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• W2023795623 abstract "The Tibetan population has adapted to the chronic hypoxia of high altitude. Tibetans bear a genetic signature in the prolyl hydroxylase domain protein 2 (PHD2/EGLN1) gene, which encodes for the central oxygen sensor of the hypoxia-inducible factor (HIF) pathway. Recent studies have focused attention on two nonsynonymous coding region substitutions, D4E and C127S, both of which are markedly enriched in the Tibetan population. These amino acids reside in a region of PHD2 that harbors a zinc finger, which we have previously discovered binds to a Pro-Xaa-Leu-Glu (PXLE) motif in the HSP90 cochaperone p23, thereby recruiting PHD2 to the HSP90 pathway to facilitate HIF-α hydroxylation. We herein report that the Tibetan PHD2 haplotype (D4E/C127S) strikingly diminishes the interaction of PHD2 with p23, resulting in impaired PHD2 down-regulation of the HIF pathway. The defective binding to p23 depends on both the D4E and C127S substitutions. We also identify a PXLE motif in HSP90 itself that can mediate binding to PHD2 but find that this interaction is maintained with the D4E/C127S PHD2 haplotype. We propose that the Tibetan PHD2 variant is a loss of function (hypomorphic) allele, leading to augmented HIF activation to facilitate adaptation to high altitude. The Tibetan population has adapted to the chronic hypoxia of high altitude. Tibetans bear a genetic signature in the prolyl hydroxylase domain protein 2 (PHD2/EGLN1) gene, which encodes for the central oxygen sensor of the hypoxia-inducible factor (HIF) pathway. Recent studies have focused attention on two nonsynonymous coding region substitutions, D4E and C127S, both of which are markedly enriched in the Tibetan population. These amino acids reside in a region of PHD2 that harbors a zinc finger, which we have previously discovered binds to a Pro-Xaa-Leu-Glu (PXLE) motif in the HSP90 cochaperone p23, thereby recruiting PHD2 to the HSP90 pathway to facilitate HIF-α hydroxylation. We herein report that the Tibetan PHD2 haplotype (D4E/C127S) strikingly diminishes the interaction of PHD2 with p23, resulting in impaired PHD2 down-regulation of the HIF pathway. The defective binding to p23 depends on both the D4E and C127S substitutions. We also identify a PXLE motif in HSP90 itself that can mediate binding to PHD2 but find that this interaction is maintained with the D4E/C127S PHD2 haplotype. We propose that the Tibetan PHD2 variant is a loss of function (hypomorphic) allele, leading to augmented HIF activation to facilitate adaptation to high altitude. Approximately 25,000 years ago, humans colonized the Tibetan plateau, which has an altitude of ∼14,000 feet. Tibetan adaptation to the extreme conditions of this plateau, particularly the marked hypoxia at this altitude (oxygen concentration is 60% of that at sea level), has provided one of the most striking examples of human adaptation (1Scheinfeldt L.B. Tishkoff S.A. Recent human adaptation: genomic approaches, interpretation and insights.Nat. Rev. Genet. 2013; 14: 692-702Crossref PubMed Scopus (82) Google Scholar, 2Beall C.M. Human adaptability studies at high altitude: Research designs and major concepts during fifty years of discovery.Am. J. Hum. Biol. 2013; 25: 141-147Crossref PubMed Scopus (46) Google Scholar). Compared with Andeans, who also reside at high altitude, Tibetans display relatively low red cell mass and pulmonary arterial pressure, and relatively high ventilation and exhaled nitric oxide (2Beall C.M. Human adaptability studies at high altitude: Research designs and major concepts during fifty years of discovery.Am. J. Hum. Biol. 2013; 25: 141-147Crossref PubMed Scopus (46) Google Scholar). A series of genome-wide investigations of the Tibetan population have recently provided convincing evidence for genetic adaptation (3Beall C.M. Cavalleri G.L. Deng L. Elston R.C. Gao Y. Knight J. Li C. Li J.C. Liang Y. McCormack M. Montgomery H.E. Pan H. Robbins P.A. Shianna K.V. Tam S.C. Tsering N. Veeramah K.R. Wang W. Wangdui P. Weale M.E. Xu Y. Xu Z. Yang L. Zaman M.J. Zeng C. Zhang L. Zhang X. Zhaxi P. Zheng Y.T. Natural selection on EPAS1 (HIF2α) associated with low hemoglobin concentration in Tibetan highlanders.