Analysis of HIF-prolyl hydroxylases binding to substrates.
Hypoxia inducible transcription factors (HIF) are mainly regulated by a group of proline hydroxylases (EGLNs) that, in the presence of oxygen, target HIF for degradation. HIFalpha contains two independent oxygen degradation domains (N-ODD and C-ODD) that are substrates for these enzymes. In this work, we employed the yeast two-hybrid assay to study the sequence determinants required for the binding of EGLN1 and 3 to HIF1alpha in a cellular context. Our results demonstrate that, while EGLN1 is able to recognize both ODDs within full length HIF1alpha protein, EGLN3 only binds to CODD. The analysis of the residue substitutions within CODD uncovered novel critical determinants for EGLN1 and 3 binding. In addition, our results show that both enzymes have a very similar, albeit not identical, residue preference at specific positions in their substrate sequences.
Landázuri
Manuel O
MO
Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain.
Vara-Vega
Alicia
A
Vitón
Mariano
M
Cuevas
Yolanda
Y
del Peso
Luis
L
D019943:Amino Acid Substitution; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049308:Dioxygenases; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D010100:Oxygen