Profiling lipid-protein interactions using non-quenched 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 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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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; DOI:10.1016/j.chemphyslip.2013.10.014 · 2.59 Impact Factor
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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.