Profiling Lipid–protein Interactions Using Nonquenched Fluorescent Liposomal Nanovesicles and Proteome Microarrays
| Autor: | Hsueh Fen Juan, Jin Ying Lu, Chia Hsien Lee, Ming Shuo Chen, Chin Yu Yang, Chien Sheng Chen, Yu Hsuan Ho, Sheng-Ce Tao, Tzu Ching Yang, Yu Yi Lin, Hsuan Cheng Huang, Heng Zhu, Chen Ching Lin, Kuan-Yi Lu |
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| Rok vydání: | 2012 |
| Předmět: |
Proteomics
Saccharomyces cerevisiae Proteins Proteome Amino Acid Motifs Molecular Sequence Data Protein Array Analysis Sulforhodamine B Saccharomyces cerevisiae Biology Biochemistry Interactome Chromatography Affinity Fluorescence GTP Phosphohydrolases Analytical Chemistry Protein–protein interaction chemistry.chemical_compound Phosphatidylinositol Phosphates Amino Acid Sequence Transport Vesicles Molecular Biology Cytoskeleton Liposome Research Vesicle Computational Biology Reproducibility of Results Lipid Metabolism Molecular Docking Simulation Protein Transport chemistry Liposomes Nanoparticles Protein Binding |
| Zdroj: | Molecular & Cellular Proteomics. 11:1177-1190 |
| ISSN: | 1535-9476 |
| DOI: | 10.1074/mcp.m112.017426 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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