Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.
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.
Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.Show MeSH
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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 , 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).
Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.