Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation Complex by O₂-Regulated Prolyl Hydroxylation

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Hypoxia (<0.1% oxygen) was obtained in a workstation with O₂ and CO₂ and temperature control (Ruskinn Technologies, Leeds, UK). RCC4-VHL.HA, labeling conditions, and coimmunoprecipitation assays have been described previously (11). We used 12.5 μM MG132 for proteasomal inhibition. For hypoxic harvest, cells were lysed in the workstation with buffers deoxygenated in the chamber overnight. For standard harvest, the cells were removed from the chamber after hypoxic exposure, before cell lysis.

pcDNA3.VHL.HA and pcDNA3.HIF-1α were used to program TNT reticulocyte lysate (Promega). When programming in hypoxia, the reaction mix was preincubated in the workstation for 10 min before addition of the DNA template. An aliquot was removed from the workstation for transcription/translation under ambient oxygenation.

Proteins were expressed in rabbit reticulocyte or wheat germ IVTT systems (Promega), in insect cells with the use of a baculoviral system, and in bacteria. IVTTs were programmed with pcDNA3-based vectors encoding subdomains of HIF-1α or pVHL.HA as indicated. For recombinant baculoviral expression, Sf9 insect cells were infected with pFastBac1 vectors (GibcoBRL) encoding PK.HIF-1α(344-698) and HIF-1α(1-826).PK and harvested 60 hours after infection. In bacteria, pVHL was expressed as glutathione-S-transferase together with elongins B and C (GST-VBC complex) and HIF-1α as a maltose-binding protein fusion [pMAL.HIF-1α(344-698)].

pGal/HIF-1α549-582/VP16 was used to program reticulocyte lysate in the presence of unlabeled methionine. The fusion protein was immunoprecipitated with beads precoated with anti-Gal4 RK5C1 (Santa Cruz). Immunoprecipitated HIF-1α fusion was washed with NETN buffer [50 mM tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, and 0.5% NP-40], and the beads were incubated with cell lysate in hypotonic extraction buffer [HEB: 20 mM tris (pH 7.5), 5 mM KCl, 1.5 mM MgCl₂, 1 mM dithiothreitol] for 60 min at 22°C. The beads were then washed with NETN containing DFO (100 μM) and incubated for 2 hours at 4°C with ³⁵S-methionine–labeled pVHL.HA.

HIF-1α(1-826).PK or PK.HIF-1α(344-698) produced from baculoviruses was immunoprecipitated with antiPK (Serotec). Bead-bound immunoprecipitates were incubated under test conditions and assayed for pVHL.HA capture.

D. R. Mole, data not shown.

For peptide-blocking assays, peptides were preincubated in cell extract or other conditions for 60 min at 30°C and then added (final concentration, 1 μM) to NETN buffer containing a mixture of HIF-1α and pVHL.HA.

Samples for mass spectroscopic analyses were biotinylated synthetic peptides 19:WT (HIF-1α residues 556 to 574) or 34:WT (HIF-1α residues 549 to 582) or baculoviral PK-tagged HIF-1α. After modification by mammalian cell lysates, the material was purified by streptavidin/biotin capture (synthetic peptides) or anti-PK immunoprecipitation and SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (baculoviral HIF). Proteolytic digestion was performed on the beads (synthetic peptides) or in-gel with trypsin and V8 protease at pH 7.8 or V8 protease at pH 4.5. Samples were lyophilized and dissolved in aqueous 0.1% trifluoroacetic acid. Peptides were concentrated, desalted on a 300-μm inside diameter/5-mm length C18 PepMap column (LC Packings, San Francisco, CA), and eluted with 80% acetonitrile. The high-performance liquid chromatography (CapLC, Waters, Milford, MA) was coupled through a Nano-LC inlet to a Q-TOF mass spectrometer (Micromass, Manchester, UK) equipped with a nanoelectrospray Z-spray source. The eluted peptide mixture was analyzed by tandem mass spectrometric sequencing with an automated MS-to-MS/MS switching protocol. Online determination of precursor-ion masses was performed over the m/z range from 300 to 1200 atomic mass units in the positive charge detection mode with a cone voltage of 30 V. The collision-induced dissociation for peptide sequencing by MS/MS was performed with argon gas at 20 to 40 eV and a three-dimensional quadrupole resolution.

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In pVHL capture assays with biotinylated peptides, the peptide was preincubated with cell extract or buffer for 30 min at 30°C and then assayed for ability to bind ³⁵S-labeled pVHL.HA.

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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; X, any amino acid; and Y, Tyr.

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Prolyl 4-hydroxylase activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo[1-¹⁴C]glutarate [

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] with recombinant human type I and II prolyl 4-hydroxylases expressed in insect cells. Each reaction contained 0.5 or 1.0 mg of peptide.

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Supported by grants from the Wellcome Trust and the Medical Research Council. P.J. is Junior Research Fellow of the Academy of Finland. We thank J. Myllyharju for performing the prolyl-4-hydroxylase assay; K. Brindle, R. Hider, K. Kivirikko, E. Maher, and R. Wolfe for advice; N. Pavletich for GST-VCB expression constructs; and E. Gibson for synthesis of 2-oxoglutarate analogs.