Metabolism Meets Met1-linked Ubiquitin: Discovery of a New Regulator of AMPK Signalling and Energy Sensing

Cells must be able to cope with energetic stress to maintain metabolic homeostasis. Cellular mechanisms for managing energetic stress are activated during periods of starvation, exercise, and in disease states such as liver disease and diabetes. Many intricate signalling pathways governing metabolic homeostasis are controlled by the master kinase 5' adenosine monophosphate-activated (AMP) protein kinase (AMPK). Ubiquitylation, the attachment of small ubiquitin proteins and formation of polyubiquitin chains, is a critical post-translational modification in numerous signalling cascades. Specifically, methionine 1 (M1)-linked polyubiquitin (M1-Ub) chains have recently been implicated in regulating metabolic pathways and protecting against liver disease and hepatocellular carcinoma (HCC). However, the molecular mechanisms by which M1-Ub controls metabolism remain largely unexplored.

In this thesis, my colleagues and I identified M1-Ub as critical for signalling by AMPK and the cellular response to energetic stress. We show that the M1-Ub ligase linear ubiquitin chain assembly complex (LUBAC) and the M1-Ub-specific deubiquitinase OTU deubiquitinase with linear specificity (OTULIN) form a complex with AMPK. The catalytic activity of LUBAC promotes AMPK activity during energetic stress, whereas OTULIN restrains AMPK signalling. Neither AMPKα nor its upstream kinase LKB1 are modified with M1-Ub, but both can be found in cellular complexes containing M1-Ub. Using multiple M1-Ub enrichment strategies coupled to mass spectrometry-based proteomics, we identified several putative LUBAC substrates during AMPK activation. Extensive phosphoproteomic analysis showed that HOIP-deficient cells have a blunted signalling response to starvation. Importantly, M1-Ub-dependent AMPK activation is critical for survival of Drosophila during starvation, and AMPK signalling is also dysregulated in HOIP- and OTULIN-deficient mouse livers and primary fibroblasts from an ORAS patient.

The findings presented in this PhD thesis demonstrate that M1-Ub serves as an important regulatory signal for cells during energetic stress by modulating AMPK activity and its subsequent phosphorylation of downstream substrates. Crucially, the data suggest that there are physiologically critical metabolic pathways that become dysregulated in the absence of LUBAC, and that dysregulated AMPK-mediated metabolic signalling and may be involved in M1-Ub-associated pathologies.