Development of covalent probes to capture Legionella pneumophila effector enzymes

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.

当嗜肺军团菌（Legionella pneumophila）感染宿主细胞时，会向宿主细胞质中释放大量效应酶，劫持宿主的多项细胞活动，其中涵盖宿主的泛素化通路。隶属于SidE家族（SidE-family）的效应因子可介导宿主底物蛋白发生非经典丝氨酸磷酸核糖基泛素化，该过程有助于形成含军团菌的空泡（Legionella-containing vacuole），而此空泡对于军团病（legionnaires disease）的发病进程至关重要。这一动态修饰过程可被名为Dups（Dups）的效应因子逆转：这类效应因子能够水解磷酸核糖基泛素化蛋白中的磷酸二酯键。我们在化学制备的核糖基化泛素上引入反应性弹头，以生成一组靶向上述军团菌酶的探针。针对重组DupA开展的体外实验表明，乙烯基磺酸酯弹头在共价复合物形成环节的效率最为突出。通过诱变实验与X射线晶体学（X-ray crystallography）技术，我们确定了共价交联的位点为一个变构半胱氨酸残基；后续对该探针的应用验证了其可从军团菌感染的细胞裂解液中选择性富集Dup酶类。