Determination and modulation of prolyl-4-hydroxylase domain oxygen sensor activity.
The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.
Wirthner
Renato
R
Institute of Physiology and Zürich Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland.
Balamurugan
Kuppusamy
K
Stiehl
Daniel P
DP
Barth
Sandra
S
Spielmann
Patrick
P
Oehme
Felix
F
Flamme
Ingo
I
Katschinski
Dörthe M
DM
Wenger
Roland H
RH
Camenisch
Gieri
G
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003653:Decarboxylation; D005977:Glutarates / Q000737:chemistry; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D014020:Tissue Extracts / Q000737:chemistry
D005977:Glutarates; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010455:Peptides; D011994:Recombinant Proteins; D014020:Tissue Extracts; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D011393:Procollagen-Proline Dioxygenase; C035736:glutaric acid; D010100:Oxygen