CHARACTERIZATION OF HOST-PATHOGEN PROTEIN-PROTEIN INTERACTIONS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 新的质谱分析方法通过鉴定给定细胞类型中表达的大多数蛋白质，彻底改变了细胞生物学。 该方法已扩展到通过亲和标签和蛋白质复合物的纯化来解剖蛋白质-蛋白质相互作用。 使用最先进的质谱法，我们将鉴定与沙门氏菌分泌的毒力因子（所谓的三型分泌效应子; TTS效应子）相互作用的细胞蛋白。 具体来说，我们将使用一种新的串联亲和标签与蛋白质-蛋白质交联的新方法来剖析炎性小体以及TTS效应子如何通过与Nod信号转导通路的相互作用抑制NFK？激活。 我们已经构建了含有串联亲和标签的载体，其适合在哺乳动物细胞以及沙门氏菌中表达。 该标签编码两个由生物素化位点分开的6x His标签，允许在完全变性条件下纯化标记的蛋白质。我们将用低浓度的甲醛交联蛋白质，并尝试使用蛋白质组学中心的能力，使用这种标签来纯化蛋白质复合物。

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.