No abstract

2-oxoglutarate (2-OG or a-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases (2-OG DDs) involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG DD function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 protects the catalytic lipoyl domain from lipid peroxidation products formed by oxidative damage, demonstrating a requirement for a lipoyl repair pathway in human cells, and highlighting how the redox sensitivity of lipoylation modulates 2-OG metabolism. Keywords2-oxoglutarate, a-ketoglutarate, 2-oxoglutarate dehydrogenase complex, a-ketoglutarate dehydrogenase complex, ABHD11, Hypoxia Inducible Factor, lipoylation, lipoic acid, lipid peroxidation.3The ability to sense and respond to nutrient abundance is a fundamental requirement for cell survival, and to achieve this, cells have evolved several strategies that link metabolic function to transcriptional adaptation. One such strategy is the coupling of 2-OG metabolism to gene transcription, whereby 2-OG, a key component of TCA cycle, can facilitate cell function by modulating the activity of 2-OG DDs involved in the Hypoxia Inducible Factor (HIF) response, DNA methylation, and histone modifications 1 .The relevance of 2-OG in modulating the activity of these dioxygenases is exemplified by changes in the relative abundance of cellular 2-OG. An increased 2-OG/succinate ratio promotes embryonic stem cell pluripotency 2 , and antagonises the growth of solid organ tumours 3 through increased hydroxymethylation of DNA (5hmC) and histone demethylation. Conversely, elevated cellular 2-OG can drive its own reduction to L-2-hydroxyglutarate (L-2-HG), which counterintuitively inhibits 2-OG DDs, leading to decreased DNA hydroxymethylation and histone demethylation, activation of the HIF response, altered T cell fate, and haematopoietic cell differentiation 4-9 .Consequently, understanding how 2-OG metabolism is regulated has broad biological implications.Central to maintaining cellular 2-OG homeostasis is the 2-oxoglutarate dehydrogenase complex (OGDHc, also known as the a-ketoglutarate dehydrogenase complex), the rate-limiting enzyme within the TCA cycle that oxidatively decarboxylates 2-oxoglutarate to succinyl-CoA. This evolutionarily conserved enzyme also requires lipoic acid, a redox sensitive cofactor that is synthesised within the mitochondria and conjugated to a single lysine within the OGDHc E2 subunit, dihydrolipoamide S...