Sự phá huỷ HIFα do pVHL qua việc hydroxyl hoá proline: Hệ quả đối với cảm nhận O 2
Tóm tắt
HIF (yếu tố tăng trưởng thiếu oxy) là một yếu tố phiên mã đóng vai trò then chốt trong việc thích nghi tế bào với sự thay đổi độ cung cấp oxy. Khi có oxy, HIF được nhắm mục tiêu phá huỷ bởi một phức hợp ubiquitin E3 chứa protein ức chế khối u von Hippel–Lindau (pVHL). Chúng tôi đã phát hiện rằng pVHL của người liên kết với một đoạn peptide bắt nguồn từ HIF khi đoạn trung gian này có một dư lượng proline bảo tồn được hydroxyl hoá. Do quá trình hydroxyl hoá proline đòi hỏi oxy phân tử và ion Fe 2+ , sự biến đổi protein này có thể đóng vai trò quan trọng trong việc nhận biết oxy ở động vật có vú.
Từ khóa
#HIF #yếu tố tăng trưởng thiếu oxy #pVHL #proline hydroxyl hoá #cảm nhận oxy #ubiquitin E3 #protein ức chế khối u von Hippel–Lindau #oxy #ion Fe2+Tài liệu tham khảo
Farwestern blot assays GST pull-down assays and in vitro ubiquitination assays were performed as described (7). There are three HIFα genes (HIF-1α HIF-2α and HIF-3α). The 786-O renal carcinoma cells used in Fig. 1 produce HIF-2α but not HIF-1α (12).
Chowdary D., Dermody J., Jha K., Ozer H., Mol. Cell. Biol. 14, 1997 (1994).
Normoxic cells were grown at restrictive (39°C) or permissive (33°C) temperature for 12 hours. Hypoxic cells were grown in the presence of 1% O 2 for 6 hours at permissive temperature and then at restrictive temperature for 12 hours. The cells were lysed by incubation in EBC [50 mM tris-HCl (pH 8.0) 120 mM NaCl and 0.5% NP-40] for 5 min at 4°C. After centrifugation at 14 000 g for 3 min the clarified lysate was boiled in SDS.
M. Ivan M. Ohh J. Asara W. S. Lane W. G. Kaelin unpublished data.
Coupled in vitro transcription-translation of 35 S-labeled proteins was performed according to the manufacturer's instructions (TNT; Promega Madison WI). In vitro translation of 3 H-P-labeled Gal4-HIF(555-575) was done similarly in a 2-ml reaction containing 450 μl of l -[2 3 4 5- 3 H]proline (New England Nuclear).
For peptide binding studies 1 μg of biotinylated peptide was bound to 30 μl of monomeric avidin agarose (Pierce). Where indicated the peptide was preincubated with 50 μl of rabbit reticulocyte lysate for 90 min at 30°C. The agarose was then washed three times with NETN [20 mM tris-HCl (pH 8.0) 100 mM NaCl 1 mM EDTA and 0.5% NP-40) and used in binding reactions containing 4 μl of 35 S-labeled HA-pVHL in 500 μl of EBC or 500 μl of 35 S-labeled cell extract (equivalent to cells from a subconfluent 100-mm dish). After a 1-hour incubation at 4°C the agarose was washed four times with NETN. Bound proteins were eluted by boiling in sample buffer containing SDS and detected by autoradiography.
C. Pugh J. O'Rourke
Xenopus egg extracts were prepared [
] and stored frozen. Degradation reactions contained 8 μl of egg extract 0.1 μl of cycloheximide (100 mg/ml) 0.25 μl of energy regeneration mix 0.25 μl of bovine ubiquitin and 0.4 μl of 35 S-labeled HIF and were carried out at room temperature. At the indicated time points 1-μl aliquots were removed and placed in sample buffer. Samples were resolved on 5 to 15% gradient gels and analyzed by autoradiography.
After purification by high-performance liquid chromatography (HPLC) peptide (1 μg) was bound to 30 μl of monomeric avidin agarose and incubated with 100 μl of rabbit reticulocyte lysate at room temperature for 1 hour with tumbling. After a brief centrifugation the reticulocyte lysate was removed fresh reticulocyte lysate was added and the cycle was repeated six times. The agarose was then washed four times with NETN and once with phosphate-buffered saline (PBS). The modified peptide was eluted in 50 μl of 20 mM ammonium acetate (pH 7.0) and 2 mM biotin. Pro 564 hydroxylation was confirmed by MS/MS using microcapillary HPLC directly coupled to a Finnigan LCQ DECA quadrupole ion trap mass spectrometer equipped with a custom nanoelectrospray source. Targeted ion MS/MS of the doubly protonated ion at mass/charge ratio ( m / z ) 1267 for the HIF(556-575) peptides was performed with an isolation width of 2.5 daltons and relative collision energy of 30%.
See Science Online (www.sciencemag.org/cgi/content/full/1059817/DC1).
We immunoprecipitated 2 ml of 3 H-P-labeled Gal4-HIF(555-575) in vitro translated with 50 μg of antibody to HA (anti-HA; 12CA5 Roche); the product was resolved on a 12% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Gal4-HIF(555-575) was visualized by autoradiography and the corresponding region of the membrane was excised and hydrolyzed by incubation in 100 μl of 10 N HCl at 105°C for 3 hours. Samples were evaporated to dryness resuspended in 20 μl of H 2 O containing 10 μg of unlabeled l -proline and trans-4-hydroxy- l -proline (Sigma) (as standards) and resolved by 2D TLC using phenol-distilled H 2 O in the first dimension and N -butanol–acetic acid–H 2 O in the second dimension [
]. After visualization of standards with ninhydrin radiolabeled proline was detected by autoradiography.
ts20 cells were transfected with pIRES-HA-VHL pIRES-HA-VHL (Y98H) or pIRES-Neo (Invitrogen) and selected in the presence of G418 (1 mg/ml). Individual G418-resistant colonies were isolated using cloning cylinders and expanded. Cells producing HA-VHL or HA-VHL (Y98H) were identified by anti-HA immunoblot analysis.
ts20 cells were grown at restrictive or permissive temperature for 14 hours methionine-starved for 90 min and then grown in methionine-free media supplemented with [ 35 S]methionine (500 μCi/ml) for 90 min. Cells were washed once with cold PBS lysed in EBC and immunoprecipitated with anti-HA (12CA5 Roche) or anti–HIF-1α (NB100-105 Novus). After five washes with NETN bound proteins were eluted by boiling in sample buffer or by incubation in 65 μl of PBS containing 7 μg of the indicated peptide. 786-O subclones were starved for 1 hour grown in methionine-free media supplemented with [ 35 S]methionine (500 μCi/ml) for 3 hours washed once with ice-cold PBS and lysed in EBC.
We thank J. Lee and P. Morrison for carrying out the MALDI-TOF analysis H. Ozer for the ts20 cells M. Kirschner for help with the Xenopus degradation assays J. Crawford for expert peptide synthesis M. Modabber for help with artwork and C. W. Park for contributions to the early stages of the work. Supported by grants from NIH and the Murray Foundation.