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.
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.
Mutagenesis genetic screens to uncover epigenetic and metabolic pathways that alter 2-OG dependent dioxygenase activity and HIF signalling
We have applied near-haploid and CRISPR/Cas9 human genome-wide mutagenesis to interrogate metabolic regulation and HIFs and to understand how HIFs activate their target genes. These screens have provided fundamental insights into the intricate relationship between mitochondrial metabolites, cellular iron metabolism 2-OG dependent dioxygenases and HIF signalling (reviewed in Ortmann et al FEBS Journal 2021).
We uncovered a novel role for (1) the Vacuolar-ATPase (V-ATPase), in controlling HIF activation by restricting cytosolic iron availability (Miles, Burr et al eLife 2017), and (2) 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).
By screening for genes required to activate the HIF response, we have uncovered that the H3K4me3 transferase, SET1B, is recruited by HIFs to facilitate selective activation of HIF target genes (Ortmann et al Nature Genetics 2021). SET1B binds the HIF-a subunit in hypoxia and translocates to the nucleus to increase transcription of specific HIF target genes.
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.
Ortmann BM, Burrows N, Lobb IT, Arnaiz E, Wit N, Bailey PSJ, Jordon LH, Lombardi O, Peñalver A, McCaffrey J, Seear R, Mole DR, Ratcliffe PJ, Maxwell PH, Nathan JA. The HIF complex recruits the histone methyltransferase SET1B to activate specific hypoxia-inducible genes. Nature Genetics 2021. DOI: 10.21203/rs.3.rs-85295/v1.
Ortmann BM, Nathan JA. Genetic approaches to understand cellular responses to oxygen availability. The FEBS journal 2021. https://doi.org/10.1111/febs.16072
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
Esther Arnaiz (postdoc)
James Bertlin (MDPhD student)
Guine Grice (postdoc)
Louise Jordon (Wellcome clinical PhD fellow)
Anthony Martinelli (Wellcome clinical PhD fellow)
Brian Ortmann (postdoc)
Tekle Pauzaite (postdoc)
Rachel Seear (research assistant and lab manager)
Niek Wit (postdoc)