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 11459-11464Crossref PubMed Scopus (590) Google Scholar, 4Simonson T.S. Yang Y. Huff C.D. Yun H. Qin G. Witherspoon D.J. Bai Z. Lorenzo F.R. Xing J. Jorde L.B. Prchal J.T. Ge R. Genetic evidence for high-altitude adaptation in Tibet.Science. 2010; 329: 72-75Crossref PubMed Scopus (799) Google Scholar, 5Yi X. Liang Y. Huerta-Sanchez E. Jin X. Cuo Z.X. Pool J.E. Xu X. Jiang H. Vinckenbosch N. Korneliussen T.S. Zheng H. Liu T. He W. Li K. Luo R. Nie X. Wu H. Zhao M. Cao H. Zou J. Shan Y. Li S. Yang Q. Asan Ni P. Tian G. Xu J. Liu X. Jiang T. Wu R. Zhou G. Tang M. Qin J. Wang T. Feng S. Li G. Huasang Luosang J. Wang W. Chen F. Wang Y. Zheng X. Li Z. Bianba Z. Yang G. Wang X. Tang S. Gao G. Chen Y. Luo Z. Gusang L. Cao Z. Zhang Q. Ouyang W. Ren X. Liang H. Huang Y. Li J. Bolund L. Kristiansen K. Li Y. Zhang Y. Zhang X. Li R. Yang H. Nielsen R. Wang J. Sequencing of 50 human exomes reveals adaptation to high altitude.Science. 2010; 329: 75-78Crossref PubMed Scopus (1044) Google Scholar, 6Bigham A. Bauchet M. Pinto D. Mao X. Akey J.M. Mei R. Scherer S.W. Julian C.G. Wilson M.J. López Herráez D. Brutsaert T. Parra E.J. Moore L.G. Shriver M.D. Identifying signatures of natural selection in Tibetan and Andean populations using dense genome scan data.PLoS Genet. 2010; 6: e1001116Crossref PubMed Scopus (411) Google Scholar, 7Peng Y. Yang Z. Zhang H. Cui C. Qi X. Luo X. Tao X. Wu T. Ouzhuluobu Basang Ciwangsangbu Danzengduojie Chen H. Shi H. Su B. Genetic variations in Tibetan populations and high-altitude adaptation at the Himalayas.Mol. Biol. Evol. 2011; 28: 1075-1081Crossref PubMed Scopus (274) Google Scholar, 8Wang B. Zhang Y.B. Zhang F. Lin H. Wang X. Wan N. Ye Z. Weng H. Zhang L. Li X. Yan J. Wang P. Wu T. Cheng L. Wang J. Wang D.M. Ma X. Yu J. On the origin of Tibetans and their genetic basis in adapting high-altitude environments.PLoS One. 2011; 6: e17002Crossref PubMed Scopus (115) Google Scholar, 9Xu S. Li S. Yang Y. Tan J. Lou H. Jin W. Yang L. Pan X. Wang J. Shen Y. Wu B. Wang H. Jin L. A genome-wide search for signals of high-altitude adaptation in Tibetans.Mol. Biol. Evol. 2011; 28: 1003-1011Crossref PubMed Scopus (232) Google Scholar). These studies consistently identified genetic signatures in the EGLN1 (also known as prolyl hydroxylase domain protein 2, or PHD2) 2The abbreviations used are: PHDprolyl hydroxylase domain proteinHIFhypoxia-inducible factorHSPheat shock proteinMEFmouse embryonic fibroblastVHLvon Hippel-Lindau proteinHTHis6-tagged. and EPAS1 (also known as hypoxia-inducible factor-2α, or HIF2A) genes in the Tibetan population (10Simonson T.S. McClain D.A. Jorde L.B. Prchal J.T. Genetic determinants of Tibetan high-altitude adaptation.Hum. Genet. 2012; 131: 527-533Crossref PubMed Scopus (97) Google Scholar). prolyl hydroxylase domain protein hypoxia-inducible factor heat shock protein mouse embryonic fibroblast von Hippel-Lindau protein His6-tagged. PHD2 and HIF2A are compelling candidate genes in this population because of the key positions that they occupy in the HIF pathway, which is the central pathway for transducing changes in oxygen tension to changes in gene expression (11Semenza G.L. Hypoxia-inducible factors in physiology and medicine.Cell. 2012; 148: 399-408Abstract Full Text Full Text PDF PubMed Scopus (2086) Google Scholar, 12Kaelin Jr., W.G. Ratcliffe P.J. Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.Mol. Cell. 2008; 30: 393-402Abstract Full Text Full Text PDF PubMed Scopus (2203) Google Scholar, 13Majmundar A.J. Wong W.J. Simon M.C. Hypoxia-inducible factors and the response to hypoxic stress.Mol. Cell. 2010; 40: 294-309Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar). Under normoxic conditions, PHD2 site-specifically prolyl hydroxylates the α subunit of HIF (of which there are three paralogues: HIF1-α, HIF-2α, and HIF-3α). In HIF-1α, the primary site of hydroxylation is Pro-564; in HIF-2α, it is Pro-531 (14Lee F.S. Percy M.J. The HIF pathway and erythrocytosis.Annu. Rev. Pathol. 2011; 6: 165-192Crossref PubMed Scopus (134) Google Scholar). This post-translational modification allows recognition by the von Hippel-Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that then targets HIF-α for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation is attenuated, leading to the stabilization of HIF-α, its dimerization with HIF-β, and activation of HIF target genes. These genes participate in a broad range of processes that promote systemic and cellular adaptation to hypoxia. HIF-1α and HIF-2α have overlapping as well as distinct gene targets. For example, HIF-1α activates many of the genes that encode for glycolytic enzymes, whereas HIF-2α is the principal regulator of the ERYTHROPOIETIN (EPO) gene (13Majmundar A.J. Wong W.J. Simon M.C. Hypoxia-inducible factors and the response to hypoxic stress.Mol. Cell. 2010; 40: 294-309Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 14Lee F.S. Percy M.J. The HIF pathway and erythrocytosis.Annu. Rev. Pathol. 2011; 6: 165-192Crossref PubMed Scopus (134) Google Scholar). The current challenge is to identify the molecular basis for how Tibetan-associated haplotypes in the PHD2 and HIF2A genes promote adaptation to high altitude. With regard to the PHD2 gene, resequencing of this gene in 46 Tibetan individuals ruled out a classic de novo mutation sweep (15Xiang K. Ouzhuluobu Peng Y. Yang Z. Zhang X. Cui C. Zhang H. Li M. Zhang Y. Bianba Gonggalanzi Basang Ciwangsangbu Wu T. Chen H. Shi H. Qi X. Su B. Identification of a Tibetan-specific mutation in the hypoxic gene EGLN1 and its contribution to high-altitude adaptation.Mol. Biol. Evol. 2013; 30: 1889-1898Crossref PubMed Scopus (112) Google Scholar). Instead, this study reported on selection from standing variation. In particular, there is significant enrichment in the Tibetan population of two nonsynonymous coding region single-nucleotide polymorphisms, originally identified by others (16Lorenzo, F. R., Simonson, T. S., Yang, Y., Ge, R., Prchal, J. T., (2010) American Society of Hematology Annual Meeting, Dec 4–7, 2010, Orlando, FL (The American Society of Hematology, Washington, D. C.), Abstract 2602Google Scholar), that reside in exon 1 of the PHD2 gene: rs186996510 and rs12097901. The single-nucleotide polymorphisms are predicted to produce D4E and C127S amino acid substitutions, respectively, in the PHD2 protein. Amino acid residues that are essential for the catalytic activity of PHD2 are encoded for largely by exons 2–5 of the PHD2 gene. Exon 1 (amino acids 1–297), in contrast, is most notable for a zinc finger of the MYND (myloid, Nervy, DEAF-1) type, which encompasses amino acids 21–58. This zinc finger shows strong phylogenetic conservation (17Loenarz C. Coleman M.L. Boleininger A. Schierwater B. Holland P.W. Ratcliffe P.J. Schofield C.J. The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens.EMBO Rep. 2011; 12: 63-70Crossref PubMed Scopus (175) Google Scholar, 18Rytkönen