Hypoxia‐Inducible Factor Prolyl‐Hydroxylase: Purification and Assays of PHD2

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Hypoxia-Inducible Factor Prolyl-Hydroxylase: Purification and Assays of PHD2 Kirsty S. Hewitson,* Christopher J. Schofield,* and Peter J. Ratcliffe† Contents 26 28 29 29 30 32 33 35 35 35 36 37 38 39

1. 2. 3. 4.

Introduction Preparation of Purified PHD2 from a Bacterial Source Assaying of PHD2 Activity Indirect Measurements of PHD2 Activity 4.1. 1-[14C]-CO2 capture assay 4.2. Fluorescence derivatization of 2OG 4.3. Oxygen consumption assay 4.4. 1-[14C]succinate quantification 5. Direct Measurements of PHD2 Hydroxylation Activity 5.1. LC/MS identification of hydroxylated HIF-1a 556 to 574 5.2. pVHL capture assay 6. Binding Assays 7. Comparison of Assay Formats References

Abstract The adaptation of animals to oxygen availability is mediated by a transcription factor termed hypoxia-inducible factor (HIF). HIF is an alpha (a)/beta (b) heterodimer that binds hypoxia response elements (HREs) of target genes, including some of medicinal importance, such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF). While the concentration of the HIF-b subunit, a constitutive nuclear protein, does not vary with oxygen availability, the abundance and activity of the HIF-a subunits are tightly regulated via oxygendependent modification of specific residues. Hydroxylation of prolyl residues (Pro402 and Pro564 in HIF-1a) promotes interaction with the von Hippel-Lindau

* {

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, United Kingdom

Methods in Enzymology, Volume 435 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)35002-7

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2007 Elsevier Inc. All rights reserved.

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E3 ubiquitin ligase and, consequently, proteolytic destruction by the ubiquitinproteasome pathway. This prolyl hydroxylation is catalyzed by the prolylhydroxylase domain (PHD) containing enzymes for which three isozymes have been identified in humans (1–3). Additionally, asparaginyl hydroxylation (Asn803 in HIF-1a) by factor-inhibiting HIF (FIH) ablates interaction of the HIF-a subunit with the coactivator p300, providing an alternative mechanism for downregulation of HIF-dependent genes. Under hypoxic conditions, when oxygenmediated regulation of the a-subunits is curtailed or minimized, dimerization of the a- and b-subunits occurs with subsequent target gene upregulation. Therapeutic activation of HIF signaling has been suggested as a potential treatment for numerous conditions, including ischemia, stroke, heart attack, inflammation, and wounding. One possible route to achieve this is via inhibition of the HIF hydroxylases. This chapter details methods for the purification and assaying of PHD2, the most abundant PHD and the most important in setting steady-state levels of HIF-a. Assays are described that measure the activity of PHD2 via direct and indirect means. Furthermore, conditions for the screening of small molecules against PHD2 are described.

1. Introduction The maintenance of oxygen homeostasis is a fundamental physiological challenge. Recent studies have revealed that a substantial component of the cell’s total complement of expressed genes (in the range of hundreds or thousands of transcripts) are modulated by changes in oxygen availability and that the majority of these are responding directly or indirectly to novel signal pathways that govern the activity of HIF by post-translational hydroxylation of specific amino acid residues. As previously stated, HIF is an a/b heterodimer that binds HREs in the cis-acting regulatory sequences of target genes that encode molecules involved in both systemic responses to hypoxia, such as enhanced erythropoiesis and angiogenesis, and cellular responses to hypoxia, such as alterations in energy metabolism and cell motility, differentiation, and survival decisions (Pugh and Ratcliffe, 2003; Semenza, 2000; Wenger, 2002). Regulation of HIF activity is provided by its a-subunits (Ivan et al., 2001; Jaakkola et al., 2001; Lando et al., 2002; Masson et al., 2001; Yu et al., 2001), which are highly inducible in hypoxic cells and appear specific to the HIF system. The b-subunits (also known as the aryl hydrocarbon nuclear translocator, ARNT) are constitutive nuclear proteins that mediate a number of different transcriptional responses in combination with other dimerization partners. HIF-a is controlled by at least two steps involving different types of hydroxylation. Thus, prolyl hydroxylation of specific residues within a central degradation domain controls interaction with the von Hippel-Lindau E3 ubiquitin ligase and proteolytic destruction by the ubiquitin-proteasome

