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 M 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)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 showed that this new tool would greatly benefit profiling lipid-protein interactions in high-throughput studies. Cell viability and physiological functions are maintained through a complex biomolecular interaction network. One of the key components in the regulatory system includes lipidprotein interactions that mediate various cell responses and metabolisms. Increasing evidence shows that such interactions have profound influences on cell polarization, the cell cycle, and other cellular processes. To date, in vitro characterizations of lipid interactions with other biomolecules are often conducted using artificial membrane models, such as liposomal nanovesicles, to mimic biological membranes. Liposomal nanovesicles, termed liposomes, are spherical vesicles that are surrounded by phospholipid bilayers in which the lipid of interest can be incorporated. An important benefit of liposomes is the ease in which a large number of fluorescent molecules can be encapsulated so that the liposome binding signals can be greatly enhanced for detection (1-4). Therefore, liposomes have become a practical and popular tool for

Mycobacterium Tuberculosis Proteome Microarray for Global Studies of Protein Function and Immunogenicity

“…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 can also be used to study post-translational modifications (PTMs), including phosphorylation (Newman et al, 2013), acetylation (Lin et al, 2009), glycosylation (Kung et al, 2009), and nitrosylation (Lee et al, 2014), providing a window into dynamic cellular responses to changes in the environment.…”

Effects of nanobubble collapse on cell membrane integrity

“…To begin with, the lipid bilayer is a ubiquitous construction in the natural world, forming the structural support for living cells and able to keep the cellular structures inside the cell while regulating the passage of molecules. The utility of the lipid bilayer has made it one of the foundations of complex organisms, and as such, this construction has received a large amount of attention and research, both for the displayed chemical and mechanical properties [Brannigan and Brown, 2004; Sumetpipat and Baowan, 2014; Lu et al , 2012]. Due to their usefulness and relative simplicity, designed nanoscopic assemblies of lipids in the form of liposomes, micelles, and model cells are used in various fields, from soft matter physics to actual synthetic biology, where lipids are used to generate systems mimicking biological structures [Tresset et al , 2007; Karlsson et al , 2004; Tresset and Takeuchi, 2005].…”

Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity

“…Proteome microarrays, usually composed of thousands of proteins from one species that are affinity purified and functionally active, are high-throughput platforms for the global profiling of thousands of molecular interactions in a single experiment (30). They provide a versatile platform for investigating many aspects at the systems-level, such as discovering serum biomarkers for various diseases (31) and globally investigating interactions with proteins (32), DNA (33), RNA (34), lipids (35), and small molecules (36,37).…”