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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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Tankryase-2 is a substrate for FIH-catalysed His hydroxylation. (A) Sequence alignment of the tankyrase-2 ARD (residues 57–798) [36] demonstrating a 4-repeat periodicity of the ARD, with insertion sequences (right) following boxed residues in the corresponding repeats to the left. Asn residues that have previously been identified as FIH substrates [8], and the two His residues located at the conserved hydroxylation position, are highlighted in bold and their residue number shown in parenthesis on the right. (B) Tankyrase-2 peptides containing His 238 and His 553 are FIH substrates in vitro. Peptides corresponding to: (I) TNKS2 223–243 (RVKIVQLLLQHGADVHAKDKG) and (II) TNKS2 538–558 (RVSVVEYLLQHGADVHAKDKG) were incubated in the presence of recombinant FIH and displayed a net +16 Da mass shift as determined by LC-MS. Subsequent MS/MS of the FIH-reacted TNKS2 538–558 peptide assigned the oxidation to His 553 (Fig. S1).
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fig01: Tankryase-2 is a substrate for FIH-catalysed His hydroxylation. (A) Sequence alignment of the tankyrase-2 ARD (residues 57–798) [36] demonstrating a 4-repeat periodicity of the ARD, with insertion sequences (right) following boxed residues in the corresponding repeats to the left. Asn residues that have previously been identified as FIH substrates [8], and the two His residues located at the conserved hydroxylation position, are highlighted in bold and their residue number shown in parenthesis on the right. (B) Tankyrase-2 peptides containing His 238 and His 553 are FIH substrates in vitro. Peptides corresponding to: (I) TNKS2 223–243 (RVKIVQLLLQHGADVHAKDKG) and (II) TNKS2 538–558 (RVSVVEYLLQHGADVHAKDKG) were incubated in the presence of recombinant FIH and displayed a net +16 Da mass shift as determined by LC-MS. Subsequent MS/MS of the FIH-reacted TNKS2 538–558 peptide assigned the oxidation to His 553 (Fig. S1).

Mentions: Previously, we have reported that various human ARD-containing proteins undergo hydroxylation at conserved Asn residues [5,6,8,9]. One of the most highly modified is tankyrase-2, which undergoes FIH-catalysed hydroxylation at eight Asn residues [8]. Alignment of the tankyrase-2 ARD revealed two His residues (His 238 and His 553) embedded within the FIH hydroxylation consensus comprising an ‘LxxxxxDVH’ motif at positions analogous to proven FIH-catalysed Asn hydroxylation sites (Fig. 1A) [8]. The positioning of these His residues within the hydroxylation consensus, coupled with the structural similarity between Asn and His residues, raised the interesting possibility that His 238 and/or His 553 of tankyrase-2 might also be hydroxylated by FIH. To test this hypothesis, we prepared synthetic 21-residue peptides encompassing the two His residues of interest and tested them as FIH substrates. Significantly, both peptides (TNKS2 223–243 and TNKS2 538–558) displayed a clear +16 Da mass increment after reaction with FIH under standard assay conditions (Fig. 1B). MS/MS analyses of the modified TNKS2 538–558 peptide after tryptic digestion assigned the site of hydroxylation to that corresponding to His 553 in the tankyrase-2 ARD (Fig. S1).


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)

Tankryase-2 is a substrate for FIH-catalysed His hydroxylation. (A) Sequence alignment of the tankyrase-2 ARD (residues 57–798) [36] demonstrating a 4-repeat periodicity of the ARD, with insertion sequences (right) following boxed residues in the corresponding repeats to the left. Asn residues that have previously been identified as FIH substrates [8], and the two His residues located at the conserved hydroxylation position, are highlighted in bold and their residue number shown in parenthesis on the right. (B) Tankyrase-2 peptides containing His 238 and His 553 are FIH substrates in vitro. Peptides corresponding to: (I) TNKS2 223–243 (RVKIVQLLLQHGADVHAKDKG) and (II) TNKS2 538–558 (RVSVVEYLLQHGADVHAKDKG) were incubated in the presence of recombinant FIH and displayed a net +16 Da mass shift as determined by LC-MS. Subsequent MS/MS of the FIH-reacted TNKS2 538–558 peptide assigned the oxidation to His 553 (Fig. S1).
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Related In: Results  -  Collection

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fig01: Tankryase-2 is a substrate for FIH-catalysed His hydroxylation. (A) Sequence alignment of the tankyrase-2 ARD (residues 57–798) [36] demonstrating a 4-repeat periodicity of the ARD, with insertion sequences (right) following boxed residues in the corresponding repeats to the left. Asn residues that have previously been identified as FIH substrates [8], and the two His residues located at the conserved hydroxylation position, are highlighted in bold and their residue number shown in parenthesis on the right. (B) Tankyrase-2 peptides containing His 238 and His 553 are FIH substrates in vitro. Peptides corresponding to: (I) TNKS2 223–243 (RVKIVQLLLQHGADVHAKDKG) and (II) TNKS2 538–558 (RVSVVEYLLQHGADVHAKDKG) were incubated in the presence of recombinant FIH and displayed a net +16 Da mass shift as determined by LC-MS. Subsequent MS/MS of the FIH-reacted TNKS2 538–558 peptide assigned the oxidation to His 553 (Fig. S1).
Mentions: Previously, we have reported that various human ARD-containing proteins undergo hydroxylation at conserved Asn residues [5,6,8,9]. One of the most highly modified is tankyrase-2, which undergoes FIH-catalysed hydroxylation at eight Asn residues [8]. Alignment of the tankyrase-2 ARD revealed two His residues (His 238 and His 553) embedded within the FIH hydroxylation consensus comprising an ‘LxxxxxDVH’ motif at positions analogous to proven FIH-catalysed Asn hydroxylation sites (Fig. 1A) [8]. The positioning of these His residues within the hydroxylation consensus, coupled with the structural similarity between Asn and His residues, raised the interesting possibility that His 238 and/or His 553 of tankyrase-2 might also be hydroxylated by FIH. To test this hypothesis, we prepared synthetic 21-residue peptides encompassing the two His residues of interest and tested them as FIH substrates. Significantly, both peptides (TNKS2 223–243 and TNKS2 538–558) displayed a clear +16 Da mass increment after reaction with FIH under standard assay conditions (Fig. 1B). MS/MS analyses of the modified TNKS2 538–558 peptide after tryptic digestion assigned the site of hydroxylation to that corresponding to His 553 in the tankyrase-2 ARD (Fig. S1).

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