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pathway (Ivan et al., 2001; Jaakkola et al., 2001; Yu et al., 2001). Asparaginyl hydroxylation at a specific residue in the C-terminal transcriptional activation domain controls coactivator recruitment and, hence, the transcriptional activity of HIF-a polypeptides that escape proteolytic destruction (Lando et al., 2002). This chapter will focus on HIF prolyl hydroxylation, which was the first oxygen-regulated step to be discovered and which exerts dominant control over HIF activity through oxygen-dependent destruction of HIF-a. HIF prolyl hydroxylation occurs at two residues that are tightly conserved within the closely related mammalian HIF-1a and HIF-2a isoforms and at a single conserved residue in mammalian HIF-3a. This process is conserved in invertebrates; and in flies and worms there appears to be a single site of prolyl hydroxylation in a single HIF-a homologue (Bacon et al., 1998; Epstein et al., 2001; Jiang et al., 2001; Lavista-Llanos et al., 2002; Wappner et al., 2003). The reactions are catalyzed by a series of dioxygenases belonging to the Fe(II)- and 2-oxoglutarate–dependent oxygenase superfamily (Clifton et al., 2006; Costas et al., 2004; Hausinger, 2004; Hewitson et al., 2005), which are represented in mammalian cells by three closely related enzymes (PHD 1, 2, and 3, also identified as HPH1–3 and EGLN1–3) (Bruick and McKnight, 2001; Epstein et al., 2001). The absolute requirement for molecular oxygen as cosubstrate confers oxygen sensitivity and, in hypoxia, hydroxylation is suppressed, allowing HIF-a to escape destruction and form a transcriptional complex. PHD1, -2, and -3 share a highly conserved catalytic domain in which the active Fe(II) center is coordinated by a 2-histidine-1 carboxylate motif (HxD ...H) aligned on the second and seventh strands of the eight-stranded b-barrel jelly-roll conformation that is common to the catalytic core of this family of enzymes (Epstein et al., 2001; McDonough et al., 2006). All three enzymes are physiological regulators of the HIF system, though they do display preferential activity for different HIF prolyl hydroxylation sites. Thus, PHD1 and -2 show activity for both of the hydroxylation sites in HIF-1a and HIF-2a, whereas PHD3 is selective for the more C-terminal of the two sites (Appelhoff et al., 2004; Hirsila et al., 2003). Cellular abundance, however, varies substantially, and PHD1 and -3 show marked tissue-specific restriction of expression (Appelhoff et al., 2004; Lieb et al., 2002; Willam et al., 2006). In most cells studied to date, PHD2 is the most abundant enzyme, while, in oxygenated cells, it is the most important in setting steady-state levels of HIF-a and hence the activity of the system (Appelhoff et al., 2004; Berra et al., 2003). For this reason, we have focused our development of the assay methods later described on PHD2. The assays are of use in the biochemical and physiological characterization of the enzymes as cellular oxygen sensors and in the development and assessment of inhibitors as a therapeutic approach to ischemic/hypoxic disease.

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2. Preparation of Purified PHD2 from a Bacterial Source Our laboratory uses the procedure described in this section for the preparation of recombinant PHD2 as a source of material for biochemical characterization studies (McNeill et al., 2005b), inhibitor screens (Hewitson et al., 2007), and crystallization (McDonough et al., 2006). Attempts to produce and purify soluble, full-length PHD2 to homogeneity in ‘‘milligram’’ quantities for the these uses, either with (e.g., maltose binding protein [MBP], His, intein-based protein-splicing vectors, glutathione S-transferase [GST]) or without affinity tags in bacterial cultures, were problematic. Sequence comparisons of PHD2 with a bacterial 2-oxyglutarate (2OG)– dependent oxygenase deacetoxycephalosporin C synthase (DAOCS) (Lloyd et al., 1999), for which crystal structures have been reported (Valega˚rd et al., 1998), identified amino acids 181 to 426 of PHD2 as being the putative PHD2 catalytic domain. An N-terminally hexahistidine-tagged fusion protein comprising these residues (PHD2181–426) was produced using the pET28a(þ) vector (Novagen, Darmstadt, Germany) to yield approximately 5% of total soluble protein when expressed in Escherichia coli BL21(DE3). Following initial growth at 37 in 2TY medium, protein expression is induced with isopropyl b-D-thiogalactoside (0.5 mM) when the OD600 reaches 0.8 to 1.0. Growth is then continued at 37 for a further 3 to 4 h before harvesting by centrifugation at 14,000 rpm. Cell pellets have been stored for at least 6 mo at –80 without any apparent deleterious effects on catalytic activity of purified PHD2181–426. Affinity purification of PHD2181–426 can be achieved using the N-terminal hexahistidine tag (McNeill et al., 2005b). His-Bind TM resin (Novagen, Darmstadt, Germany) is commonly used in our laboratory for such purification and utilizes Ni2þ ions held by tridentate chelation to the resin by iminodiacetic acid (IDA). A typical purification involves the resuspension of 20 g of PHD2181–426/BL21(DE3) in 100-ml binding buffer (40 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, pH 7.9) with subsequent lysis by sonication. After centrifugation at 14,000 rpm (20 min), the cleared lysate is applied to a column (10 ml) containing Ni-IDA His-Bind resin preequilibrated with binding buffer. Once the lysate is loaded, a further 10 column volumes of binding buffer is flowed through the column to remove any unbound protein. After washing with six column volumes of wash buffer (40 mM Tris-HCl, 0.5 M NaCl, 60 mM imidazole, pH 7.9), the PHD2181–426 is eluted with six column volumes of elute buffer (40 mM Tris-HCl, 0.5 M NaCl, 1 M imidazole, pH 7.9). At this stage, the PHD2181–426 can be stored frozen as the His-tagged protein, once desalted (note that, if the protein is stored without the removal of imidazole,

