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James Nathan

Cellular mechanisms of oxygen and metabolite sensing

A fundamental requirement for cell survival is the ability to respond to the local oxygen and nutrient environment. 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.

                              

Nathan web 2018
2-OG dependent dioxygenases as oxygen/metabolite sensors. 2-OG-dependent dioxygenases are a diverse group of around 60 enzymes involved in processes such as collagen synthesis, ribosome modifications, HIF signalling and chromatin remodelling. Our studies focus on 2-OG dependent dioxygenases involved in HIF signalling and chromatin remodelling: PHDs- prolyl hydroxylases, FIH – factor inhibiting HIF, TETs - ten-eleven translocation enzymes (hydroxylate 5-methylcytosine), and JHDMs - Jumonji-domain containing histone demethylases.

 

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 been 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.

Key papers

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 12(4):545-53 (2015). PMC4533228. http://www.cell.com/cell-reports/abstract/S2211-1247(15)00687-7

Nathan JA, Spinnenhirn V, Schmidtke G, Basler M, Groettrup M and Goldberg AL. Immuno- and constitutive proteasomes do not differ in ability to degrade ubiquitinated proteins. Cell (2013) 152,1184-94. PMC3791394. http://www.cell.com/abstract/S0092-8674(13)00129-3

Nathan JA, Kim HT, Ting L, Gygi S, and Goldberg A. Why do cell proteins linked to K63-polyubiquitin chains not associate with proteasomes? EMBO Journal (2013) 32, 552-65. PMC3579138. http://emboj.embopress.org/content/32/4/552

Research highlight in Nature Reviews Molecular Cell Biology. http://www.nature.com/nrm/journal/v14/n3/full/nrm3540.html

Link to full list of publications

https://www.ncbi.nlm.nih.gov/pubmed/?term=nathan+ja

 

James Nathan

Dr James Nathan

Wellcome Trust Senior Clinical Fellow

Honorary Consultant in Respiratory Medicine, Addenbrooke's Hospital

Department: Medicine

contact: jan33@cam.ac.uk

01223 748483

Plain English

Cells have to be able to sense oxygen and nutrient availability to allow growth, differentiation or survival when nutrients are scarce. Our aims are to explore the links between oxygen sensing and nutrient levels. Through an improved understanding of these pathways, we hope that we will be able to provide new therapeutic targets for cancers and inflammatory disease.

Group members

Peter Bailey · Michal Chojnacki · Anna Dickson · Guinevere Grice · Brian Ortmann · Annie Miles · William Sherwood · Sandra Stefanovic-Barrett

Funding

Wellcome Trust

Medical Research Council

Lister Institute

Pfizer ITEN Partnership

 

 

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