Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-13939
Wirthner, R; Balamurugan, K; Stiehl, D P; Barth, S; Spielmann, P; Oehme, F; Flamme, I; Katschinski, D M; Wenger, R H; Camenisch, G (2007). Determination and modulation of prolyl-4-hydroxylase domain oxygen sensor activity. Methods in Enzymology, 435:43-60.
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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.
|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||04 Faculty of Medicine > Center for Integrative Human Physiology|
04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
|DDC:||570 Life sciences; biology|
610 Medicine & health
|Deposited On:||23 Mar 2009 16:49|
|Last Modified:||27 Nov 2013 20:25|
|Citations:||Web of Science®. Times Cited: 14|
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