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it may precipitate upon thawing), or the His tag can be cleaved by treatment with thrombin (Novagen, Darmstadt, Germany). Efficient cleavage of the His-tag is possible with 1 U of thrombin to 20 mg of PHD2181–426 follow ing a 16-h incubation at 4 . As a final step, separation of the His-tag from PHD2181–426 is achieved by using a 300-ml Superdex S75 gel filtration column, which also serves to desalt the protein. The resulting PHD2181–426 is greater than 95% pure by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and has been stored for at least 6 mo at –80 with no resulting loss in enzymatic activity. Typical yields of protein are 30 to 40 mg of PHD2181–426 from 20 g of cells. As an alternative to Ni-IDA resin, Ni-NTA and TALONTM Resin (BD Biosciences, San Jose, CA; where cobalt [II] is substituted for nickel [II]) have also been used successfully with similar protocols to those previously detailed to purify PHD2181–426. Although not observed for PHD2181–426, care must be taken with the use of metal affinity chromatography and the 2OG-dependent oxygenases. Since these enzymes bind iron at the active site, as required for catalysis, any leaching of metal from the affinity column and subsequent coordination at the active site can lead to a reduction in enzymatic activity (Searls et al., 2005).

3. Assaying of PHD2 Activity A variety of techniques can be used to assay PHD2 activity in vitro. Those detailed here are procedures used routinely in our laboratories that yield reproducible results. A brief comparison of the relative merits and disadvantages of each of the assay formats is given at the end of this section. The assays can themselves, broadly speaking, be classified into two types: those that are generic to the Fe(II) and 2OG-dependent oxygenase family (of which PHD2 is a member) (Bruick and McKnight, 2001; Epstein et al., 2001) and those that are specific to the PHDs. While soluble PHD2181–426 is currently the only readily available PHD isoform reported to be prepared in milligram quantities, these techniques could be applied to all PHD family members.

4. Indirect Measurements of PHD2 Activity PHD2 is a member of the Fe(II) and 2OG-dependent oxygenase superfamily in which almost all members appear to follow the generalized reaction scheme shown in Scheme 2.1 (Costas et al., 2004; Hausinger, 2004; Hewitson et al., 2005).

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Substrate + O2 + 2OG (HIF-a)

Oxidised + CO2 + succinate Product (hydroxylated HIF-a)

Scheme 2.1 Generalized reaction scheme for the 20G-dependent oxygenase family.

Oxidative decarboxylation of 2OG to give CO2 and succinate accompanies hydroxylation of HIF-a substrate by PHD2. Indirect measurement of PHD2 activity can therefore be achieved by quantification of the amount of 2OG or dioxygen consumed or by the amount of CO2 or succinate formed. It should be noted that, for the 2OG-dependent oxygenase family, uncoupling of 2OG decarboxylation from substrate formation can occur (i.e., turnover of 2OG does not always correlate to prime substrate hydroxylation) (Costas et al., 2004; Hausinger, 2004; Hewitson et al., 2005). Care must be taken in the following procedures to control for such a possibility.