K.T. Williams T.A. Renshaw G.M. Primmer C.R. Nikinmaa M. Molecular evolution of the metazoan PHD-HIF oxygen-sensing system.Mol. Biol. Evol. 2011; 28: 1913-1926Crossref PubMed Scopus (103) Google Scholar) and in fact is present in the PHD2 orthologue in the simplest metazoan, Trichoplax adhaerens (17Loenarz C. Coleman M.L. Boleininger A. Schierwater B. Holland P.W. Ratcliffe P.J. Schofield C.J. The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens.EMBO Rep. 2011; 12: 63-70Crossref PubMed Scopus (175) Google Scholar). We have recently discovered that the function of this zinc finger is to couple PHD2 to the HSP90 pathway (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Specifically, it binds to a Pro-Xaa-Leu-Glu (PXLE) motif that is present in select HSP90 cochaperones such as p23, and this allows recruitment of PHD2 to HSP90 to facilitate prolyl hydroxylation of HIF-1α, which itself is an HSP90 client protein (20Isaacs J.S. Jung Y.J. Mimnaugh E.G. Martinez A. Cuttitta F. Neckers L.M. Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1α-degradative pathway.J. Biol. Chem. 2002; 277: 29936-29944Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 21Katschinski D.M. Le L. Heinrich D. Wagner K.F. Hofer T. Schindler S.G. Wenger R.H. Heat induction of the unphosphorylated form of hypoxia-inducible factor-1α is dependent on heat shock protein-90 activity.J. Biol. Chem. 2002; 277: 9262-9267Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 22Minet E. Mottet D. Michel G. Roland I. Raes M. Remacle J. Michiels C. Hypoxia-induced activation of HIF-1: role of HIF-1α-Hsp90 interaction.FEBS Lett. 1999; 460: 251-256Crossref PubMed Scopus (279) Google Scholar). The presence of the Tibetan-associated rs186996510 (D4E) and rs12097901 (C127S) single-nucleotide polymorphisms in exon 1 of the PHD2 gene led us to explore the possibility that this haplotype might affect PHD2 function, potentially through its zinc finger. Here, we provide evidence that the Tibetan D4E/C127S haplotype impairs PHD2 function and that mechanistically, it leads to a dramatically decreased ability of the PHD2 zinc finger to associate with the HSP90 cochaperone p23. Remarkably, this impairment depends on both the D4E and C127S amino acid substitutions. On the basis of these results, we conclude that the Tibetan PHD2 D4E/C127S haplotype leads to a loss of function (hypomorphic) PHD2 allele, and we propose that this is a key mechanism by which the HIF pathway has been reconfigured for adaptation to chronic hypoxia in this population. pcDNA3-HA-PHD2 (1–131) was constructed by subcloning the 0.4-kb BamHI/NotI (partial digest) fragment encoding residues 1–131 of pcDNA3-HA-PHD2 (1–196) (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) into the BamHI/NotI site of pcDNA3-HA. pcDNA3-HA-PHD2 (1–173) was constructed by digesting pcDNA3-HA-PHD2 (1–196) with BlpI/XhoI, blunting the ends with the Klenow fragment of Escherichia coli DNA polymerase I, and then religating. pcDNA3-HA-PHD2 (1–131) D4E/C127S was constructed by amplifying by PCR a 0.4-kb band using pcDNA3-HA-PHD2 (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) as a template and the following primers: PHD2 D4E 5′, 5′-AAG GGG ATC CGA ATT CTT AAG CTC GAC ATG GCC AAT GAG AGC GGC GGT CCC GGC GGG CCG AGC CCG AGC GAG-3′; and PHD2 C127S 3′, 5′-GCC GGC GGC CGC TCT GGA GGG GCT AGC AGC CGC CGC TGG GTC GGC CGG-3′. The product was digested with BamHI and NotI and subcloned into the BamHI/NotI site of pcDNA3-HA to give pcDNA3-HA-PHD2 (1–131) D4E/C127S. pcDNA3-HA-PHD2 (1–196) D4E/C127S was constructed by subcloning the 0.23-kb NotI fragment pcDNA3-HA-PHD2 (1–196) into the NotI site of pcDNA3-HA-PHD2 (1–131) D4E/C127S. pcDNA3-HA-PHD2 (1–173) D4E/C127S was constructed by