CHARACTERIZATION OF HOST-PATHOGEN PROTEIN-PROTEIN INTERACTIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 新的质谱学方法通过识别在特定细胞类型中表达的大多数蛋白质，使细胞生物学发生了革命性的变化。该方法已经扩展到通过亲和标记和蛋白质复合体的纯化来剖析蛋白质-蛋白质相互作用。利用最先进的质谱仪，我们将鉴定与沙门氏菌分泌的毒力因子(所谓的第三型分泌效应器；TTS效应器)相互作用的细胞蛋白。具体地说，我们将使用一个新的串联亲和标签和新的蛋白质-蛋白质交联的方法来剖析炎症体，以及一个TTS效应器如何通过与NOD信号转导通路的相互作用来抑制NFK的激活。我们已经构建了包含串联亲和标签的载体，该标签适合在哺乳动物细胞和沙门氏菌中表达。该标签编码两个由生物素化位点隔开的6x His标签，允许在完全变性的条件下纯化标记的蛋白质。我们将利用蛋白质组学中心的能力，将蛋白质与低浓度的甲醛进行交联，并尝试使用这个标签来纯化蛋白质复合体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. New mass spectrometry approaches have revolutionized cell biology by identifying the majority of proteins expressed in a given cell type. The methodology has been extended to the dissection of protein-protein interactions through affinity tags and purification of protein complexes. Using state of the art mass spectrometry we will identify cellular proteins that interact with Salmonella secreted virulence factors (so called type three secreted effectors; TTS effector). Specifically we will use a new tandem affinity tag together with new methods of protein-protein cross-linking to dissect the inflammasome and how one TTS effector inhibits NFK¿ activation via interaction with the Nod signal transduction pathway. We have constructed vectors containing a tandem affinity tag that are suitable for expression in mammalian cells as well as in Salmonella. The tag encodes two 6x His tags separated by a biotinylation site allowing purification of the tagged proteins under fully denaturing conditions. We will cross-link proteins with low concentrations of formaldehyde and attempt to use this tag to purify protein complexes using capabilities in the Proteomics Center.