Host-pathogen interaction profiling using self-assembling human protein arrays.
Host-pathogen interaction profiling using self-assembling human protein arrays.
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DOI：
10.1021/pr5013015
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发表时间：
2015-04-03
影响因子：
4.4
通讯作者：
Machner, Matthias P.
中科院分区：
文献类型：
--
作者：
Yu, Xiaobo;Decker, Kimberly B.;Barker, Kristi;Neunuebel, M. Ramona;Saul, Justin;Graves, Morgan;Westcott, Nathan;Hang, Howard;LaBaer, Joshua;Qiu, Ji;Machner, Matthias P.
宿主-病原体蛋白质相互作用是每种微生物感染的基础，但由于缺乏简单的检测工具来避免丰度偏差，同时为实验修改提供开放格式，因此它们的鉴定仍然具有挑战性。在这里，我们应用核酸可编程蛋白阵列和HaloTag-Halo配体检测系统来确定嗜肺军团菌效应子（SidM和利达）与10，000种独特的人类蛋白质的相互作用网络。我们确定了这些L的已知目标。pneumophila蛋白和潜在的新型相互作用候选物。此外，我们应用我们的基于点击化学的NAPPA平台来鉴定SidM的底物，SidM是一种具有腺苷酰转移酶结构域的效应物，其催化腺苷酸化（腺苷酸化），即一磷酸腺苷（AMP）的共价加成。我们在独立的体外下拉和体内基于细胞的测定中证实了新型SidM和利达靶标的子集，并进一步了解了这些效应物如何区分不同的宿主Rab GTP酶。我们的方法避免了数千种人类和病原体蛋白的纯化，并且不需要针对查询蛋白的抗体或预标记。该系统适用于来自多种人类病原体的效应物的高通量分析，所述效应物可以结合和/或后修饰人类蛋白质组内的靶标。
Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10,000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or pre-labeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.
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DOI：
10.1126/science.1227026
发表时间：
2012-11-23
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Choy A;Dancourt J;Mugo B;O'Connor TJ;Isberg RR;Melia TJ;Roy CR
通讯作者：
Roy CR
影响因子：
4.9
作者：
Gibson DS;Qiu J;Mendoza EA;Barker K;Rooney ME;LaBaer J
通讯作者：
LaBaer J
影响因子：
6.7
作者：
Cheng, Wei;Yin, Kun;Gu, Lichuan
通讯作者：
Gu, Lichuan
影响因子：
3.6
作者：
Kubori, Tomoko;Hyakutake, Akihiro;Nagai, Hiroki
通讯作者：
Nagai, Hiroki
影响因子：
3.4
作者：
Lomma, M.;Dervins-Ravault, D.;Buchrieser, C.
通讯作者：
Buchrieser, C.