Gaining Insights into Non-Canonical Ubiquitination through MALDI-TOF MS

Activity: Talk or presentation typesInvited talk

Description

GAINING INSIGHTS INTO NON-CANONICAL UBIQUITINATION THROUGH MALDI-TOF/MS


Syed Arif Abdul Rehman1, Chiara Cazzaniga1, Elena Di Nisio1-3, Odetta Antico1, Axel Knebel1, Clare Johnson2, Alp T. Şahin4, Peter E. G. F. Ibrahim5, Frederic Lamoliatte1, Rodolfo Negri3,6, Miratul Muqit MK1, Virginia De Cesare1*


1MRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, UK ([email protected]).

2MRC PPU Reagents and Services, School of Life Sciences, University of Dundee, UK.

3Department of Biology and Biotechnologies “C. Darwin”, Sapienza University of Rome, Italy.

4Computational Biology, School of Life Sciences, University of Dundee, Dundee, UK.

5Drug Discovery Unit, Division of Biological Chemistry & Drug Discovery, University of Dundee, UK.

6Institute of Molecular Biology and Pathology, CNR, Via degli Apuli 4, 00185 Rome, Italy.


Ubiquitination is a crucial and complex post-translational modification essential for cellular homeostasis, mediated by ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), and deubiquitinating enzymes (DUBs). Recapitulating these enzymatic activities in vitro is vital for understanding their cellular functions and supporting drug discovery efforts.


We developed Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF/MS) based assays to assess E2[1], E3[2, 3], and DUB activities[4]. This technology supports foundational research and translational applications by identifying diverse ubiquitin products and unexpected, non-canonical enzymatic activities.


In this talk, we highlight our discovery of the non-canonical activities of the UBE2Q family of E2 ubiquitin-conjugating enzymes. E2s are pivotal in ubiquitination and interact with multiple E3 ligases to shape the cellular ubiquitination landscape. Although ubiquitination typically targets lysine residues, our research reveals that UBE2Qs can conjugate ubiquitin to serine, threonine, and other hydroxyl-containing biomolecules independently of E3 ligases.


This discovery, supported by structural and biochemical analyses, broadens the landscape of ubiquitin-mediated modifications. We anticipate that the discovery of novel E2s with non-canonical activity will have profound and wide-ranging impacts on the expansion of the ubiquitination landscape and, consequently, toward the understanding of ubiquitin-mediated biological processes.


References

1. Abdul Rehman, S.A., et al., Discovery and characterization of noncanonical E2-conjugating enzymes. Sci Adv, 2024. 10(13): p. eadh0123.

2. Traynor, R., et al., Design and high-throughput implementation of MALDI-TOF/MS-based assays for Parkin E3 ligase activity. Cell Rep Methods, 2024. 4(2): p. 100712.

3. De Cesare, V., et al., The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway. Cell Chem Biol, 2018. 25(9): p. 1117-1127 e4.

4. De Cesare, V., et al., High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry-based deubiquitylating enzyme assay for drug discovery. Nat Protoc, 2020. 15(12): p. 4034-4057.
Period9 Jul 2024
Event title3rd Ubiquitin Function in Health and Disease Conference #Ubiq24
Event typeConference
LocationLisbon, PortugalShow on map