Abstract

Cells respond to decreased oxygen with the induction of an adaptive gene expression programme aimed at restoring oxygen supply and maintaining cell viability during oxygen restriction. Most of the genes induced during low oxygen exposure are under control of the hypoxia inducible factor.
The transcription factor Hif-1 (hypoxia-inducible factor 1) plays an essential role in the maintenance of oxygen homeostasis in metazoan organisms. It is a heterodimer composed of an oxygen regulated Hif-1 alpha subunit and a constituvely expressed Hif-1 beta subunit. Under normoxic conditions, Hif-1 alpha is continuously synthesized and degraded, while under hypoxic conditions it accumulates, dimerizes with Hif-1 beta and activates its target gene transcription. In normoxia, Hif-1 alpha is hydroxylated at P402 and P564 (human numbering) by prolyl hydroxylases. This allows binding of von Hippel-Lindau protein (pVHL) which leads to ubiquitination and proteosomal degradation of the Hif-1 alpha . Under hypoxic conditions, hydroxylase activity is reduced due to substrate limitation, inhibition of the catalytic centre or both, this leads to accumulation of Hif-1 alpha which can enter the nucleus, dimerize with Hif-1 beta and activate transcription of hypoxic genes.
Activated genes include erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and others whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Hif-1 alpha is often over expressed in the majority of common human cancers and their metastases, due to the presence of intratumoral hypoxia and as a result of mutation in genes encoding oncoproteins and tumor suppressors. Solid tumors require vascularization and so the hypoxia pathway is seen as a major therapeutic target.
Three isoforms of Hif alpha (Hif-1 alpha, Hif-2 alpha and Hif-3 alpha) are encoded by distinct genetic loci. Sites from all isoforms align into a conserved LxxLAP motif. This sequence is evolutionarily conserved in Hif alpha proteins from nematodes to mammals (Mircea I. et al. 2001, Jaakkola P. et al. 2001). However, several studies suggest the absence of a rigid consensus sequence for binding of Hypoxia-inducible factor prolyl hydroxylases (Huang J. et al. 2002, Li D. et al. 2004). Also, it was shown that downstream residue L574 is required for prolyl hydroxylation and binding of Hif-prolyl hydroxylase 2 (Kageyama et al. 2004). Moreover, the X-ray structure of the complex between Hif-1 alpha peptide bound to pVHL reveals a bipartite binding site, namely, residues 560-567 that include hydroxylated P564 and residues 571-577 that include L574 (Min J.H., 2002, Hon W.C., 2002).
Two other proteins were also suggested to have the LxxLAP motif. I-kappa-B kinase-beta (IKK beta) protein contains an evolutionarily conserved LxxLAP sequence on a loop of its protein kinase domain. It was suggested that hypoxia releases repression of NF-kappa-B activity through prolyl-hydroxylase dependent hydroxylation of IKK beta. The second protein is the Rpb1 subunit of RNA polymerase II. It was reported that pVHL specifically binds the hyperphosphorylated Rpb1 in a proline-hydroxylation-dependent manner, targeting it for ubiquitination. These finding are difficult to reconcile with the enzymatic studies of prolyl hydroxylase specificity cited above.
It is possible that only the Hif1 proteins will prove to have the hypoxia hydroxyproline modification site.