4.1. 1-[14C]-CO2 capture assay The activity of PHD2 can be assayed by measuring the release of 1-[14C]CO2 from 1-[14C]-2OG (PerkinElmer, Waltham, MA) (Epstein et al., 2001). This measurement is a well-established assay technique for 2OGdependent oxygenases and has been used successfully in our laboratory for a range of other enzymes from this family, including FIH (Hewitson et al., 2002), AlkB (Welford et al., 2003), phytanoyl CoA hydroxylase (PAHX) (Mukherji et al., 2001), and DAOCS (Lloyd et al., 1999). It has also been used by additional groups for other 2OG-dependent oxygenases, notably the procollagen hydroxylating enzymes, for which the assay was originally developed (Myllyharju and Kivirikko, 1997). 4.1.1. Reagents A standard PHD2 assay contains the following components in 50 mM Tris-HCl, pH 7.5: 5.7 mM PHD2181–426 57 mM HIF-a substrate (discussed later) 160 mM 2OG (5% [14C]-2OG, 95% [12C]-2OG) 80 mM Fe(II) 4 mM ascorbate 1 mM dithiothreitol (DTT) 0.6 mg/ml catalase

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All incubations are carried out in a final volume of 100 ml. Care must be taken in preparing the Fe(II) solution since aerobic oxidation to Fe(III) readily occurs. To minimize this, a 500-mM Fe(II) stock is first prepared in 20 mM HCl, which is then subsequently diluted with Milli-Q water (SynthesisTM, Millipore, Billerica, MA) to 4 mM. Fe(II) sulphate is normally used, but it can be replaced by Fe(II) chloride without adverse effect. Several different sources of the HIF-a substrate have been utilized, but the most widely used is a 19mer peptide, HIF-1a 556 to 574 (DLDLEMLAPYIPMDDDFQL, Peptide Protein Research Ltd, Fareham, UK). Peptides of alternative HIF-a isoforms have also been used successfully with this assay, as have recombinant preparations of HIF-a protein (e.g., HIF-1a 530–698). The DTT, ascorbate, and 2OG solutions should be prepared fresh each time in 50 mM Tris-HCl, pH 7.5 to avoid inconsistent results. All the aforementioned components are known to degrade with time and repeated freeze-thawing. These considerations also apply for the additional assays detailed later and shall not be repeated again within this manuscript. The 2OG solution comprises 5% [14C]-2OG and 95% [12C]2OG. The radiolabeled 2OG is used as a tool to monitor the reaction since, ultimately, [14C]-CO2 is captured and quantified by scintillation. Samples to be assayed are at least duplicated. 4.1.2. Protocol The reaction vessel of choice is a 5-ml polycarbonate tube that can be obtained from numerous commercial suppliers (e.g., Thermo Fisher Scientific, Waltham, MA; International Scientific Supplies Ltd., Bradford, UK). A master mix is freshly made that contains the 2OG, Fe(II), ascorbate, DTT, and any additional Tris-HCl buffer required to reach the final 100-ml volume. This mix is placed as a spot at the bottom of the 5-ml tube. Additional separate spots containing the PHD2181–426 and HIF-a substrate are also placed at the bottom of the tube. Care must be taken to ensure that no mixing of these spots ensues before the samples are placed in the incubator; otherwise, reaction will occur prematurely in an uncontrolled manner. PHD2181–426 is capable of hydroxylating HIF-a at room temperature, albeit at a slower rate than at 37 . A 0.5-ml Eppendorf tube (with the lid removed) containing 200 ml of hyamine hydroxide (MP Biomedicals, Solon, OH) is then carefully placed inside the 5-ml tube, which is then sealed by the use of a rubber septum. The hyamine hydroxide is used to trap any CO2 released from the 2OG following decarboxylation. Reaction is initiated by shaking the tubes at 200 rpm in an orbital shaker at 37 . After 30 min, the reaction is quenched by the addition of 200 ml of methanol through the rubber septum with a needle and syringe. Samples are subsequently incubated on ice for a further 30 min to enable collection of any released CO2. In order to quantify CO2 release, the 0.5-ml Eppendorf tube containing the hyamine hydroxide is carefully removed with tweezers and

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wiped with a tissue to remove any [14C]-2OG that may be adhered to the outside of the Eppendorf vial. The entire Eppendorf vial (containing the hyamine hydroxide) is then placed in a scintillation vial, OptiPhase Safe scintillation fluid is added, and the amount of [14C]-CO2 is quantified with the use of a scintillation counter. 4.1.3. Inhibitor testing The discussed protocol can be modified readily to screen small molecules or other inhibitors of the enzyme (Hewitson et al., 2007). PHD2181–426 can tolerate a dimethyl sulphoxide (DMSO) concentration of 10%, with all substrates tested under the previously mentioned assay conditions. Results from our laboratory actually show that this DMSO concentration is weakly stimulatory to PHD2181–426 activity. Compounds to be assayed are dissolved to the required concentration in DMSO (if they are not soluble in aqueous solution) and included with the ‘‘master mix’’ spot previously described. The assay then proceeds as detailed. Methanol can also be used as an alternative small molecule solvent to 10% final concentration with PHD2181–426 with no adverse effect.

