Many amino acid substitutions in a hypoxia-inducible transcription factor (HIF)-1alpha-like peptide cause only minor changes in its hydroxylation by the HIF prolyl 4-hydroxylases: substitution of 3,4-dehydroproline or azetidine-2-carboxylic acid for the proline leads to a high rate of uncoupled 2-oxoglutarate decarboxylation.
Three human prolyl 4-hydroxylases (P4Hs) regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylating a Leu-Xaa-Xaa-Leu-Ala-Pro motif. We report here that the two leucines in the Leu-Glu-Met-Leu-Ala-Pro core motif of a 20-residue peptide corresponding to the sequence around Pro(564) in HIF-1alpha can be replaced by many residues with no or only a modest decrease in its substrate properties or in some cases even a slight increase. The glutamate and methionine could be substituted by almost any residue, eight amino acids in the former position and four in the latter being even better for HIF-P4H-3 than the wild-type residues. Alanine was by far the strictest requirement, because no residue could fully substitute for it in the case of HIF-P4H-1, and only serine or isoleucine, valine, and serine did this in the cases of HIF-P4Hs 2 and 3. Peptides with more than one substitution, having the core sequences Trp-Glu-Met-Val-Ala-Pro, Tyr-Glu-Met-Ile-Ala-Pro, Ile-Glu-Met-Ile-Ala-Pro, Trp-Glu-Met-Val-Ser-Pro, and Trp-Glu-Ala-Val-Ser-Pro were in most cases equally as good or almost as good substrates as the wild-type peptide. The acidic residues present in the 20-residue peptide also played a distinct role, but alanine substitution for any six of them, and in some combinations even three of them, had no negative effects. Substitution of the proline by 3,4-dehydroproline or l-azetidine-2-carboxylic acid, but not any other residue, led to a high rate of uncoupled 2-oxoglutarate decarboxylation with no hydroxylation. The data obtained for the three HIF-P4Hs in various experiments were in most cases similar, but in some cases HIF-P4H-3 showed distinctly different properties.
Li
Dongxia
D
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014, Oulu, Finland.
Hirsilä
Maija
M
Koivunen
Peppi
P
Brenner
Mitchell C
MC
Xu
Leon
L
Yang
Charles
C
Kivirikko
Kari I
KI
Myllyharju
Johanna
J
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D001383:Azetidinecarboxylic Acid / Q000737:chemistry; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D002853:Chromatography, Liquid; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007313:Insecta; D007656:Ketoglutaric Acids / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry*; D011392:Proline / Q000031:analogs & derivatives* / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry*
D016895:Culture Media, Serum-Free; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D010455:Peptides; D014157:Transcription Factors; C002195:3,4-dehydroproline; D018698:Glutamic Acid; D001383:Azetidinecarboxylic Acid; D011392:Proline; D008715:Methionine; D011393:Procollagen-Proline Dioxygenase; D007930:Leucine; D000409:Alanine