digesting pcDNA3-HA-PHD2 (1–196) D4E/C127S with BlpI/XhoI, blunting the ends with the Klenow fragment of E. coli DNA polymerase I, and then religating. pcDNA3-HA-PHD2 D4E/C127S was constructed by subcloning the 0.6-kb BamHI/XhoI fragment from pcDNA3-HA-PHD2 (1–196) D4E/C127S into the BamHI/XhoI site of pcDNA3-HA-PHD2 (130–426) (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). pcDNA5/FRT/TO-FlagPHD2 D4E/C127S was constructed by subcloning the 0.6-kb BamHI/XhoI fragment of pcDNA3-HA-PHD2 (1–196) D4E/C127S into the BamHI/XhoI site of pcDNA5/FRT/TO-FlagPHD2 (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). pcDNA3-FlagPHD2 D4E/C127S was constructed by subcloning the 0.6 kb BamHI/XhoI fragment of pcDNA3-HA-PHD2 (1–196) D4E/C127S into the BamHI/XhoI site of pcDNA3-FlagPHD2 (23Huang J. Zhao Q. Mooney S.M. Lee F.S. Sequence determinants in hypoxia-inducible factor-1α for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3.J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). pcDNA3-HA-PHD2 (1–196) D4E was constructed by amplifying by PCR a 0.6-kb band using pcDNA3-HA-PHD2 (1–196) as a template and the following primers, PHD2 D4E 5′ (see above) and BGH rev: 5′-TAG AAG GCA CAG TCG AGG-3′. The product was digested with BamHI and XhoI and subcloned into the BamHI/XhoI site of pcDNA3-HA to give pcDNA3-HA-PHD2 (1–196) D4E. pcDNA3-HA-PHD2 D4E was constructed by subcloning the 0.6-kb BamHI/XhoI fragment from pcDNA3-HA-PHD2 (1–196) D4E into the BamHI/XhoI site of pcDNA3-PHD2 (130–426). pcDNA3-HA-PHD2 (1–131) C127S was constructed by amplifying by PCR a 0.4-kb band using pcDNA3-HA-PHD2 as a template and the following primers: CMV 5′, 5′-GCG TGT ACG GTG GGA GGT C-3′; and PHD2 C127S 3′ (see above). The product was digested with BamHI and NotI and subcloned into the BamHI/NotI site of pcDNA3-HA to give pcDNA3-HA-PHD2 (1–131) C127S. pcDNA3-HA-PHD2 (1–196) C127S was prepared by subcloning the 0.23-kb NotI fragment pcDNA3-HA-PHD2 (1–196) into the NotI site of pcDNA3-HA-PHD2 (1–131) C127S. pcDNA3-HA-PHD2 C127S was constructed by subcloning the 0.6-kb BamHI/XhoI fragment from pcDNA3-HA-PHD2 (1–196) C127S into the BamHI/XhoI site of pcDNA3-PHD2 (130–426). pFastBac-HT-FlagPHD2 D4E/C127S was prepared by subcloning the 0.4-kb BamHI/NotI fragment of pcDNA3-HA-PHD2 (1–196) D4E/C127S into the BamHI/NotI site of pFastBac-HT-FlagPHD2 (23Huang J. Zhao Q. Mooney S.M. Lee F.S. Sequence determinants in hypoxia-inducible factor-1α for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3.J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). pcDNA3-Flag-p23 W106A was constructed by overlapping PCR. In the first round of PCR, we performed two PCRs. We amplified a 0.32-kb band using pcDNA5/FRT/TO-3×Flag-p23 as a template and the following primers: CMV 5′, 5′-GCG TGT ACG GTG GGA GGT C-3′; and P23 W106A 3′, 5′-GAA TCA TCT TCC CAG TCT TTC GCA TTA TTG AAA TCG ACA CTA AGC CAA TTA AG-3′. We amplified a 0.2-kb band using pcDNA5/FRT/TO-3×Flag-p23 as a template and the following primers: P23 W106A 5′, 5′-CTT AAT TGG CTT AGT GTC GAT TTC AAT AAT GCG AAA GAC TGG GAA GAT GAT TC-3′; and BGH rev, 5′-TAG AAG GCA CAG TCG AGG-3′. The two PCR products were mixed and employed as template in a second round of PCR using the CMV 5′ and BGH rev primers. The product was digested with BamHI and XhoI and subcloned into the BamHI/XhoI site of pcDNA5/FRT/TO-3×Flag. The plasmid Hsp90 HA (β isoform) (24García-Cardeña G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Dynamic activation of endothelial nitric oxide synthase by Hsp90.Nature. 