Development of covalent probes to capture Legionella pneumophila effector enzymes

ABSTRACT: Upon infection of host cells Legionella pneumophila releases a multitude of effector enzymes into the hosts cytoplasm that hijack a plethora of cellular activities, including the hosts ubiquitination pathways. Effectors belonging to the SidE-family are involved in non-canonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole which is crucial in the onset of legionnaires disease. This dynamic process is reversed by effectors called Dups that hydrolyse the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targetting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and x-ray crystallography approaches were used to identify the site of covalent crosslinking to be an allosteric cysteine residue and subsequent application of this probe highlight the potential to selective enrich Dup enzymes from Legionella infected cell lysates.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Legionella Pneumophila Subsp. Pneumophila Lp02

TISSUE(S): Cell Culture

SUBMITTER: Rayman Tjokrodirijo  

LAB HEAD: Peter A. van Veelen

PROVIDER: PXD049797 | Pride | 2025-05-06

REPOSITORIES: Pride