Neutrophils are the most abundant cell type among circulating white cells and constitute the first line of host defense against invading pathogens. Various neutrophil functions such as chemotaxis, phagocytosis, superoxide production, death/survival, and bacteria killing, can be regulated by PtdIns(3,4,5)P3, an inositol phospholipid localized on the plasma membrane. PtdIns(3,4,5)P3 exerts its function by mediating protein translocation via binding to their pleckstrin homolog (PH)-domains. Thus, PH domain translocation provides an attractive target for developing modulators of neutrophil function. The ultimate goal of this study is to identify PtdIns(3,4,5)P3 pathway activators that specifically target PH domain plasma membrane translocation via conducting a high throughput chemical genetic screening. We have recently established an experimental system for visualizing this process in live cells. We utilized the PH-domain of Akt (PHAkt) fused with green fluorescent protein (PHAkt-GFP) as a marker for this event. A HL60 cell line stably expressing PHAkt-GFP has been generated. HL60 cells can be induced to differentiate towards morphologically mature neutrophils. PHAkt-GFP translocation from cytosol to the plasma membrane could be easily detected in differentiated HL60 cells after chemoattractant stimulation. While PHAkt-GFP translocates to the leading edge of chemotaxing cells in chemotactic gradient, during uniform treatment with chemoattractant it transiently translocates from cytosol to the plasma membrane. Elevating PtdIns(3,4,5)P3 signal not only augments the initial cytoplasm-to-plasma membrane translocation of PHAkt-GFP, but also dramatically delays its subsequent """"""""reverse translocation"""""""" from the plasma membrane to cytosol, providing an excellent readout for high throughput screening (HTS). A method for quantifying PHAkt-GFP plasma membrane translocation has also been established. In addition, our preliminary data demonstrated the selectivity and reproducibility of our assay for detecting chemoattractant- elicited PH domain membrane translocation. In this proposed research, we will adapt this cell-based system to a high-throughput format and examine whether the same selectivity and reproducibility can be achieved (Aim I). In addition, several secondary screening assays will be established to confirm the effect of each positive hit compound identified from the primary screening and to identify the most specific and potent ones for future characterization (Aim II). Finally, a plan to evaluate the biological activities of identified compounds is proposed. We will investigate whether elevating PH domain membrane translocation by identified compounds is able to enhance various neutrophil functions (Aim III).
Project Narrative The ultimate goal of our research is to identify PtdIns(3,4,5)P3 pathway activators that specifically target PH domain plasma membrane translocation via performing a high throughput screening of small molecule libraries. Discovery of these activators will greatly facilitate our research on PtdIns(3,4,5)P3 signaling in neutrophil function and innate immunity. Moreover, the identified activators can be directly utilized as starting chemical compounds for novel immune enhancer drug development, particularly for severe infections due to lack of enough pathogen killing capability.
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