4.2. Fluorescence derivatization of 2OG An alternative assay developed in this laboratory (McNeill et al., 2005a) for measuring PHD2 activity is based upon the derivatization of 2OG with o-phenylenediamine (OPD; Acros Organics, Geel, Belgium) to produce a fluorescent adduct. This methodology was originally used for the identification of different 2-oxo acids in mixtures and required high performance liquid chromatography (HPLC) separation of the subsequent OPD derivatives (Muhling et al., 2003; Singh et al., 1993). However, for assays of PHD2 (and other 2OG-dependent oxygenases), the only 2-oxo acid present in the reaction mixture is 2OG itself, and, therefore, chromatography separation is not required. This assay has also been used successfully with the 2OGdependent oxygenases FIH (McNeill et al., 2005a) and PAHX (unpublished results). 4.2.1. Reagents A standard PHD2 assay combines the following components in 50 mM HEPES, pH 7.0: 4 mM PHD2181–426 60 mM HIF-a substrate (discussed later) 300 mM 2OG 150 mM Fe(II) 1 mM DTT 0.6 mg/ml catalase

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All concentrations described are final in a 50-ml volume. Both peptide and recombinant protein preparations as HIF-a substrate have been used successfully with this assay. Ascorbate, a stimulatory cofactor for the PHDs (Hewitson et al., 2007; Hirsila et al., 2003), must be omitted from the list of reagents for use in this assay due to its reactivity with OPD. OPD is  recrystallized from petroleum ether (120–140 ) before use. OPD for use in the assay must be white and flaky. Any color still present after recrystallization will adversely affect the results. 4.2.2. Protocol PHD2181–426 activity is measured by mixing the DTT, 2OG, catalase, substrate, and buffer to a final volume of 44 ml and incubating for 5 min at 37 . Simultaneously, the PHD2181–426 is mixed with the Fe(II) solution (final volume 6 ml) and kept at room temperature for 3 min. Reaction is initiated by addition of the PHD2:Fe(II) solution to the assay mix and is stopped by the addition of 100 ml of 0.5-M HCl after 12 min at 37 . Derivatization is achieved by the addition of 50 ml 10-mg/ml OPD in 0.5-M HCl with subsequent heating at 95 for 10 min. Following centrifugation for 5 min, 50 ml of the supernatant is removed and made basic with 30 ml of the 1.25-M NaOH. Reaction of the OPD with the 2-oxo acid moiety of 2OG gives rise to a fluorescent product (3-[2-carboxyethyl]-2 [1H]-quinoxalinone). The resulting fluorescence is measured with a Novostar (BMG LABTECH, Offenburg, Germany) with the excitation filter at 340 nm and the emission filter at 420 nm. As with the 1-[14C]-CO2 assay, inhibitors can be assayed by inclusion in the reaction mix with DMSO/MeOH to a final concentration of 10%.

4.3. Oxygen consumption assay A further assay for PHD2 involves the continuous measurement of oxygen consumption (Ehrismann et al., 2007). This assay has also been successfully used in this laboratory with other members of the 2OG-dependent oxygenase family, including FIH (Ehrismann et al., 2007), PAHX (unpublished results), and taurine dioxygenase (TauD., unpublished results). 4.3.1. Reagents A standard PHD2 assay combines the following components in 50 mM Tris-HCl, pH 7.5: 50 mM PHD2181–426 50 to 250 mM HIF-a substrate (discussed later) 750 mM 2OG 50 mM Fe(II)

