Comparison of phosphoribosyl ubiquitin probes targeting legionella dup enzymes

Kloet, M.S.; Mukhopadhyay, R.; Mukherjee, R.; Misra, M.; Ormeño, C.M.P.; Tjokrodirijo, R.T.N.; Hensbergen, P.J.; Veelen, P.A. van; Dikic, I.; Sapmaz, A.; Noort, G.J.V. van

doi:doi:10.1021/acs.bioconjchem.4c00541

In order to effectively replicate within a host cell, the Legionella pneumophila bacterium secretes effector enzymes into the cytoplasm in order to manipulate cellular host pathways including host ubiquitination. Some of these effectors, the so-called SidE-family, mediate noncanonical phosphoribosyl serine ubiquitination (PR-ubiquitination) of host substrate proteins, contributing to the recruitment of ER-remodeling proteins and the formation of a Legionella-containing vacuole, which is crucial in the early stages of bacterial infection. PR-ubiquitination is a dynamic process that is reversed by other Legionella effectors called deubiquitinases for PR-ubiquitination (Dups). We recently discovered a reactive allosteric cysteine in close proximity to the catalytic triad of DupA, which can be exploited as a target for covalent probe development. We here report on the synthesis of vinyl-sulfonate and fluoro-sulfonate warhead-containing phosphoribosyl ubiquitin probes, where the Arg42...Show moreIn order to effectively replicate within a host cell, the Legionella pneumophila bacterium secretes effector enzymes into the cytoplasm in order to manipulate cellular host pathways including host ubiquitination. Some of these effectors, the so-called SidE-family, mediate noncanonical phosphoribosyl serine ubiquitination (PR-ubiquitination) of host substrate proteins, contributing to the recruitment of ER-remodeling proteins and the formation of a Legionella-containing vacuole, which is crucial in the early stages of bacterial infection. PR-ubiquitination is a dynamic process that is reversed by other Legionella effectors called deubiquitinases for PR-ubiquitination (Dups). We recently discovered a reactive allosteric cysteine in close proximity to the catalytic triad of DupA, which can be exploited as a target for covalent probe development. We here report on the synthesis of vinyl-sulfonate and fluoro-sulfonate warhead-containing phosphoribosyl ubiquitin probes, where the Arg42 position of ubiquitin is linked to the C1 of ribose via a native guanidinium group, and compare them to triazole-linked probes. In vitro tests on recombinant DupA and SdeAPDE revealed that these probes are able to capture the enzymes covalently. In a pull-down proteomics experiment, DupA and DupB enzymes are enriched from Legionella-infected cell lysates, highlighting the potential of native Arg-riboside linked probes to capture Legionella effector enzymes in a complex proteome.Show less

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Comparison of phosphoribosyl ubiquitin probes targeting legionella dup enzymes