Discovery and characterization of non-canonical E2 conjugating enzymes

Abdul Rehman, S. A.; Di Nisio, E.; Cazzaniga, C.; Antico, O.; Knebel, A.; Johnson, C.; Lamoliatte, F.; Negri, R.; Muqit, M.; De Cesare, V.

doi:10.1101/2023.03.05.531151

bioRxiv · 10.1101/2023.03.05.531151

Discovery and characterization of non-canonical E2 conjugating enzymes

Abdul Rehman, S. A.·Di Nisio, E.·Cazzaniga, C.·Antico, O.·Knebel, A.·Johnson, C.·Lamoliatte, F.·Negri, R.·Muqit, M.·De Cesare, V.

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Abstract

E2 conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation is fundamental for maintaining cellular homeostasis and impacts almost all cellular process. By interacting with multiple E3 ligases, E2s direct the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a post-translational modification that specifically targets lysine side chains (canonical ubiquitylation). We used MALDI-TOF Mass Spectrometry to discover and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We employed protein modelling and prediction tools to identify the catalytic determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results join a stream of recent literature that challenges the definition of ubiquitylation as an exquisitely lysine-specific modification and provide crucial insights into the missing E2 element responsible for non-canonical ubiquitylation. TeaserE2 conjugating enzymes (E2s) play a fundamental role in the attachment of ubiquitin to its substrate. Most E2s can form an isopeptide bond between the ubiquitin C- terminus and a lysine present on the substrate. We identified a family of E2s, UBE2Q1 and UBE2Q2, able to target amino acids other than lysine. Currently nothing is known about their mechanism of action and what substrates they are targeting, even though genetic ablation of UBE2Q1 produce substantial infertility in mice. Here we answer the question about what the key residues beneath their peculiar activity are. We discovered that UBE2Q1 target the lysine-free cytoplasmic domain of the Golgi resident protein Beta-1,4-galactosyltransferase 1, providing an interesting precedent for the role of non-canonical ubiquitylation in eukaryotic cells.

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Abdul Rehman, S. A., Di Nisio, E., Cazzaniga, C., Antico, O., Knebel, A., Johnson, C., Lamoliatte, F., Negri, R., Muqit, M., De Cesare, V.. 2023-03-05. Discovery and characterization of non-canonical E2 conjugating enzymes. https://doi.org/10.1101/2023.03.05.531151

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