Monitoring dynamic protein-protein interactions (PPIs) in situ has been a topic of interest due to the importance of PPIs in cell signaling regulation and implications in therapeutic interventions. In this proposal, we introduce a new PPI method based on nanotechnology-assisted magnetic co-concentration (MCo-C). The key component of this technology that enables this PPI investigation method is the ability of the nanoprobe-microtweezer system to magnetically co-concentrate the targeted protein and its interacting partners, enabling significant amplification of weak and transient PPI signals to be detectable with robust signals. MCo-C can induce signal amplifications more than 100 times (in vitro; >10 times for live cells) and hence promises to identify weak PPIs that are not detectable with existing PPI methods. Additionally, this method does not involve any harsh post- treatments (e.g. cell lysis, chemical membrane crosslinking) and hence allows in situ real-time monitoring of dynamic PPIs in live cells during the course of cell signaling. To demonstrate the capacity of MCo-C, we will first carry out PPI assays in in vitro model reaction systems using DNA duplex formation/dissociation. Once the system is optimized, as initial studies, we propose to investigate PPIs of Notch with potential interaction partners, including Notch (homodimerization), cis-ligand (e.g. Dll4), and ?-catenin. Ultimately, we aim to provide a platform technology for the systematic investigation of PPIs, accelerating our understanding of the dynamic regulation of interactome networks in cell signaling.
Blocking protein-protein interactions (PPIs) via small molecule inhibitors has emerged as an attractive therapeutic strategy and several clinical trials have been reached. Additionally, aberrant PPIs implicate in diseases including Alzheimer's diease and cancer. This proposed study will greatly accelerate our understanding of PPIs in cell signaling and thus will be beneficial for dissecting disease development and providing therapeutic strategy. Also, the proposed nanotool to detect dynamic PPIs will provide a method for sensitive screening tool for evaluating drug efficacy to the respective molecular target.