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.
"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 "
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide lipids (PIPns) control numerous critical biological pathways, typically through the regulation of protein function driven by non-covalent protein-lipid binding interactions. Despite the importance of these systems, the unraveling of the full scope of protein-PIPn interactions has represented a significant challenge due to the massive complexity associated with these events, including the large number of diverse proteins that bind to these lipids, variations in the mechanisms by which proteins bind to lipids, and the presence of multiple distinct PIPn isomers. As a result of this complexity, global methods in which numerous proteins that bind PIPns can be identified and characterized simultaneously from complex samples, which have been enabled by key technological advancements, have become popular as an efficient means for tackling this challenge. This review article provides an overview of advancements in large-scale methods for profiling protein-PIPn binding, including experimental methods, such as affinity enrichment, microarray analysis and activity-based protein profiling, as well as computational methods, and combined computational/experimental efforts.
Chemistry and Physics of Lipids 11/2013; 182. DOI:10.1016/j.chemphyslip.2013.10.014 · 2.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria.
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