The role of ABHD11 in the regulation of the hypoxia inducible transcription factors

Hypoxia inducible transcription factors (HIFs) mediate a highly conserved cellular oxygen sensing system, driving a diverse set of transcriptional changes that allow adaptation to hypoxic conditions. However, HIFs can also be activated independently of oxygen by changes in levels of mitochondrial metabolites, a mechanism which is important in the development of certain HIF-driven cancers. Critical to understanding this metabolic axis is the recognition that the prolyl hydroxylase enzymes (PHDs), the oxygen sensors within the HIF pathway, are members of a diverse group of enzymes termed 2-oxoglutarate-dependent dioxygenases. These enzymes all require oxygen, iron and the Tricarboxylic Acid (TCA) cycle metabolite, 2-oxoglutarate (2-OG/a-ketoglutarate) for catalytic activity. Therefore, understanding how 2-OG levels are regulated is essential to dissect the relative contribution of metabolism to activation of HIFs. A key determinant of 2-OG metabolism is the 2-oxoglutarate dehydrogenase complex (OGDHc) – a rate limiting step for conversion of 2-OG to succinyl-CoA within the TCA cycle. Genetic disruption of the OGDHc leads to HIF stabilisation through the accumulation of 2-OG and formation of L-2-hydroxyglutarate, an inhibitor of PHDs. Patients with hereditary mutations in the OGDHc develop tumour syndromes, typical of HIF activation. However, how the OGDHc is regulated is not known. In this thesis, I optimise CRISPR/Cas9 forward genetic screens in oxygen replete conditions to identify genes involved in HIF metabolic activation, focusing on the OGDHc. These screens successfully identify known determinants of 2-OG metabolism, including core components of the OGDHc. In addition, they identified ABHD11 as an uncharacterised mitochondrial protein that, on depletion, leads to metabolic stabilisation of HIF-1⍺. Using a combination of cell biology, LC-MS metabolomics and in vitro enzymatic assays, I demonstrate that ABHD11 localises to the mitochondrial matrix and is required for normal TCA cycle function. ABHD11 depletion decreases the activity of the OGDHc, leading to 2-OG accumulation and L-2-hydroxyglutarate formation, consistent with a central role for OGDHc function in HIF metabolic activation. ABHD11 associates with the OGDHc, but ABHD11 depletion does not alter protein levels of the subunits. Instead, ABHD11 is required to maintain an essential post-translational fatty acid modification on the OGDHc E2 subunit (DLST), lipoylation, which is sensitive to damage from reactive oxygen species and lipid peroxidation products. Together, these studies identify ABHD11 as a new component maintaining TCA cycle function through regulating the lipoylation of the OGDHc.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2019

Author dc:creator

• Bailey, Peter Stephen John

Advisor dc:contributor.advisor

• Nathan, James

• hypoxia
• oxygen sensing
• HIF
• HIF1a
• TCA cycle
• 2-oxoglutarate
• 2-OG
• OGDH complex
• L-2-hydroxyglutarate
• lipoate
• lipoylation
• ABHD11
• lipid peroxidation

Statement dc:rights

• Figure 1.2 (page 4) has been adapted by permission from Islam et al., 2018 (licence number 1012532-1): Annual Reviews, Annual Review of Biochemistry; 2-Oxoglutarate-Dependent Oxygenases. Islam, M.S., Leissing, T.M., Chowdhury, R., Hopkinson, R.J., and Schofield, C.J. 87, 585-620 (2018). Figure 1.3 (page 9) has been adapted by permission from Burr et al., 2016 (licence number 4744271150088): Elsevier, Cell Metabolism; Mitochondrial Protein Lipoylation and the 2-Oxoglutarate Dehydrogenase Complex Controls HIF1a Stability in Aerobic Conditions. Burr, S.P., Costa, A.S.H., Grice, G.L., Timms, R.T., Lobb, I.T., Freisinger, P., Dodd, R.B., Dougan, G., Lehner, P.J., Frezza, C., and Nathan, J.A. 24, 740-752 (2016). Figure 6.1 (page 109) was adapted from the HNE CLUB website https://sites.unimi.it/HNECLUB/?page_id=490, and has been redacted from the publicly available version of this thesis. Figure 7.1 (page 129) was adapted from the NCBI genome browser website https://ncbi.nlm.nih.gov.uk/genome (National Library of Medicine). As a work of the US government it is in the public domain.

Licence

• https://www.repository.cam.ac.uk/bitstreams/441696c3-a62e-4a89-a264-6871ca85a306/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.49081

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/302005