Erythrocytosis-inducing PHD2 mutations implicate biological role for N-terminal prolyl-hydroxylation in HIF1α oxygen-dependent degradation domain
- Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, Canada
- Department of Biochemistry, Temerty Faculty of Medicine, University of Toronto, Canada
- Princess Margaret Cancer Centre, University Health Network, Canada
- Department of Medical Biophysics, Temerty Faculty of Medicine, University of Toronto, Canada
Figures
Figure 1
Analysis of disease-causing PHD2 mutants.
(A) Distribution of mutation type calculated from clinical case reports. Case reports have been compiled and summarized in Supplementary file 1B. Linear map of mutant location and frequency on PHD2. The zinc finger is comprised of residues 21–58 while the catalytic core ranges from 181 to 426. Each dot represents a clinical report of a disease-causing mutation at the given residue. Mutations selected for analysis have been highlighted in red. (C) Structure of PHD2 HIF1αCODD complex (PDB:5L9B) with PHD2 mutant locations highlighted (yellow). PHD2 (gray) and HIF1αCODD (red) are depicted as ribbons. PHD2 mutants selected for analysis are highlighted in green and labeled. The structure was turned 260° on the y-axis to highlight all mutant locations.
Figure 2 with 1 supplement
Methods in HEK293A cells detect defects in some PHD2 mutants but not all.
(A) Dual luciferase reporter assays were performed to measure HIF1α transcriptional activity in the presence of PHD2 mutants in PHD2 -/- HEK293A cells. Individual data points are plotted. Loading accuracy was evaluated via immunoblotting for FLAG-tagged PHD2 and vinculin. (B) PHD2 mutant stability was measured via cycloheximide chase assay. HEK293A cells were transfected with wild-type or mutant PHD2 constructs, with the amount of transfected adjusted to ensure equal expression at 0 hr. After 24 hr, the transfected cells were treated with cycloheximide (CHX) to halt protein production and monitor the stability of PHD2. Cells were harvested at various time points up to 24 hr and lysates were immunoblotted to measure PHD2 levels. (C) FLAG immunoblot density was quantified at each time point and normalized with vinculin density to yield a relative density. 24 hr time points were compared to determine significance. For A and C, bars represent mean values, and standard error is represented by error bars (n=3, * indicates p<0.0332, ** indicates p<0.0021, *** indicates p<0.0002, and **** indicates p<0.0001 [two-tailed t-test]).
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Figure 2—source data 1
Original membranes corresponding to Figure 2A.
Red line indicates FLAG-PHD2 and vinculin. A BLUelf prestained protein ladder was employed, and the corresponding molecular weights are labeled.
- https://cdn.elifesciences.org/articles/107121/elife-107121-fig2-data1-v1.zip
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Figure 2—source data 2
Original membrane image files corresponding to Figure 2A.
- https://cdn.elifesciences.org/articles/107121/elife-107121-fig2-data2-v1.zip
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Figure 2—source data 3
Original membranes corresponding to Figure 2C.
The membranes used in Figure 2C are noted by a red box. Anti-FLAG and anti-vinculin antibodies were used to detect FLAG-PHD2 and vinculin, respectively. A BLUelf prestained protein ladder was employed, and the corresponding molecular weights are labeled.
- https://cdn.elifesciences.org/articles/107121/elife-107121-fig2-data3-v1.zip
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Figure 2—source data 4
Original membrane image files corresponding to Figure 2C.
- https://cdn.elifesciences.org/articles/107121/elife-107121-fig2-data4-v1.zip
Figure 2—figure supplement 1
Validation of PHD2 CRISPR knockout (KO) in HEK293A cells.
Homozygous PHD2 KO HEK293A cells were generated via CRISPR. PHD2 KO was confirmed through immunoblotting. pX330 represents an empty plasmid without EGLN1 gRNA and is shown here as a positive control. Each lane represents single-cell clones that were selected for PHD2 KO screening. HIF1α levels were also monitored. F4, D8, and E8 displayed complete loss of PHD2 along with HIF1α stabilization. F4, as indicated by asterisk, was chosen for use in further experiments.
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Figure 2—figure supplement 1—source data 1
Original, replicate membranes run to monitor PHD2 KO.
The membranes used in Figure 2—figure supplement 1 are noted by a red box. Red dashes indicate PHD2, HIF1α, and vinculin according to each indicated antibody. A BLUelf prestained protein ladder was employed, and the corresponding molecular weights are labeled.
