Synthesis and biological applications of phospholipid-based chemical probes

© 2010 Dr. Meng-Xin YinPhosphoinositides play significant roles as secondary messengers to regulate a broad range of intracellular processes in a spatio and temporal manner, including signal transduction pathways, cell motility and cytokinesis, excocytosis and endocytosis, vesicular trafficking, as well as control of ion channels, pumps, and transporters. Their regulatory functions are achieved by directly recognition and interaction with a variety of phosphoinositide-binding proteins. Synthetic chemistry has played a defining role in further understanding of phosphoinositide interactome. Total syntheses of the palmitic acid and amino-terminal PI(3,4)P2 derivatives were performed. The dipalmitoyl derivatives were incorporated to form liposomes, and the amino-terminal analogues were conjugated to Affi-Gel 10 resins, both of which were used as affinity probes for proteomics studies via affinity experiments to investigate phosphoinositide interacting proteins/protein complexes. With colorectal carcinoma cell cytosolic extracts, 282proteins/protein complexes were identified as PI(3,4,5)P3 binding proteins. A morecomprehensive proteomic study was carried out for PI(3,4)P2 with cell extracts fromcytosol, membrane, and nucleus of colorectal carcinoma cells, giving 1134 proteins as PI(3,4)P2 interacting proteins/protein complexes including 126 proteins containing known phosphoinositide binding domains. Additionally, a number of novel proteins were identified as potential phosphoinositide binding proteins in our studies. The molecular functions, protein networks, and biological processes of identified proteins were analysed by submission to various databases to provide better knowledge of the involvement of phosphoinositides in intracellular signalling pathways. Therefore, our bioinformatics studies using phosphoinositide affinity probes provide initial detailed assessment of PI(3,4,5)P3 and PI(3,4)P2 interactome and suggest potential phosphoinositide functions and specificities for further biochemical characterisation by using other alternative biological techniques. Cardiolipin (diphosphatidylglycerol) is a unique phospholipid predominantly existing in the mitochondrial membranes throughout the eukaryotic and prokaryotic kingdom. It has been subsequently studied as the key phospholipid stimulating mitochondrial enzymes and regulating the mitochondria energy metabolism and cell apoptosis by interactions with a number of mitochondrial proteins. An amino-terminal cardiolipin derivative was totally synthesised and immobilised to Affi-Gel 10 beads to afford a novel cardiolipin affinity matrix. Pull-down experiments were undertaken; the results confirmed the binding interaction between cardiolipin and the HR1 domain of PRK2 protein. Furthermore, a novel cardiolipin biosensor chip has been developed by immobilisation of the amino-terminal cardiolipin derivative to the gold surface, and used as a powerful biological tool for clinical diagnosis of antiphospholipid syndrome (APS) via surface plasmon resonance (SPR) experiments. In order to facilitate protein co-crystallisation processes, a novel PI(4,5)P2 derivative containing adamantanecarbonyl groups as replacements of acyl side chains has been synthesised; synthesis towards metabolically stabilised phosphatidylinositol phosphorothioate derivatives was also undertaken