UZH-Logo

Determination and modulation of prolyl-4-hydroxylase domain oxygen sensor activity


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.

Abstract

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.

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.

Citations

14 citations in Web of Science®
14 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

335 downloads since deposited on 23 Mar 2009
68 downloads since 12 months
Detailed statistics

Additional indexing

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
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2007
Deposited On:23 Mar 2009 15:49
Last Modified:05 Apr 2016 13:01
Publisher:Elsevier
ISSN:0076-6879
Publisher DOI:10.1016/S0076-6879(07)35003-9
PubMed ID:17998048
Permanent URL: http://doi.org/10.5167/uzh-13939

Download

[img]Filetype: PDF (Verlags-PDF) - Registered users only
Size: 1MB
View at publisher

[img]
Preview
Content: Accepted Version
Filetype: PDF
Size: 1MB

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations