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Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.

Yang M, Chowdhury R, Ge W, Hamed RB, McDonough MA, Claridge TD, Kessler BM, Cockman ME, Ratcliffe PJ, Schofield CJ - FEBS J. (2011)

Bottom Line: However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues.NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the β-position.The results further expand the scope of FIH-catalysed hydroxylations.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.

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LC/MS spectra illustrating the effect of FIH intervention on tankyrase-2 hydroxylation at His 238 and His 553. 293T cells were treated with siRNAs against FIH (‘FIH siRNA’) or a control sequence (‘Endogenous FIH’/‘FIH overexpression’). After siRNA treatment, cells were transfected with FLAG–TNKS2 and either pcDNA3 FIH (‘FIH overexpression’) or empty vector (‘FIH siRNA’/‘Endogenous FIH’). FLAG–TNKS2 was immunopurified, digested and analysed by LC/MS to quantify hydroxylation. The efficacy of the siRNA and plasmid transfections were confirmed by anti-FIH and anti-FLAG immunoblotting (data not shown). (A) Representative LC/MS spectra of the 226–240 tryptic fragment containing His 238. Hydroxylation (∼ 30%) was observed for His 238 under conditions where the level of FIH was not limiting. (B) Representative LC/MS spectra of the 538–555 tryptic fragment containing His 553. Hydroxylation was observed at endogenous level of FIH (∼ 15%) and when FIH was overexpressed (∼ 70%).
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fig03: LC/MS spectra illustrating the effect of FIH intervention on tankyrase-2 hydroxylation at His 238 and His 553. 293T cells were treated with siRNAs against FIH (‘FIH siRNA’) or a control sequence (‘Endogenous FIH’/‘FIH overexpression’). After siRNA treatment, cells were transfected with FLAG–TNKS2 and either pcDNA3 FIH (‘FIH overexpression’) or empty vector (‘FIH siRNA’/‘Endogenous FIH’). FLAG–TNKS2 was immunopurified, digested and analysed by LC/MS to quantify hydroxylation. The efficacy of the siRNA and plasmid transfections were confirmed by anti-FIH and anti-FLAG immunoblotting (data not shown). (A) Representative LC/MS spectra of the 226–240 tryptic fragment containing His 238. Hydroxylation (∼ 30%) was observed for His 238 under conditions where the level of FIH was not limiting. (B) Representative LC/MS spectra of the 538–555 tryptic fragment containing His 553. Hydroxylation was observed at endogenous level of FIH (∼ 15%) and when FIH was overexpressed (∼ 70%).

Mentions: To determine whether the His hydroxylation observed on tankyrase-2 was FIH dependent, we quantified hydroxylation at His 238 and His 553 by LC/MS in the presence and absence of small interfering RNA (siRNA) for FIH. 293T cells were transfected with siRNA duplexes directed against FIH or a nontargeting control, then transfected with tankyrase-2 plus empty vector. As an additional control, and to ensure that FIH levels were not limiting, tankyrase-2 was cotransfected with FIH. LC/MS data of one representative experiment are presented in Fig. 3. Following coexpression with FIH, the two hydroxylation sites displayed different levels of hydroxylation; His 238 was hydroxylated to ∼ 30%, whereas His 553 was hydroxylated to ∼ 70%. The preference for His 553 was also observed under physiological levels of FIH with detectable levels of hydroxylated peptide (∼ 15%) observed at His553, but no appreciable hydroxylation (< 4%) on the His 238 peptide. Importantly, siRNA-mediated knockdown of FIH suppressed His 553 hydroxylation to below the limit of detection, indicating a nonredundant role for FIH in the catalysis of hydroxyhistidine in the ARD of tankyrase-2. Consistent with previous work [8], the relative hydroxylation levels for some previously identified Asn hydroxylation sites in tankyrase-2 expressed in the presence of endogenous level of FIH were approximately: Asn 427, 12%; Asn 586, 42%; Asn 671, 5%; and Asn 739, 60% (tryptic fragments containing the Asn 203, Asn 271, Asn 518 and Asn 706 hydroxylation sites were not detected, data not shown).


Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.

