title: Profiling lipid-protein interactions using nonquenched fluorescent liposomal nanovesicles and proteome microarrays.
abstract: 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 nonquenched fluorescent (NQF)1 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 show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-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)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid-protein interactions in high-throughput studies.
authors: Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli 32001, Taiwan.
authors: Kuan-Yi
authors: KY
authors: Lu
authors: Sheng-Ce
authors: SC
authors: Tao
authors: Tzu-Ching
authors: TC
authors: Yang
authors: Yu-Hsuan
authors: YH
authors: Ho
authors: Chia-Hsien
authors: CH
authors: Lee
authors: Chen-Ching
authors: CC
authors: Lin
authors: Hsueh-Fen
authors: HF
authors: Juan
authors: Hsuan-Cheng
authors: HC
authors: Huang
authors: Chin-Yu
authors: CY
authors: Yang
authors: Ming-Shuo
authors: MS
authors: Chen
authors: Yu-Yi
authors: YY
authors: Lin
authors: Jin-Ying
authors: JY
authors: Lu
authors: Heng
authors: H
authors: Zhu
authors: Chien-Sheng
authors: CS
authors: Chen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D019295:Computational Biology; D003599:Cytoskeleton / Q000378:metabolism; D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000378:metabolism; D050356:Lipid Metabolism; D008081:Liposomes / Q000378:metabolism*; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D021381:Protein Transport; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D008081:Liposomes; D018129:Phosphatidylinositol Phosphates; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; C106336:phosphatidylinositol 3,5-diphosphate; D020558:GTP Phosphohydrolases