TY - JOUR
T1 - Covalent Probes To Capture Legionella pneumophila Dup Effector Enzymes
AU - Kloet, Max S.
AU - Mukhopadhyay, Rishov
AU - Mukherjee, Rukmini
AU - Misra, Mohit
AU - Jeong, Minwoo
AU - Talavera Ormeño, Cami M.P.
AU - Moutsiopoulou, Angeliki
AU - Tjokrodirijo, Rayman T.N.
AU - van Veelen, Peter A.
AU - Shin, Donghyuk
AU - Đikić, Ivan
AU - Sapmaz, Aysegul
AU - Kim, Robbert Q.
AU - van der Heden van Noort, Gerbrand J.
N1 - Publisher Copyright:
© 2024 The Authors. Published by American Chemical Society.
PY - 2024/10/2
Y1 - 2024/10/2
N2 - Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires’ disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting 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 cross-linking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.
AB - Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires’ disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting 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 cross-linking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.
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U2 - 10.1021/jacs.4c08168
DO - 10.1021/jacs.4c08168
M3 - Article
C2 - 39288007
AN - SCOPUS:85205604295
SN - 0002-7863
VL - 146
SP - 26957
EP - 26964
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 39
ER -