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Covalent Probes To Capture Legionella pneumophila Dup Effector Enzymes
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2024-09-17 , DOI: 10.1021/jacs.4c08168 Max S Kloet 1 , Rishov Mukhopadhyay 1 , Rukmini Mukherjee 2 , Mohit Misra 2 , Minwoo Jeong 3 , Cami M P Talavera Ormeño 1 , Angeliki Moutsiopoulou 1 , Rayman T N Tjokrodirijo 4 , Peter A van Veelen 4 , Donghyuk Shin 3 , Ivan Đikić 2 , Aysegul Sapmaz 1 , Robbert Q Kim 1 , Gerbrand J van der Heden van Noort 1
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2024-09-17 , DOI: 10.1021/jacs.4c08168 Max S Kloet 1 , Rishov Mukhopadhyay 1 , Rukmini Mukherjee 2 , Mohit Misra 2 , Minwoo Jeong 3 , Cami M P Talavera Ormeño 1 , Angeliki Moutsiopoulou 1 , Rayman T N Tjokrodirijo 4 , Peter A van Veelen 4 , Donghyuk Shin 3 , Ivan Đikić 2 , Aysegul Sapmaz 1 , Robbert Q Kim 1 , Gerbrand J van der Heden van Noort 1
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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.
中文翻译：
捕获嗜肺军团菌 Dup 效应酶的共价探针
感染宿主细胞后，嗜肺军团菌会向细胞质中释放大量效应酶，劫持大量细胞活动，包括宿主泛素化途径。属于 SidE 家族的效应子参与宿主底物蛋白的非经典丝氨酸磷酸核糖泛素化，有助于形成含有军团菌的液泡，这对于军团病的发病至关重要。这种动态过程被称为 Dups 的效应子逆转，该效应子水解磷酸核糖泛素化蛋白中的磷酸二酯。我们在化学制备的核糖基化泛素上安装了反应弹头，以生成一组针对这些军团菌酶的探针。对重组 DupA 的体外测试表明，乙烯基磺酸盐弹头在共价复合物形成方面最有效。使用诱变和 X 射线晶体学方法来鉴定变构半胱氨酸残基的共价交联位点。该探针的后续应用凸显了从军团菌感染的细胞裂解物中选择性富集 Dup 酶的潜力。
更新日期：2024-09-17
中文翻译：
捕获嗜肺军团菌 Dup 效应酶的共价探针
感染宿主细胞后，嗜肺军团菌会向细胞质中释放大量效应酶，劫持大量细胞活动，包括宿主泛素化途径。属于 SidE 家族的效应子参与宿主底物蛋白的非经典丝氨酸磷酸核糖泛素化，有助于形成含有军团菌的液泡，这对于军团病的发病至关重要。这种动态过程被称为 Dups 的效应子逆转，该效应子水解磷酸核糖泛素化蛋白中的磷酸二酯。我们在化学制备的核糖基化泛素上安装了反应弹头，以生成一组针对这些军团菌酶的探针。对重组 DupA 的体外测试表明，乙烯基磺酸盐弹头在共价复合物形成方面最有效。使用诱变和 X 射线晶体学方法来鉴定变构半胱氨酸残基的共价交联位点。该探针的后续应用凸显了从军团菌感染的细胞裂解物中选择性富集 Dup 酶的潜力。
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