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All concentrations described are final in a 200-ml volume with assays typically carried out at either 25 or 37 . Both peptide and recombinant protein preparations as HIF-a substrate have been used successfully with this assay. The addition of DTT and catalase made no significant difference to initial rate measurements and, consequently, were omitted from the final reaction conditions. The inclusion of ascorbate stimulates oxygen consumption in a manner independent of PHD2. Since ascorbate is a reducing agent, molecular oxygen is likely reduced to water in a reaction that is promoted by the presence of transition metals, such as Fe(III) (Xu and Jordan, 1990). Ascorbate is thus excluded from those reagents suitable for use with this assay format. 4.3.2. Protocol It is essential that the assays are performed under conditions of reduced light due to interference with the fiberoptic probe. Rubber septum-sealed (Wilmad-Labglass, Buena, NJ; NMR rubber caps) reaction vials (Supelco, Bellefont, PA; 0.35-mm HPLC vial glass inserts) are used to allow buffer exchange with the appropriate oxygen gas mixture (to give the required oxygen concentration) without contamination by atmospheric oxygen. The reaction vial is placed in a water bath (water-jacketed holder taken from a Clark-type oxygen electrode from Qubit Systems, Kingston, Ontario), and a FOXY AL-300 probe (Ocean Optics, Dunedin, FL; no additional silicone coating was used, in order to improve response time) is inserted through the vial into a solution containing the 2OG. PHD2 reconstituted with Fe(II) is then injected using a Hamilton syringe into this 2OG solution, which has been equilibrated to the required oxygen concentration. This mixture is allowed to equilibrate for 1 to 2 min, and the reaction is initiated by the injection of appropriate amounts of substrate. Oxygen levels are monitored using a Fiberoptic Oxygen Sensor System (FOXY, Ocean Optics, Dunedin, FL) previously calibrated with both oxygen-saturated and oxygen-depleted (by addition of crystals of Na2SO3) aqueous solutions. The initial rate (5–20% conversion) is plotted against the substrate concentration, and the Michaelis-Menton equation is fitted directly to the data using a primary Hanes plot or Systat SigmaPlot 2000. Values are typically quoted as a mean of at least three to five independent measurements. Different oxygen concentrations are obtained by bubbling the 2OG and Tris-HCl buffer solution with either oxygen or nitrogen gas via a Hamilton syringe. Results with this assay demonstrated the preference of PHD2181–426 for longer substrate fragments. Km values of 21.6 and 1.8 mM were obtained for HIF-1a 556 to 574 and HIF-1a 530 to 698, respectively (Ehrismann et al., 2007). Consequently, a range of substrate concentrations (see ‘‘Reagents,’’ Section 4.3.1) dependent upon the identity of the substrate are recommended for use with this assay.

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Since both DMSO and methanol are incompatible with the FOXY AL-300 probe, inhibitor screening is limited to those soluble in aqueous solution.

4.4. 1-[14C]succinate quantification Although not an assay routinely used in our laboratories, PHD2 activity can also be measured by quantifying the amount of 1-[14C]succinate produced from 5-[14C]-2OG decarboxylation (Cunliffe et al., 1986). This assay requires HPLC (equipped with an online radiochemical detector) separation of the generated succinate from any remaining unreacted 2OG .

5. Direct Measurements of PHD2 Hydroxylation Activity All of the previously described assays rely upon indirect measurement of PHD2 activity, namely quantification of co-substrate/product concentration. The following two assays measure the amount of hydroxylated HIF-a produced after reaction of substrate with PHD2.

5.1. LC/MS identification of hydroxylated HIF-1a 556 to 574 The molecular weight of HIF-1a 556 to 574 (HIF-1a 19mer) is 2254.5 Da and, as such, is amenable to mass spectrometric analyses (Hewitson et al., 2007; McNeill et al., 2005b). Reactions, conditions, and components used with PHD2181–426 and this substrate are identical to those described for the 1-[14C]-CO2 capture assay. However, the reaction is quenched by immediate freezing at –80 rather than the addition of methanol, since this organic molecule is not compatible with the chromatography column used for separation purposes. Following quenching, the samples can be stored at –80 until analysis. A Micromass (now Waters) Q-TOFmicro (quadrupole time-of-flight) mass spectrometer (MS) coupled to an Agilent 1100 capillary liquid chromatography (LC) system with autosampler is used for peptide observation, while a Phenomenex Jupiter C4 column (150  4.6 mm, 5-mm particle size) pre-equilibrated with water/0.1% formic acid/5% acetonitrile is used with a gradient of acetonitrile/0.1% formic acid for separation of peptide from the other assay components. The modified and unmodified HIF-1a 19mer peptides can be separated by this method, although long gradients are required for baseline separation, and the þ16Da shift is easily seen in the mass spectrum. For both the modified and unmodified HIF-1a 19mers, a potassium adduct can be observed and is associated with a mass shift

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of þ39 Da on the respective hydroxylated and non-hydroxylated peptide masses. Reactions using different assay components (e.g.,  ascorbate) and inhibitor testing can all be compared to a standard reaction mixture to determine the relative effects of such variations. The position of hydroxylation can be verified by tandem mass spectrometry (MS/MS) analyses if required; the stereochemistry of hydroxylation (trans-hydroxyproline) has been defined by nuclear magnetic resonance (NMR) analyses.

