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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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FIH-catalysed His hydroxylation of ARDs may be common. (A) clustalw nongapped multiple sequence alignment of ankyrin repeat sequences containing a target histidine residue at the conserved hydroxylation position. Corresponding peptides spanning the potential histidinyl hydroxylation sites were tested as FIH substrates in vitro, among which peptides derived from TNKS1, GABPB2 and TRPV4 demonstrate FIH-dependent hydroxylation. (B–D) LC/MS analyses demonstrating FIH-catalysed His hydroxylation of the following peptides: (B) TNKS1 381–400; (C) TNKS1 696–715; and (D) GABPB2 115–135. (E) MALDI spectra showing the hydroxylation of the TRPV4 249–269 peptide.
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fig06: FIH-catalysed His hydroxylation of ARDs may be common. (A) clustalw nongapped multiple sequence alignment of ankyrin repeat sequences containing a target histidine residue at the conserved hydroxylation position. Corresponding peptides spanning the potential histidinyl hydroxylation sites were tested as FIH substrates in vitro, among which peptides derived from TNKS1, GABPB2 and TRPV4 demonstrate FIH-dependent hydroxylation. (B–D) LC/MS analyses demonstrating FIH-catalysed His hydroxylation of the following peptides: (B) TNKS1 381–400; (C) TNKS1 696–715; and (D) GABPB2 115–135. (E) MALDI spectra showing the hydroxylation of the TRPV4 249–269 peptide.

Mentions: To investigate whether FIH-catalysed His hydroxylation can occur in other AR sequences, we searched for naturally occurring AR sequences containing an ‘LxxxxxDVH’ motif with the His residue located at the conserved hydroxylation position, and tested the corresponding peptides as FIH substrates (Fig. 6). In addition to tankyrase-2, peptides derived from tankyrase-1, GA-binding protein subunit beta-2 (GABPB2) and the transient receptor potential vanilloid-4 (TRPV4) ARD all displayed +16 Da mass shifts after reaction with FIH (Fig. 6B). MS/MS analyses assigned sites of hydroxylation to His 711 in the tankyrase-1 sequence (Fig. S3) and His 265 in the TRPV4 sequence (Fig. S4), both of which are located within the β-hairpin loop at the structurally conserved hydroxylation position.


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)

FIH-catalysed His hydroxylation of ARDs may be common. (A) clustalw nongapped multiple sequence alignment of ankyrin repeat sequences containing a target histidine residue at the conserved hydroxylation position. Corresponding peptides spanning the potential histidinyl hydroxylation sites were tested as FIH substrates in vitro, among which peptides derived from TNKS1, GABPB2 and TRPV4 demonstrate FIH-dependent hydroxylation. (B–D) LC/MS analyses demonstrating FIH-catalysed His hydroxylation of the following peptides: (B) TNKS1 381–400; (C) TNKS1 696–715; and (D) GABPB2 115–135. (E) MALDI spectra showing the hydroxylation of the TRPV4 249–269 peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3569879&req=5

fig06: FIH-catalysed His hydroxylation of ARDs may be common. (A) clustalw nongapped multiple sequence alignment of ankyrin repeat sequences containing a target histidine residue at the conserved hydroxylation position. Corresponding peptides spanning the potential histidinyl hydroxylation sites were tested as FIH substrates in vitro, among which peptides derived from TNKS1, GABPB2 and TRPV4 demonstrate FIH-dependent hydroxylation. (B–D) LC/MS analyses demonstrating FIH-catalysed His hydroxylation of the following peptides: (B) TNKS1 381–400; (C) TNKS1 696–715; and (D) GABPB2 115–135. (E) MALDI spectra showing the hydroxylation of the TRPV4 249–269 peptide.
Mentions: To investigate whether FIH-catalysed His hydroxylation can occur in other AR sequences, we searched for naturally occurring AR sequences containing an ‘LxxxxxDVH’ motif with the His residue located at the conserved hydroxylation position, and tested the corresponding peptides as FIH substrates (Fig. 6). In addition to tankyrase-2, peptides derived from tankyrase-1, GA-binding protein subunit beta-2 (GABPB2) and the transient receptor potential vanilloid-4 (TRPV4) ARD all displayed +16 Da mass shifts after reaction with FIH (Fig. 6B). MS/MS analyses assigned sites of hydroxylation to His 711 in the tankyrase-1 sequence (Fig. S3) and His 265 in the TRPV4 sequence (Fig. S4), both of which are located within the β-hairpin loop at the structurally conserved hydroxylation position.

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