Cellular mechanisms of oxygen and metabolite sensing:
A fundamental requirement for cell survival is the ability to respond to local oxygen and nutrient environments. Our goal is to gain novel insights in oxygen and metabolite sensing pathways, providing potential new therapeutic targets for inflammatory disease and cancers.

Oxygen and metabolite sensing by 2-oxoglutarate dependent dioxygenases:
The ability to sense and respond to changes in oxygen availability is conserved in all metazoans. Central to this process are a group of enzymes that require oxygen, 2-oxoglutarate (2-OG), and iron for catalytic activity – the 2-OG dependent dioxygenases. The most well-described function of these enzymes relates to the prolyl hydroxylases (PHDs) which sense intracellular oxygen, controlling the stability of Hypoxia Inducible transcription Factors (HIFs). However, 2-OG dependent dioxygenases have diverse functions, including altering cell phenotypes by remodelling chromatin. We aim to (i) uncover new metabolic pathways that influence the activity of 2-OG dependent dioxygenases (ii) gain insights into their biological relevance, focusing on HIF signalling and chromatin remodelling.

Using forward genetic screens to uncover metabolic pathways that alter 2-OG dependent dioxygenase activity:
We have applied near-haploid and CRISPR/Cas9 human forward genetic screens to identify genes required for the metabolic regulation of HIFs. This approach uncovered a novel role for the 2-oxoglutarate dehydrogenase complex (OGDHc) – a key TCA cycle enzyme, in the control of the HIF response (Burr et al Cell Metabolism 2016). Loss of the OGDHc drives the formation of the metabolite L-2-Hydroxyglutarate (L-2-HG), which accumulates in cells and directly inhibits PHDs and 2-OG dependent dioxygenases involved in modifying chromatin (e.g. TETs). We have also shown that defects in mitochondrial lipoylation activate HIFs by reducing OGDHc activity and promoting L-2-HG formation. Activation of this metabolic HIF response can also be seen in patients with germline mutations in lipoylation (Burr et al Cell Metabolism 2016).

Our forward genetic approaches uncovered an unexpected role for the Vacuolar-ATPase (V-ATPase), in controlling HIF activation by restricting cytosolic iron availability (Miles, Burr et al eLife 2017). The V-ATPase is the main proton pump for acidifying endo-lysosomal compartments and is therefore required for lysosomal protein degradation. However, rather than preventing the lysosomal degradation of HIF1a, V-ATPase inhibition prevents the uptake of ferric iron and conversion to the ferric form, thereby inhibiting PHD activity.

Therefore, these screens provide fundamental insights into the intricate relationship between mitochondrial metabolites, cellular iron metabolism and 2-OG dependent dioxygenases.

Protein degradation in oxygen and metabolite sensing pathways:
We have developed biochemical approaches to explore the role of the ubiquitin enzymes in regulating the turnover of proteins involved in oxygen-sensing/metabolic pathways. This work has led to the identification that lysine-11 (K11) linked ubiquitin chains, a ubiquitin linkage implicated in cell-cycle regulation and HIF signalling can mediate proteasome-mediated degradation dependent on whether they are pure (homotypic) or mixed with other linkages (heterotypic) (Grice et al, Cell Reports 2015). In combination with our forward genetic approaches, we have identified a role for two ER-associated E3 ligases in a protein quality control pathway for heme-oxygenase 1 (HO-1) (Stefanovic-Barrett et al, EMBO Reports 2018), a tail-anchored protein which metabolises heme and releases free iron. Understanding the role of ubiquitin enzymes in non-canonical regulation of HIFs and metabolic pathways is a focus of our current studies.