Oxygen Promotes Hydroxylation

Abstract

Under normal oxygen conditions, hypoxia-inducible factor-1α (HIF-1α) is rapidly degraded through interaction with the E3 ubiquitin ligase von Hippel-Lindau tumor suppressor protein (VHL), which targets HIF-1α for degradation by the proteasome. Hypoxic conditions or treatment of cells with cobalt or desferrioxamine (a chelating agent) stabilizes HIF-1α and promotes its dissociation from VHL. Using a combination of in vitro assays (incubating purified protein with cytosolic extracts) and experiments with transfected cells, Yu et al. show that HIF-1α undergoes a posttranslational modification in the region of amino acids 549 to 575 that promotes VHL binding under normal oxygen conditions. Systematic mutation of the amino acids in this region showed that the regulated interaction with VHL was only disrupted if the sole proline (P 564 ) or the adjacent alanine (A 563 ) were mutated. Matrix-assisted laser desorption time of flight (MALDI-TOF) analysis (see the Protocol by Pandey et al. ) demonstrated that an HIF-1α fragment isolated from transfected cells showed a higher mass (consistent with a proline hydroxylation) under normal conditions than that isolated from cells exposed to cobalt. Final confirmation that proline hydroxylation promoted binding to VHL was provided by experiments with a peptide synthesized to contain 4-hydroxyl-L-proline, which bound VHL with or without incubation with cytoplasmic extracts, indicating that this modified peptide no longer required posttranslational modification to promote binding. Thus, reversible and regulated proline hydroxylation can be added to the list of posttranslational modifications controlling proteins involved in signal transduction. F. Yu, S. B. White, Q. Zhao, F. S. Lee, HIF-1α binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc. Natl. Acad. Sci. U.S.A. 98 , 9630-9635 (2001). [Abstract] [Full Text] A. Pandey, J. S. Andersen, M. Mann, Use of mass spectrometry to study signaling pathways. Science's STKE (2001), http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2000/37/pl1. [Full Text]