| Autor: |
Renato, Wirthner, Kuppusamy, Balamurugan, Daniel P, Stiehl, Sandra, Barth, Patrick, Spielmann, Felix, Oehme, Ingo, Flamme, Dörthe M, Katschinski, Roland H, Wenger, Gieri, Camenisch |
| Rok vydání: |
2007 |
| Předmět: |
|
| Zdroj: |
Methods in enzymology. 435 |
| ISSN: |
0076-6879 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
