A Conserved Family of Prolyl-4-Hydroxylases That Modify HIF
Tóm tắt
Mammalian cells respond to changes in oxygen availability through a conserved pathway that is regulated by the hypoxia-inducible factor (HIF). The alpha subunit of HIF is targeted for degradation under normoxic conditions by a ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF. We identified a conserved family of HIF prolyl hydoxylase (HPH) enzymes that appear to be responsible for this posttranslational modification. In cultured mammalian cells, inappropriate accumulation of HIF caused by forced expression of the HIF-1α subunit under normoxic conditions was attenuated by coexpression of HPH. Suppression of HPH in cultured
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Tài liệu tham khảo
N. C. Bacon et al. Biochem. Biophys. Res. Commun. 249 811 (1998).
P. J. Kallio W. J. Wilson S. O'Brien
K. I. Kivirikko T. Pihlajaniemi in Advances in Enzymology and Related Areas of Molecular Biology D. L. Purich Ed. (Wiley New York 1998) vol. 72 pp. 325–398.
Coding regions were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from total RNA prepared from human cell lines with oligonucleotides derived from the following sequences (GenBank accession number): HPH-1 (XM_012332) HPH-2 () HPH-3 () candidate A (NM_017732) candidate B () candidate C () and candidate D (). A splice variant of HPH-2 was used in which residues 76 to 177 were omitted. Point mutations in HPH-1 were generated by PCR. Each cDNA was cloned into the pcDNA3.1/V5-HIS vector (Invitrogen) in frame with the COOH-terminal V5-HIS tag.
Candidate polypeptides were synthesized with the TNT Coupled Reticulocyte Lysate System (Promega) for 1 hour at 30°C. Expression of each gene product was confirmed by Western blot analysis with an antibody specific for the COOH-terminal V5 tag. 12.5 μl of each in vitro transcription/translation reaction was incubated for 30 min at 30°C in a reaction buffer containing 20 mM tris-Cl (pH 7.5) 5 mM KCl 1.5 mM MgCl 2 1 mM dithiothreitol 2 mM 2-oxoglutarate 2 mM ascorbate and 250 μM FeSO 4 in the presence of 30 μl of ImmunoPure Immobilized Streptavidin beads that had previously been incubated with 1 μg of peptide for 30 min at room temperature and washed three times to remove excess peptide. After incubation the beads were washed three times with 1 ml of cold NTEN buffer [20 mM tris-Cl (pH 8.0) 100 mM NaCl 1 mM EDTA and 0.5% NP-40] and incubated for 10 min at 4°C with about 35 kcpm of [ 35 S]-labeled human VHL in 500 μl of EBC buffer [50 mM tris-Cl (pH 8.0) 120 mM NaCl and 0.5% NP-40]. The beads were washed three times with cold NTEN buffer and bound [ 35 S]-VHL was measured by scintillation counting. [ 35 S]-labeled human VHL was synthesized from the human VHL cDNA cloned into the pcDNA3.1/V5-HIS vector (Invitrogen) with the TNT Coupled Reticulocyte Lysate System (Promega) and [ 35 S]- l -Met (Amersham Pharmacia Biotech) and desalted with a PD-10 column (Amersham Pharmacia Biotech).
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; and Y Tyr.
HPH-2 was cloned into the pMBP-parallel1 vector (36) and expressed in the BL21-CodonPlus-RIL E. coli strain (Stratagene). Recombinant protein was purified by virtue of the NH 2 -terminal MBP protein with amylose resin (New England Biolabs) and eluted in the presence of 10 mM maltose.
One microgram of peptide was incubated with 1 μg of recombinant HPH-2 in reaction buffer for 1 hour at 30°C. Peptide mass was determined by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry.
Transfection experiments were performed as in (24) with DNA amounts indicated in the text. Western blot analysis with an antibody specific for the COOH-terminal V5 tag was used to confirm expression of each polypeptide after transfection of 1 μg of each expression vector.
KC167 cells were maintained at 24°C in CCM 3 media (HyClone). Cells were treated as follows: initially 4 × 10 5 KC167 cells were incubated for 7 days in the presence of 25 μg of double-stranded RNAs of about 700 base pairs in length generated with the T7 MEGAscript Kit (Ambion) with double-stranded RNA refreshed daily. Cells were incubated under normoxic (20% O 2 ) or hypoxic (1% O 2 99% N 2 ) conditions for an additional 15 hours and total RNA was prepared with RNA STAT-60 (Tel-Test). RNAs were resolved by electrophoresis in a 1.2% agarose gel in the presence of 1.8% formaldehyde transferred to nitrocellulose filters and hybridized with the indicated 32 P-labeled DNA probes.
M. Detmar et al. J. Invest. Dermatol. 111 1 (1998).
We thank members of the McKnight Abrams and Wang laboratories for advice and encouragement; L. Wu C. Michnoff and F. Hirani for technical assistance; L. Huang for peptide synthesis; Y. Zhao for assistance with mass spectrometry; and N. Grishin for assistance with database searches. Funded by a National Research Service Award from the NIH (R.K.B.) NIH grants DK52031 and MH59388 (S.L.M.) and endowment funds provided to S.L.M. by an anonymous donor.