- https://cdn.elifesciences.org/articles/107121/elife-107121-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original membrane image files corresponding to Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/107121/elife-107121-fig2-figsupp1-data2-v1.zip
Figure 3 with 1 supplement
PHD2 mutants display instability through aggregation and decreased thermostability.
(A) Chromatograms of PHD2 catalytic cores were acquired via size exclusion chromatography (SEC) on a Superdex200 column. Curves have been normalized according to molecular concentrations. Dashed red lines indicate mutants that were not purified (n = 2). (B) Circular dichroism (CD) was performed on PHD2 mutants to predict secondary structure variations. The far-UV spectra (190–260 nm) of the purified catalytic cores were measured and converted to molar ellipticity (n=3). (C) Molar ellipticity of PHD2 mutants was monitored at 220 nm from 25 to 95°C to evaluate thermal stability. Melting curves were generated from the CD melt (n=3). Molar ellipticity values were normalized and transformed into a fraction of folded protein and fitted with a sigmoidal curve. Melting temperatures (Tm) were determined using the EC50 of each curve. Tm and R2 are listed in Table 1.
Figure 3—figure supplement 1
Purification of PHD2 181–426.
His6-PHD2 (181–426) was expressed in BL21 (DE3) Escherichia coli cells and purified using a Ni-NTA agarose and size exclusion chromatography (SEC). 10 μl samples were taken throughout the purification and analyzed via Coomassie-stained SDS-PAGE gel. The expected final size of His6-PHD2 (181–426) is 27.76 kDa. (1) BLUelf prestained protein ladder, 15 μl. (2) Lysate from BL21 (DE3). (3) Ni-NTA agarose flowthrough. (4) Ni-NTA agarose 5 mM imidazole wash. (5) Ni-NTA agarose 30 mM imidazole wash 1. (6) Ni-NTA agarose 30 mM imidazole wash 2. (7) PHD2 elution from Ni-NTA agarose. (8) Thrombin cleavage to remove His6 tag. (9) Reverse Ni-NTA agarose flowthrough. (10) Reverse Ni-NTA agarose wash. (11) Reverse Ni-NTA agarose elution. (12) Pooled SEC fractions containing PHD2. (13) BLUelf prestained protein ladder, 5 μ.
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Figure 3—figure supplement 1—source data 1
Unedited image of PHD2 purification SDS-PAGE gels stained with Coomassie blue corresponding to Figure 3—figure supplement 1.
The top gel corresponds to a purification of PHD2 wild-type (WT), while the bottom gel corresponds to a purification of PHD2 P317R. A BLUelf prestained protein ladder was employed, and the corresponding molecular weights are labeled.
- https://cdn.elifesciences.org/articles/107121/elife-107121-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original image file corresponding to Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/107121/elife-107121-fig3-figsupp1-data2-v1.zip
Figure 4
PHD2 mutants have minor binding defects to HIF1α peptides.
Microscale thermophoresis was performed on fluorescently labeled PHD2 and HIF1α 555-574 CODD (A) or HIF2α 522-542 CODD (B) peptides. PHD2 P317R displayed a severe binding defect, whereas the other three mutants had minor binding defects. It is suspected that amine-reactive fluorescent labeling induced a binding defect on PHD2 P317R. Data points represent normalized mean value, and standard error is represented by error bars (n=3). Kd values with standard deviation are listed in Table 2.
Figure 5 with 3 supplements
PHD2 P317R does not hydroxylate HIFα N-terminal oxygen-dependent degradation domain (NODD), while PHD2 A228S has very minor enzymatic defects.
An assay measuring hydroxylation of HIF1α oxygen-dependent degradation domain (ODD) (394–574) by PHD2 via nuclear magnetic resonance (NMR) was performed. (A) Resonance shifting was monitored in real time to compare hydroxylation rates of A228S (blue), P317R (red), and wild-type (WT) (black) PHD2. PHD2 A228S displayed minorly impaired hydroxylation of both ODDs. PHD2 P317R displayed no activity on the P402 (NODD), while retaining near WT activity on P564 (C-terminal ODD [CODD]). Data points represent mean value, and standard deviation is represented by error bars (n=2). (B) HSQC spectra display the resonance shifting pattern of HIF1a ODD upon prolyl-hydroxylation catalyzed by PHD2 (WT, P317R, A228S) over the course of 20.2 hr. Neighboring residues, A403 and I566, were used to monitor hydroxylation of P402 and P564, respectively. Spectra recorded at 0 hr are shown in black while spectra recorded at the endpoint (20.2 hr) are shown in red.