1998; 392: 821-824Crossref PubMed Scopus (864) Google Scholar) was obtained from Addgene (plasmid 22487). pFastBac-HT-HA-HSP90β was constructed by subcloning the 2.2-kb NotI/XbaI fragment of Hsp90 HA into the NotI/XbaI site of pFastBacHTc. pFastBac-HT-HA-HSP90β P709A/L711A/E712A was constructed by standard recombinant DNA methods. pFastBac-HT-FKBP38 (FK506-binding protein 38) was constructed by subcloning the 1.2-kb BamHI/NotI fragment of pcDNA5/FRT/TO-3×Flag-FKBP38 (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) into the BamHI/NotI site of pFastBacHTc. pFastBac-HT-FlagHIF-1α was prepared by subcloning the 3.2-kb BamHI/XbaI fragment of pcDNA3-FlagHIF-1α (25Yu F. White S.B. Zhao Q. Lee F.S. Dynamic, site-specific interaction of hypoxia-inducible factor-1α with the von Hippel-Lindau tumor suppressor protein.Cancer Res. 2001; 61: 4136-4142PubMed Google Scholar) into the BamHI/XbaI site of pFastBacHTb. pFastBac-HT-HA-VHL was prepared by subcloning the 0.7-kb NcoI/XhoI fragment of pcDNA3-HA-VHL (23Huang J. Zhao Q. Mooney S.M. Lee F.S. Sequence determinants in hypoxia-inducible factor-1α for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3.J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) into the NcoI/XhoI site of pFastBacHTc. pFastBac-HT-p23 was constructed by subcloning the 0.5-kb BamHI/XhoI fragment of pcDNA5/FRT/TO-3×Flag-p23 (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) into the BamHI/XhoI site of pFastBac-HTc. pFastBac-HT-HA-p23 was constructed by subcloning the 0.5-kb BamHI/XhoI fragment of pcDNA5/FRT/TO-3×Flag-p23 into the BamHI/XhoI site of pFastBac-HT-HA-VHL. The sources of pGEX-HIF-1α (531–575), pGEX-HIF-2α (516–549), and all other plasmids have been described (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 26Percy M.J. Furlow P.W. Beer P.A. Lappin T.R. McMullin M.F. Lee F.S. A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove.Blood. 2007; 110: 2193-2196Crossref PubMed Scopus (124) Google Scholar). Baculoviruses for HT-FlagPHD2 D4E/C127S, HT-p23, HT-HA-p23, HT-HA-HSP90β, HT-HA-HSP90β P709A/L711A/E712A, HT-FKBP38, HT-FlagHIF-1α, and HT-HA-VHL were prepared using the appropriate pFastBac vectors and the Bac-To-Bac system (Invitrogen). GST, GST-HIF-1α (531–575), and GST-HIF-2α (516–549) were purified as described (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). His6-tagged (HT)-FlagPHD2, HT-FlagPHD2 D4E/C127S, HT-p23, HT-HA-p23, HT-HA-HSP90β, HT-HA-HSP90β P709A/L711A/E712A, HT-FKBP38, and HT-FlagHIF-1α were purified from Sf9 cells infected with the appropriate baculovirus using nickel-nitrilotriacetic agarose (Qiagen). HT-HA-VHL was purified as a complex with ElonginB and ElonginC following coinfection of Sf9 cells with a baculovirus for HT-HA-VHL and one for ElonginB/ElonginC (23Huang J. Zhao Q. Mooney S.M. Lee F.S. Sequence determinants in hypoxia-inducible factor-1α for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3.J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). WT and D4E/C127S HT-FlagPHD2 purified from baculovirus-infected Sf9 cells were subjected to filter-aided sample preparation (27Wiśniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5099) Google Scholar). Mass spectrometry was then performed as described (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) with the following modifications: fragmentation spectra (MS2s) were selected from within a reduced parent mass scan range (600–800 m/z) and using a targeted parent mass list designed to selectively acquire spectra for the indicated peptides. Masses selected for fragmentation included both fully and semitryptic peptides in addition to mass shifts associated with zero, one, or two serine phosphorylation events (+79.966 Da). The data were analyzed using the MaxQuant analytical package (28Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar) version 1.2.2.5 and included N-terminal acetylation, oxidation of methionine, and phosphorylation of serine (with