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

National Central University, Taiwan
Molecular &amp 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.

Full-text preview

Available from:
  • Source
    • "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: Poor understanding of the basic biology of Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis, hampers development of much-needed drugs, vaccines, and diagnostic tests. Better experimental tools are needed to expedite investigations of this pathogen at the systems level. Here, we present a functional MTB proteome microarray covering most of the proteome and an ORFome library. We demonstrate the broad applicability of the microarray by investigating global protein-protein interactions, small-molecule-protein binding, and serum biomarker discovery, identifying 59 PknG-interacting proteins, 30 bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding proteins, and 14 MTB proteins that together differentiate between tuberculosis (TB) patients with active disease and recovered individuals. Results suggest that the MTB rhamnose pathway is likely regulated by both the serine/threonine kinase PknG and c-di-GMP. This resource has the potential to generate a greater understanding of key biological processes in the pathogenesis of tuberculosis, possibly leading to more effective therapies for the treatment of this ancient disease. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 12/2014; 9(6). DOI:10.1016/j.celrep.2014.11.023 · 8.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein microarray technology is one of the most powerful tools presently available for proteomic studies. Numerous types of protein microarrays have been widely and successfully applied for both basic biological studies and clinical researches, including those designed to characterize protein-protein, protein-nucleic acid, protein-drug/small molecule and antibody-antigen interactions. In the past decade, a variety of protein microarrays have been developed, including those spotted with whole proteomes, smaller peptides, antibodies, and lectins. Featured as high-throughput, miniaturized, and capable of parallel analysis, the power of protein microarrays has already been demonstrated many times in both basic research and clinical applications. In this review, we have summarized the latest developments in the production and application of protein microarrays. We discuss several of the most important applications of protein microarray, ranging from proteome microarrays for large scale identification of protein-protein interactions to lectin microarrays for live cell surface glycan profiling, with special emphasis on their use in studies of drug mechanisms and biomarker discovery. Already with tremendous success, we envision protein microarrays will become an indispensible tool for any systems-wide studies, fostering the integration of basic research observations to clinically useful applications.
    Current pharmaceutical design 03/2013; 20(1). DOI:10.2174/138161282001140113123707 · 3.45 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Unlabelled: A phosphatidylinositol-phosphate affinity chromatographic approach combined with mass spectrometry was used in order to identify novel PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana suspension cell extracts. Most of the phosphatidylinositol-phosphate interacting candidates identified from this differential screening are characterized by lysine/arginine rich patches. Direct phosphoinositide binding was identified for important membrane trafficking regulators as well as protein quality control proteins such as the ATG18p orthologue involved in autophagosome formation and the lipid Sec14p like transfer protein. A pentatricopeptide repeat (PPR) containing protein was shown to directly bind to PI(3,5)P2 but not to PI(3)P. PIP chromatography performed using extracts obtained from high salt (0.4M and 1M NaCl) pretreated suspensions showed that the association of an S5-1 40S ribosomal protein with both PI(3)P and PI(3,5)P2 was abolished under salt stress whereas salinity stress induced an increase in the phosphoinositide association of the DUF538 domain containing protein SVB, associated with trichome size. Additional interacting candidates were co-purified with the phosphoinositide bound proteins. Binding of the COP9 signalosome, the heat shock proteins, and the identified 26S proteasomal subunits, is suggested as an indirect effect of their interaction with other proteins directly bound to the PI(3)P and the PI(3,5)P2 phosphoinositides. Biological significance: PI(3,5)P2 is of special interest because of its low abundance. Furthermore, no endogenous levels have yet been detected in A. thaliana (although there is evidence for its existence in plants). Therefore the isolation of novel interacting candidates in vitro would be of a particular importance since the future study and localization of the respective endogenous proteins may indicate possible targeted compartments or tissues where PI(3,5)P2 could be enriched and thereafter identified. In addition, PI(3,5)P2 is a phosphoinositide extensively studied in mammalian and yeast systems. However, our knowledge of its role in plants as well as a list of its effectors from plants is very limited.
    Journal of proteomics 09/2013; 91. DOI:10.1016/j.jprot.2013.08.020 · 3.89 Impact Factor
Show more