cez structure. The UBA^Cez is presented as a ribbon diagram. Residues with intermediately (δ ≤ CSP ≤ 2×δ) and strongly (CSP ≥ 2×δ) perturbed backbone HN resonances are marked in yellow and red, respectively. Residues with anomalous exchange behavior (described above) are marked in blue. H, K_D calculated for the selected UBA^Cez residues upon titration with linear diubiquitin. Normalized CSP values of residues Ser¹⁰, Leu²⁷, Glu⁴², Gln⁴³, and Gly⁵⁰, showing significant CSP in the fast exchange mode, were used in the global fit.