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, Bailey et al Nature Communications 2020). 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 patient defects in mitochondrial lipoylation activate HIFs by reducing OGDHc activity and promoting L-2-HG formation (Burr et al Cell Metabolism 2016), and identified an orphan serine hydrolase, ABHD11, which is required for normal OGDHc activity by preserving functional lipoylation of the complex (Bailey et al Nature Communications 2020).
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.
Bailey PSJ, Ortmann BM, Martinelli AW, Houghton JW, Costa ASH, Burr SP, Antrobus R, Frezza C, Nathan JA. ABHD11 maintains 2-oxoglutarate metabolism by preserving functional lipoylation of the 2-oxoglutarate dehydrogenase complex. Nature Communications 2020. https://rdcu.be/b6e1h
Stefanovic-Barrett S, Dickson AS, Burr SP, Williamson JC, Lobb IT, van den Boomen DJH, Lehner PJ, and Nathan JA. MARCH6 and TRC8 facilitate the quality control of cytosolic and tail anchored proteins. EMBO Reports 2018. http://embor.embopress.org/content/early/2018/03/08/embr.201745603
Miles AL*, Burr SP*, Grice GL, and Nathan JA. The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1alpha prolyl hydroxylation by regulating cellular iron levels. eLife 2017. http://dx.doi.org/10.7554/eLife.22693. *equal contribution. Recommended on F1000
Burr SP, Costa ASH, Grice GL, Timms RT, Lobb IT, Freisigner P, Dodd RB, Dougan G, Lehner PJ, Frezza C, and Nathan JA. Mitochondrial protein lipoylation and the 2-oxoglutarate dehydrogenase complex controls HIF1α stability in aerobic conditions. Cell Metabolism 2016.http://dx.doi.org/10.1016/j.cmet.2016.09.015
Grice GL, Lobb IT, Weekes MP, Gygi SP, Antrobus R, and Nathan JA. The proteasome distinguishes between heterotypic and homotypic lysine-11 linked polyubiquitin chains. Cell Reports 2015. 12(4):545-53. PMC4533228. http://www.cell.com/cell-reports/abstract/S2211-1247(15)00687-7