5.2. pVHL capture assay The pVHL assay (Tuckerman et al., 2004) is based upon the tight interaction between pVHL (von Hippel-Lindau protein) and the hydroxylated Pro564 of HIF-1a (Epstein et al., 2001; Ivan et al., 2001; Jaakkola et al., 2001). Studies have demonstrated that an HIF-1a 19mer peptide (HIF-1a 556–574) is of sufficient length for recognition by pVHL and the PHDs (Hirsila et al., 2003; Hon et al., 2002). The pVHL capture assay is of sufficient sensitivity to allow the measurement of PHD activity in crude cell extracts and recombinant protein preparations. Since mammalian extracts can be used with this procedure, it has been possible to assay all of the full-length PHD enzymes with this format (Tuckerman et al., 2004). 5.2.1. Reagents A standard PHD assay combines the following components in 100 mM Tris-HCL, pH 7.5: PHD2 extract (discussed later) 1 mM biotinylated-HIF-1a 19mer peptide (HIF-1a 556–574) 2 mM 2OG 50 mM Fe(II) 2 mM ascorbate 1 mM DTT 0.3 mg/ml catalase 1 mg/ml bovine serum albumin (BSA) Stop buffer: 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.5% Igepal, 300 mM desferrioxamine mesylate (DFO), 200 mM ethylenediamine tetraacetic acid (EDTA) Capture buffer: 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.5% Igepal, 100 mM DFO All concentrations described are final, typically in a 50-ml volume. [35S]-Methionine-labeled proteins are produced by in vitro transcription/ translation (IVTT) reactions using TNT Quick-Coupled Rabbit Reticulocyte Lysate (Promega, Madison, WI). The system is calibrated by mixing known amounts of hydroxylated and non-hydroxylated biotinylated HIF-1a 19mer peptide with [35S]pVHL.

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The signal obtained from captured [35S]-pVHL can then be correlated with the known amounts of hydroxylated HIF-1a 19mer peptide. Linear calibration curves are obtained and can be used for subsequent calculation. Determination of PHD2 concentration in lysates is via use of purified FLAG-PHD2 from Sf9 cells. Monoclonal antibodies to PHD2 are used to calibrate the immunoblot signal with this purified material and then compared with the signal obtained using a PHD2 lysate. For PHD1 and -3 however, this procedure cannot be followed, since production of highly purified enzyme has so far proved problematic in this laboratory. Instead, [35S]methionine preparations of PHD1 and -3 are compared to known amounts of PHD2 to determine the absolute concentration of each. Using this methodology, the concentration of very small quantities of PHD enzyme can be determined (as low as 4 fmol). 5.2.2. Protocol A reaction mix is made that contains the biotinylated-HIF-1a 19mer peptide, 2OG, Fe(II), ascorbate, DTT, BSA, catalase, and any additional Tris-HCl buffer required to reach the final 50-ml volume. Reactions are initiated by the addition of PHD enzyme followed by incubation at 37 for 6 min. Initial reaction rates are linear over this time period. The reaction is quenched by dilution in an equal volume of ice-cold stop buffer. The DFO and EDTA present in this solution are known iron chelators, and, hence, further reaction of the PHDs is prevented, since iron is a crucial cofactor for these enzymes. Biotinylated-HIF-1a 19mer peptide (5 ml of the stopped reaction) is captured using 2.7  106 streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin, Dynal Biotech, Carlsbad, CA) in capture buffer. Following incubation on ice for 30 min, the stopped reaction mix is removed and the beads washed once with 500 ml of capture buffer. The peptide-coated beads are then incubated with excess [35S]pVHL at 4 for 60 min. Following two washes (750 ml capture buffer), the captured [35S]pVHL is quantified by SDS-PAGE and autoradiography using a phosphorimager. Currently, only water-soluble inhibitors have been screened with this assay technique made by dissolving in 100 mM Tris-HCl, pH 7.5 and inclusion with the main reaction mix. The DMSO/MeOH tolerance with this assay format is currently unknown.

