Oxygen Sensing and Hypoxic Response
The Peet Laboratory is interested in characterising the molecular mechanisms by which cells are able to sense and respond to hypoxia in normal physiology and disease.
The ability of essentially all cells in the body to sense and respond to changes in oxygen, specifically low oxygen (or hypoxia), is crucial for survival, but also involved in most major human diseases. These changes may be environmental, such as high altitude, or part of the normal developmental process. However, hypoxia is also an important factor in many major human diseases, such as heart attack, stroke, vascular disease and cancer.
A key way that cells respond to hypoxia is by modulating gene expression. Central to hypoxic gene regulation are the hypoxia inducible factors (HIFs). These essential transcription factors are regulated by a family of oxygen-sensing hydroxylases that control both the protein level and transcriptional activity of the HIFs, resulting in activation when oxygen levels decrease. We currently use the latest techniques in molecular biology, cell culture, protein chemistry and metabolic analysis to understand the role of this pathway in normal physiology and disease.
For available PhD and Honours projects please contact Dr Dan Peet
Non-HIF Substrates of FIH
Factor inhibiting HIF (FIH) is an oxygen-sensing asparaginyl hydroxylase involved in regulating the activity of HIF.
We and others have investigated whether these oxygen-sensing enzymes can modify other cellular proteins, thus mediating others aspects of the cellular response to hypoxia. Consequently we now know that FIH is able to specifically hydroxylate a number of proteins containing ankyrin repeat domains (ARDs), including Notch, IkBα and others. However, the consequence of these modifications of ARDs in most cases remains unclear.
We have recently identified the TRP ion channels as likely substrates of the enzyme FIH. These cation channels have a number of different functions, including sensing temperature and pain. Our preliminary data demonstrate that at least one of these channels, TRPV3, can be hydroxylated by FIH. Importantly, we have also demonstrated that this modification influences channel activity (current flow) in an oxygen dependent manner. This project seeks to identify and characterise the regulation of TRP channels, and other non-HIF substrates, by FIH-dependent hydroxylation.
Differential functions of HIF-1 α and HIF-2 α
HIF-1α and HIF-2α are the two major oxygen-regulated HIF-α subunits responsible for HIF-dependent changes in gene expression. They can both be regulated by hypoxia via prolyl and asparaginyl hydroxylation, have overlapping expression patterns and some shared target genes, and contribute to normal physiology, development and disease. However, they are not redundant and have distinct physiological roles.
We are interested in how HIF-1α and HIF-2α are differentially regulated, and their distinct contributions to human disease. This project aims to characterise the differential functions and regulation of each of these proteins in specific disease models, such as multiple myeloma.
Cancer-like metabolism of the mammalian retina
Glucose metabolism has long been of interest to Biochemists, but has undergone a major resurgence in the last few years due to a growing understanding of its important role in cancer. This project examines how cells in the mammalian retina display similar metabolic characteristics to cancer cells, and explores whether they are controlled by a common mechanism.
In the presence of oxygen, differentiated cells usually generate ATP very efficiently from oxygen using oxidative phosphorylation. However cancer and other rapidly proliferating cells favour anaerobic glycolysis, even in the presence of abundant oxygen, which is commonly known as the Warburg effect. This is now a very active field of cancer research, to help understand, diagnose and even therapeutically target cancer cells. The underlying cause is postulated to be a function of the high energetic and biosynthetic needs of these proliferating cells.
Interestingly, photoreceptor cells in the mammalian retina also display the Warburg Effect, even though they do not proliferate. One possible explanation is their high biosynthetic requirements stemming from the constant turnover of the outer segments of these cells. Photoreceptor degeneration and visual impairment have also been linked to cellular metabolism. In addition, new therapeutic approaches designed to target the Warburg effect in cancer may also target the retina, having negative consequences on vision. This project aims to determine the mechanisms controlling the Warburg effect in mammalian retina, and compare them to the mechanisms controlling metabolism in cancer cells.
For a list of publications, please visit the researcher profile of Dr Dan Peet.
Underlying hypoxic gene regulation are the hypoxia inducible transcription factors (HIFs). The HIFs consist of a dimer of HIF-alpha and HIF-beta subunits. The HIF-alpha subunits are regulated by oxygen, with the activity and abundance of the HIF-alpha subunit increased in hypoxia, whereas the HIF-beta subunit (better known as ARNT) is oxygen-independent (Figure1). Oxygen regulated control of HIF-alpha abundance is mediated by three homologous oxygen-dependent prolyl hydroxylases, PHD1-3. They modify distinct proline residues in the HIF-alpha proteins in normoxia resulting in the recruitment of the Von Hippel Lindau protein (pVHL), polyubuiquitylation and rapid proteosomal degradation of the HIF-alpha proteins. The control of HIF-alpha activity is mediated by asparaginyl hydroxylation by the oxygen-dependent asparaginyl hydroxylase, FIH (Factor Inhibiting HIF). Asparaginyl hydroxylation by FIH represses transcriptional activity by preventing the interaction of HIF-alpha proteins 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. Research by ourselves and others have demonstrated that these hydroxylases act as cellular oxygen sensors.
Figure 1: Oxygen-dependent regulation of gene expression mediated by the hypoxia inducible transcription factors (HIFs). The 3 HIF prolyl hydroxylases (PHD 1-3) and single HIF asparaginyl hydroxylase (factor inhibiting HIF, FIH) are oxygen dependent enzymes. The von hippel lindau protein (VHL) is part of an E3 ubiquitin ligase complex that promotes proteosomal degradation of HIF, and CBP and p300 are transcriptional coactivators. Examples of HIF target genes include erythropoietin (Epo), vascular endothelial growth factor (VEGF) and glucose transporter 1 (Glut1).