Yang M, Chowdhury R, Ge W, Hamed RB, McDonough MA, Claridge TD, Kessler BM, Cockman ME, Ratcliffe PJ, Schofield CJ - FEBS J. (2011)

LC/MS spectra illustrating the effect of FIH intervention on tankyrase-2 hydroxylation at His 238 and His 553. 293T cells were treated with siRNAs against FIH (‘FIH siRNA’) or a control sequence (‘Endogenous FIH’/‘FIH overexpression’). After siRNA treatment, cells were transfected with FLAG–TNKS2 and either pcDNA3 FIH (‘FIH overexpression’) or empty vector (‘FIH siRNA’/‘Endogenous FIH’). FLAG–TNKS2 was immunopurified, digested and analysed by LC/MS to quantify hydroxylation. The efficacy of the siRNA and plasmid transfections were confirmed by anti-FIH and anti-FLAG immunoblotting (data not shown). (A) Representative LC/MS spectra of the 226–240 tryptic fragment containing His 238. Hydroxylation (∼ 30%) was observed for His 238 under conditions where the level of FIH was not limiting. (B) Representative LC/MS spectra of the 538–555 tryptic fragment containing His 553. Hydroxylation was observed at endogenous level of FIH (∼ 15%) and when FIH was overexpressed (∼ 70%).
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fig03: LC/MS spectra illustrating the effect of FIH intervention on tankyrase-2 hydroxylation at His 238 and His 553. 293T cells were treated with siRNAs against FIH (‘FIH siRNA’) or a control sequence (‘Endogenous FIH’/‘FIH overexpression’). After siRNA treatment, cells were transfected with FLAG–TNKS2 and either pcDNA3 FIH (‘FIH overexpression’) or empty vector (‘FIH siRNA’/‘Endogenous FIH’). FLAG–TNKS2 was immunopurified, digested and analysed by LC/MS to quantify hydroxylation. The efficacy of the siRNA and plasmid transfections were confirmed by anti-FIH and anti-FLAG immunoblotting (data not shown). (A) Representative LC/MS spectra of the 226–240 tryptic fragment containing His 238. Hydroxylation (∼ 30%) was observed for His 238 under conditions where the level of FIH was not limiting. (B) Representative LC/MS spectra of the 538–555 tryptic fragment containing His 553. Hydroxylation was observed at endogenous level of FIH (∼ 15%) and when FIH was overexpressed (∼ 70%).
Mentions: To determine whether the His hydroxylation observed on tankyrase-2 was FIH dependent, we quantified hydroxylation at His 238 and His 553 by LC/MS in the presence and absence of small interfering RNA (siRNA) for FIH. 293T cells were transfected with siRNA duplexes directed against FIH or a nontargeting control, then transfected with tankyrase-2 plus empty vector. As an additional control, and to ensure that FIH levels were not limiting, tankyrase-2 was cotransfected with FIH. LC/MS data of one representative experiment are presented in Fig. 3. Following coexpression with FIH, the two hydroxylation sites displayed different levels of hydroxylation; His 238 was hydroxylated to ∼ 30%, whereas His 553 was hydroxylated to ∼ 70%. The preference for His 553 was also observed under physiological levels of FIH with detectable levels of hydroxylated peptide (∼ 15%) observed at His553, but no appreciable hydroxylation (< 4%) on the His 238 peptide. Importantly, siRNA-mediated knockdown of FIH suppressed His 553 hydroxylation to below the limit of detection, indicating a nonredundant role for FIH in the catalysis of hydroxyhistidine in the ARD of tankyrase-2. Consistent with previous work [8], the relative hydroxylation levels for some previously identified Asn hydroxylation sites in tankyrase-2 expressed in the presence of endogenous level of FIH were approximately: Asn 427, 12%; Asn 586, 42%; Asn 671, 5%; and Asn 739, 60% (tryptic fragments containing the Asn 203, Asn 271, Asn 518 and Asn 706 hydroxylation sites were not detected, data not shown).

Bottom Line: However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues.NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the β-position.The results further expand the scope of FIH-catalysed hydroxylations.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.

Show MeSH
Related in: MedlinePlus