Peet Laboratory

The human body is able to sense and respond to changes in oxygen levels, most importantly low oxygen (hypoxia), to maintain oxygen homeostasis. These changes may be environmental such as high altitude, but are even more important in many major human diseases, such as heart attack and stroke, where blood flow is disrupted and consequently oxygen delivery is compromised. In this case an immediate response to this localised hypoxia is crucial for minimising damage. In contrast, in cancer this process is exploited with hypoxic regions deep inside tumours stimulating the growth of new blood vessels and promoting tumour growth and metastasis.

The crucial genomic response to hypoxia involves switching on numerous genes to increase oxygen delivery and metabolically adapt to reduced oxygen availability. For example, genes such as erythropoietin (Epo) increase red blood cell production, vascular endothelial growth factor (VEGF) stimulates vascular development, and other genes increase glucose transport and glycolysis to produce energy in the absence of oxidative phosphorylation.

The Hypoxia Inducible Factors (HIFs) are oxygen regulated transcription factors that are central to this hypoxic response and act as master switches to directly turn on these and other essential genes in response to hypoxia. It is these transcription factors, particularly how they sense and respond to changes in oxygen, and their role in human disease, that are the focus of my research. The HIFs consist of a dimer of HIF-alpha and HIF-beta subunits, where the HIF-alpha subunits are regulated by oxygen levels, whereas the HIF-beta subunit is not. Under normoxia the HIF-alpha proteins are rapidly degraded and transcriptionally repressed, rendering them essentially inactive. When oxygen is limiting the HIF-alpha subunits are stablised and transcriptionally active, translocate to the nucleus where they dimerise with HIF-1beta, and bind to hypoxic response elements (HREs) in the regulatory regions of target genes to upregulate transcription. However, although the HIF-alpha proteins are activated by hypoxia they themselves do not directly sense changes in oxygen levels. The identities of the cellular oxygen sensors that regulate the HIFs have long remained a mystery. Recent research by ourselves and others internationally has identified these oxygen sensors as oxygen-dependent hydroxylases.

At normoxia with ample oxygen available these enzymes directly modify the HIF-alpha proteins and keep them inactive. One group of these oxygen sensing enzymes, the prolyl hydroxylases (PHDs), modify distinct proline residues in the HIF proteins at normoxia resulting in the recruitment of the Von Hippel Lindau protein (pVHL), polyubuiquitylation and rapid proteosomal degradation of the HIF-alpha proteins. A second enzyme, an asparaginyl hydroxylase called FIH-1 that was first characterised by our laboratory, also modifies the HIF proteins at normoxia. This modification represses their transcriptional activity by preventing the interaction with transcriptional coactivators such as CBP/p300. When oxygen is limiting both prolyl and asparaginyl hydroxylases are unable to modify the HIFs, resulting in stable, transcriptionally active HIFs activating their target genes in response to hypoxia (Figure 1).

The first key area of current research is characterizing the function of FIH-1, the novel oxygen-sensing asparaginyl hydroxylase. The second key area is understanding the different roles and mechanisms of regulation between HIF-1alpha and HIF-2alpha. This information is invaluable for our understanding of how the body is able to sense and respond to changes in oxygen, and the role of HIFs in major human diseases, and may also provide therapeutic targets for these same diseases. We currently use the latest techniques in molecular biology, cell culture and protein analysis, including PCR, cDNA cloning, siRNA, DNA microarrays, cell culture models of hypoxic response, transcription assays, animal models of development, protein expression and purification, in vitro enzyme assays, immunoblotting, chromatography, and mass spectrometry.