Cell membrane lipids have been identified as key participants in cell signaling activities. One important role is their involvement as site-specific ligands in protein-membrane binding interactions, which result in the anchoring of peripheral proteins onto cellular membranes. These events generally regulate protein function and localization and have been implicated in both normal physiological processes and those pertaining to disease state onset. Thus, it is important to elucidate the details of interactions at the molecular level, such as lipid-binding specificities and affinities, the location of receptor binding domains and multivalency in binding. For this purpose, we have designed and developed azido-tagged lipid analogues as conveniently functionalizable lipid probe scaffolds. Herein, we report the design and synthesis of the initial structure of this type, diacylglycerol analogue 2, which contains an azide tag at the sn-1 position of the lipid headgroup. Direct functionalization of this compound with a range of reporter groups has been performed to illustrate the facile access to probes of use for characterizing binding. Quantitative lipid-binding studies using protein kinase C, a known DAG-binding receptor, demonstrate that these probes are active mimetics of natural DAG. Thus, these DAG probes will serve as robust sensors for studies aimed at understanding binding interactions and as precursors for the development of analogous probes of more complex phospholipids and glycolipids.
Interdisciplinary research focused on biological membranes has revealed them as signaling and trafficking platforms for processes fundamental to life. Biomembranes harbor receptors, ion channels, lipid domains, lipid signals, and scaffolding complexes, which function to maintain cellular growth, metabolism, and homeostasis. Moreover, abnormalities in lipid metabolism attributed to genetic changes among other causes are often associated with diseases such as cancer, arthritis and diabetes. Thus, there is a need to comprehensively understand molecular events occurring within and on membranes as a means of grasping disease etiology and identifying viable targets for drug development. A rapidly expanding field in the last decade has centered on understanding membrane recruitment of peripheral proteins. This class of proteins reversibly interacts with specific lipids in a spatial and temporal fashion in crucial biological processes. Typically, recruitment of peripheral proteins to the different cellular sites is mediated by one or more modular lipid-binding domains through specific lipid recognition. Structural, computational, and experimental studies of these lipid-binding domains have demonstrated how they specifically recognize their cognate lipids and achieve subcellular localization. However, the mechanisms by which these modular domains and their host proteins are recruited to and interact with various cell membranes often vary drastically due to differences in lipid affinity, specificity, penetration as well as protein-protein and intramolecular interactions. As there is still a paucity of predictive data for peripheral protein function, these enzymes are often rigorously studied to characterize their lipid-dependent properties. This review summarizes recent progress in our understanding of how peripheral proteins are recruited to biomembranes and highlights avenues to exploit in drug development targeted at cellular membranes and/or lipid-binding proteins.
Phosphatidic acid (PA) is an important signaling lipid that plays roles in a range of biological processes including both physiological and pathophysiological events. PA is one of a number of signaling lipids that can act as site-specific ligands for protein receptors in binding events that enforce membrane-association and generally regulate both receptor function and subcellular localization. However, elucidation of the full scope of PA activities has proven problematic, primarily due to the lack of a consensus sequence among PA-binding receptors. Thus, experimental approaches, such as those employing lipid probes, are necessary for characterizing interactions at the molecular level. Herein, we describe an efficient modular approach to the synthesis of a range of PA probes that employs a late stage introduction of reporter groups. This strategy was exploited in the synthesis of PA probes bearing fluorescent and photoaffinity tags as well as a bifunctional probe containing both a photoaffinity moiety and an azide as a secondary handle for purification purposes. To discern the ability of these PA analogues to mimic the natural lipid in protein binding properties, each compound was incorporated into vesicles for binding studies using a known PA receptor, the C2 domain of PKCα. In these studies, each compound exhibited binding properties that were comparable to those of synthetic PA, indicating their viability as probes for effectively studying the activities of PA in cellular processes.
Ribonuclease P (RNase P) is a Mg2+-dependent endoribonuclease responsible for the 5′-maturation of transfer RNAs. It is a ribonucleoprotein complex containing an essential RNA and a varying number of protein subunits depending on the source: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Since bacterial RNase P is required for viability and differs in structure/subunit composition from its eukaryal counterpart, it is a potential antibacterial target. To elucidate the basis for our previous finding that the hexa-arginine derivative of neomycin B is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, we synthesized hexa-guanidinium and -lysyl conjugates of neomycin B and compared their inhibitory potential. Our studies indicate that side-chain length, flexibility and composition cumulatively account for the inhibitory potency of the aminoglycoside-arginine conjugates (AACs). We also demonstrate that AACs interfere with RNase P function by displacing Mg2+ ions. Moreover, our finding that an AAC can discriminate between a bacterial and archaeal (an experimental surrogate for eukaryal) RNase P holoenzyme lends promise to the design of aminoglycoside conjugates as selective inhibitors of bacterial RNase P, especially once the structural differences in RNase P from the three domains of life have been established.
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