potential accompanying neutral losses) as variable modifications and carbamidomethylation of cysteine as a fixed modification. Recombinant WT or D4E/C127S HT-FlagPHD2 was incubated with substrate in a buffer consisting of 20 mm Hepes, pH 8.0, 100 mm KCl, 10 μm FeCl2, 1 mm 2-oxoglutarate, 5 mm ascorbate, 1 mm DTT, and 1 μm ZnCl2 at 37 °C. For hypoxic conditions, the reactions were conducted in either a Ruskinn In Vivo 200 hypoxia work station or a Billups Rothenberg modular incubator perfused with a gas mixture containing the appropriate oxygen concentration. The products were subjected to SDS-PAGE, transferred to Immobilon membranes, blocked with PBS containing 0.5% Tween 20, and 1% bovine serum albumin for 30 min, and then incubated with recombinant HT-HA-VHL (in complex with ElonginB and ElonginC) for 1 h in the same buffer at 4 °C. Following washing, HT-HA-VHL was visualized using anti-HA antibodies conjugated to alkaline phosphatase. Peptides corresponding to HSP90β (703–712) and HSP90α (711–720) were synthesized by Genscript. These peptides, as well as one corresponding to p23 (151–160) possessed an N-terminal tyrosine residue to allow spectrophotometric quantitation (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) as well as an N-terminal biotin. Peptides (0.5 μg) were prebound to 15-μl aliquots of streptavidin-agarose (Sigma). The resins were incubated with Sf9 lysates containing baculovirus-expressed HT-FlagPHD2 (23Huang J. Zhao Q. Mooney S.M. Lee F.S. Sequence determinants in hypoxia-inducible factor-1α for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3.J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) for 1 h with rocking at 4 °C in buffer A (20 mm Tris, pH 7.6, 150 mm NaCl, 10% glycerol, 1% Triton X-100). The resins were washed four times with buffer A and eluted, and the eluates were subjected to SDS-PAGE and Western blotting. HEK293 FT cells were maintained and transfected with plasmids as described (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Luciferase assays were performed as described (19Song D. Li L.S. Heaton-Johnson K.J. Arsenault P.R. Master S.R. Lee F.S. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.J. Biol. Chem. 2013; 288: 9662-9674Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Hypoxia experiments were performed in an In Vivo 200 hypoxia work station (Ruskinn Technologies). Phd2+/− mice (29Arsenault P.R. Pei F. Lee R. Kerestes H. Percy M.J. Keith B. Simon M.C. Lappin T.R. Khurana T.S. Lee F.S. A knock-in mouse model of human PHD2 gene-associated erythrocytosis establishes a haploinsufficiency mechanism.J. Biol. Chem. 2013; 288: 33571-33584Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) were crossed, and MEFs were obtained from embryonic day 11.5 Phd2−/− embryos by the primary explant technique (30Xu J. Preparation, culture, and immortalization of mouse embryonic fibroblasts.in: Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology, Chapter 28, Unit 28.21. John Wiley & Sons, Hoboken, NJ2005Crossref Google Scholar). MEFs were immortalized by transfection with pSG5-T, which contains the coding sequence for the SV40 large T antigen (31Yu Y. Alwine J.C. Human cytomegalovirus major immediate-early proteins and simian virus 40 large T antigen can inhibit apoptosis through activation of the phosphatidylinositide 3′-OH kinase pathway and the cellular kinase Akt.J. Virol. 2002; 76: 3731-3738Crossref PubMed Scopus (138) Google Scholar), and maintained in Dulbecco's modified Eagle's medi" @default.
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• W2023795623 title "Defective Tibetan PHD2 Binding to p23 Links High Altitude Adaption to Altered Oxygen Sensing" @default.
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