Figure 5—figure supplement 1
Tracking proline hydroxylation using CON versus HSQC nuclear magnetic resonance (NMR).
HSQC NMR was performed to monitor resonance shifting of oxygen-dependent degradation domain (ODD) neighboring residues, I566 and A403, which were used as reporters for P564 and P402 hydroxylation, respectively. This method yields similar results as directly tracking proline hydroxylation using CON NMR, while requiring only HIF1α ODD to be isotopically labeled. CON results are red, while HSQC results are black. Residues associated with C-terminal ODD (CODD) are represented with a circle and those associated with N-terminal ODD (NODD) are represented with a triangle.
Figure 5—figure supplement 2
Bio-layer interferometry (BLI) shows PHD2 P317R does not have a severe binding defect for HIF1α C-terminal oxygen-dependent degradation domain (CODD) or HIF1α N-terminal oxygen-dependent degradation domain (NODD).
BLI was performed to confirm the binding defect of PHD2 P317R observed via microscale thermophoresis (MST). Measurements with HIF1αCODD and HIF1αNODD peptides indicated that PHD2 P317R did not display a severe binding defect compared to PHD2 wild-type (WT). While a minor defect was observed with HIF1αCODD and PHD2 P317R, no binding defect was observed with HIF1α NODD. Binding measurements were performed in technical triplicates.
Figure 5—figure supplement 3
Validation of bio-layer interferometry (BLI) using hydroxylated peptides as a negative control.
BLI was performed with hydroxylated peptides as a negative control. As expected, no binding is observed with the hydroxylated peptides compared to unhydroxylated. Hydroxylated peptides are colored in red while unhydroxylated are black. The concentrations used for each experiment are as follows: PHD2 WT+HIF1α CODD: 14.6 μM, PHD2 WT+HIF1α NODD: 21.9 μM, PHD2 P317R+HIF1α CODD: 21.9 μM, PHD2 P317R+: 7.3 μM.
Tables
Table 1
PHD2 mutant melting temperatures calculated via circular dichroism (CD).
| Tm (°C) | R2 | |
|---|---|---|
| WT | 51.9 | 0.871 |
| A228S | 48.3 | 0.707 |
| P317R | 46.2 | 0.748 |
| F366L | 43.8 | 0.878 |
| R371H | 45.1 | 0.843 |
Table 2
Microscale thermophoresis (MST) determined dissociation constants between PHD2 mutants and HIFα C-terminal oxygen-dependent degradation domain (CODD) peptides.
| Kd (µM) | ||
|---|---|---|
| PHD2 | HIF1α | HIF2α |
| WT | 7.5±0.52 | 15±1.1 |
| A228S | 9.3±0.57 | 22±1.6 |
| R371H | 13±1.6 | 39±4.4 |
| F366L | 16±1.8 | 43±4.7 |
| P317R | 320±20 | 580±38 |
Table 3
Bio-layer interferometry (BLI) determined binding constants between PHD2 wild-type (WT) and P317R and HIF1α N-terminal oxygen-dependent degradation domain (NODD) and C-terminal oxygen-dependent degradation domain (CODD) peptides.
| Kd (µM) | ka (×104) (1/Ms) | kd (×10–3) (1/s) | |
|---|---|---|---|
| PHD2 WT+HIF1α CODD | 2.1±0.8 | 5.2±1.4 | 101±20 |
| PHD2 P317R+HIF1α CODD | 6.0±0.7 | 0.21±0.11 | 12.3±5 |
| PHD2 WT+HIF1α NODD | 2.0±0.8 | 0.26±0.093 | 4.7±0.2 |
| PHD2 P317R+HIF1α NODD | 2.0±1.0 | 0.17±0.049 | 3.0±0.7 |
Additional files
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Source data 1
- https://cdn.elifesciences.org/articles/107121/elife-107121-data1-v1.xlsx
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Supplementary file 1
Clinical case reports of disease-causing PHD2 mutations.
- https://cdn.elifesciences.org/articles/107121/elife-107121-supp1-v1.xlsx
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Supplementary file 2
HIFα peptide sequences.
- https://cdn.elifesciences.org/articles/107121/elife-107121-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107121/elife-107121-mdarchecklist1-v1.docx
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Erythrocytosis-inducing PHD2 mutations implicate biological role for N-terminal prolyl-hydroxylation in HIF1α oxygen-dependent degradation domain
eLife 14:RP107121.
https://doi.org/10.7554/eLife.107121.3
