Profiling Lipid-protein Interactions Using Nonquenched Fluorescent Liposomal Nanovesicles and Proteome Microarrays

National Central University, Taiwan
Molecular & Cellular Proteomics (Impact Factor: 6.56). 07/2012; 11(11). DOI: 10.1074/mcp.M112.017426
Source: PubMed


Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a non-quenched fluorescent (NQF) liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 μM of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To demonstrate their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P(2)-specific binding proteins (PI(3,5)P(2)-BPs). We not only recovered many proteins that possessed known PI(3,5)P(2)-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P(2)-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P(2)-BPs were similar to those associated with PI(3,5)P(2), including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P(2)-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P(2) interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P(2)-binding kinases. Our study demonstrated that this new tool will greatly benefit profiling lipid-protein interactions in high-throughput studies.

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Available from: Sheng-Ce Tao, Dec 12, 2015
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    • "Proteome microarrays, usually composed of thousands of proteins from one species that are affinity purified and functionally active, are powerful highly parallel, high-throughput platforms for globally profiling thousands of molecular interactions in a single experiment (Chen et al., 2008; Zhu et al., 2001). Their use in the discovery of serum biomarkers for various diseases (Gnjatic et al., 2010) and global investigations of protein interactions with other proteins (PPI) (Chen et al., 2013), with DNA (Lin et al., 2009), with RNA (Zhu et al., 2007), with lipids (Lu et al., 2012), and with a range of small molecules (Huang et al., 2004) demonstrate the power of this approach. Furthermore, they "
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