6. Binding Assays As an alternative to catalysis-based assays, we have developed a binding assay (Ehrismann et al., 2007) that utilizes the technique of surface plasmon resonance (SPR) to investigate the binding properties of different HIF-a substrates to PHD2181–426. Both His6-HIF-1a 344 to 503 (NODD) and

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His6-HIF-1a 530 to 698 (CODD) can be covalently coupled to a CM5 sensor chip using the BIACore (Piscataway, NJ) Amine Coupling kit at pH 4.5 for such a purpose. Binding studies on a BIAcore 2000TM sensor are initiated by the injection of 15 ml of PHD2181–426 at 50 ml/min in 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactant-P20, over both control and experimental  cells. The temperature is held constant at 25 . Experiments with PHD2181–426 have been performed over a range of concentrations to allow binding data analysis.

7. Comparison of Assay Formats The assays can essentially be divided into those that directly measure HIF-1a hydroxylation (LC/MS and pVHL capture assay), those that use indirect means to quantify PHD2 activity (1-[14C]-CO2 capture, fluorescence derivatization, oxygen consumption assay), and binding assays (SPR). While the first is normally the preferred option for assays aimed at investigating substrate specificity or inhibition studies, limitations do exist with this type of assay (described later); hence, indirect measurements of PHD2 activity are still routinely used. A main drawback with the use of indirect measurements is the potential for (partial) uncoupling of 2OG oxidation from hydroxylation with this family of enzymes. Under appropriate circumstances (e.g., misfolded protein, a suboptimal substrate) the 2OGdependent oxygenases can catalyze the oxygen-dependent turnover of 2OG that is not directly linked to prime substrate hydroxylation (Costas et al., 2004; Hausinger, 2004; Hewitson et al., 2005). It may appear that the enzyme is displaying activity, but, in fact, each cycle is nonproductive with respect to hydroxylation. This uncoupled turnover can, however, be partly controlled for in the previously described assay formats by the use of a ‘‘no substrate control.’’ Confirmation of product formation (e.g., by MS analyses) is usually required, even if controls are used. The 1-[14C]-CO2 and pVHL capture assays are well-established means for assaying the PHD enzymes and have been used by a variety of groups successfully. In both cases, radiochemicals are employed, which may limit use in some laboratories. The pVHL assay is particularly useful in that, not only can small quantities of PHD be assayed (4–10 fmols in 10–25 ml), but non-purified material (i.e., lysates) can also be used. However, this assay is limited by the number of samples that can be processed in a given time (10–20 per day). Note also that peptide-derived substrates are required when the assay is used in fully quantitative format, and, to allow calibration of the system, both the hydroxylated and non-hydroxylated peptides must be synthesized. This assay is, however, potentially suitable for modification to enable its use in a high-throughput format.

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All other assay formats require the use of purified components, both in terms of the enzyme and the substrate. High background readings in 2OG turnover, 2-oxo acid content, and oxygen consumption may result from use of lysates with the 1-[14C]-CO2 capture, fluorescence derivatization, and oxygen consumption assay. Ascorbate is incompatible with two of the current assay formats: the fluorescence derivatization and oxygen consumption assays. PHD2 requires ascorbate (Hewitson et al., 2007; Hirsila et al., 2003) to stimulate optimal in vitro activity as observed with several other members of the 2OGdependent oxygenase family (e.g., procollagen prolyl hydroxylase [Myllyla et al., 1984]). It must be assumed that, with these assay techniques, the activity of PHD2 is less than maximal, which could potentially affect the measurement of kinetic parameters. It is also why, in the case of the oxygen consumption assay, such high (millimolar) concentrations of PHD2 and HIF-1a are required with the current sensors. In terms of screening for inhibitors with purified enzymes, the 1-[14C]-CO2 capture assay can be routinely used. This assay is less reliable with partially purified material due to the potential presence of interfering enzymes that utilize 2OG as a substrate. Thirty inhibitors at a single point concentration can be screened in a day, which is higher throughput than can be achieved with most of the other reported techniques. Furthermore, small molecules containing oxo-acid functionality cannot be used with the fluorescence derivatization assay, while both the oxygen consumption and pVHL capture assays are currently limited to those molecules soluble in aqueous solution. The LC/MS assay has proved to be extremely useful in terms of validating peptides as substrates for PHD2. However, this technique can be timeconsuming when considering the long (>30 min) run times required per sample. Inhibitor studies using this technique have yet to be reported, but should be a possibility with improving MS techniques. In conclusion, still no definitive, readily-usable assay has been described for the PHDs nor, in fact, for the 2OG-dependent oxygenase family as a whole. A useful advance would be the development of a generic continuous assay employing routine spectroscopic methodology and without the use of coupled enzymes. Despite the lack of such an assay, in the case of the PHDs, careful consideration of the assay formats described here should allow for the selection of the technique most appropriate for the required use.

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