Protocol for identifying physiologically relevant binding proteins of G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are important therapeutic targets expressed on the cell surface. Here, we present a protocol for identifying physiologically relevant binding proteins of adhesion GPCR GPR110. We describe steps for in-cell chemical crosslinking, immunoprecipitation, and quantitative high-resolution mass spectrometry.

Interaction between GPR110 (ADGRF1) and tight junction protein occludin implicated in blood-brain barrier permeability.

Activation of adhesion receptor GPR110 by the endogenous ligand synaptamide promotes neurogenesis, neurite growth, and synaptogenesis in developing brains through cAMP signal transduction. However, interacting partners of GPR110 and their involvement in cellular function remain unclear. Here, we demonstrate using chemical crosslinking, affinity purification, and quantitative mass spectrometry that GPR110 interacts with the tight junction adhesion protein occludin.

Molecular and Signaling Mechanisms for Docosahexaenoic Acid-Derived Neurodevelopment and Neuroprotection.

The neurodevelopmental and neuroprotective actions of docosahexaenoic acid (DHA) are mediated by mechanisms involving membrane- and metabolite-related signal transduction. A key characteristic in the membrane-mediated action of DHA results from the stimulated synthesis of neuronal phosphatidylserine (PS). The resulting DHA-PS-rich membrane domains facilitate the translocation and activation of kinases such as Raf-1, protein kinase C (PKC), and Akt.

Synaptamide activates the adhesion GPCR GPR110 (ADGRF1) through GAIN domain binding.

Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid.

Current advances in screening for bioactive components from medicinal plants by affinity ultrafiltration mass spectrometry.

Introduction: Medicinal plants have played an important role in maintaining human health for thousands of years. However, the interactions between the active components in medicinal plants and some certain biological targets during a disease are still unclear in most cases.

Objective: To conduct the high-throughput screening for small active molecules that can interact with biological targets, which is of great theoretical significance and practical value.

Identification of 4-phenylquinolin-2(1H)-one as a specific allosteric inhibitor of Akt.

Akt plays a major role in tumorigenesis and the development of specific Akt inhibitors as effective cancer therapeutics has been challenging. Here, we report the identification of a highly specific allosteric inhibitor of Akt through a FRET-based high-throughput screening, and characterization of its inhibitory mechanism. Out of 373,868 compounds screened, 4-phenylquinolin-2(1H)-one specifically decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC = 6 µM) and downstream signaling.

Orphan GPR110 (ADGRF1) targeted by N-docosahexaenoylethanolamine in development of neurons and cognitive function.

Docosahexaenoic acid (DHA, 22:6n-3) is an omega-3 fatty acid essential for proper brain development. N-docosahexaenoylethanolamine (synaptamide), an endogenous metabolite of DHA, potently promotes neurogenesis, neuritogenesis and synaptogenesis; however, the underlying molecular mechanism is not known. Here, we demonstrate orphan G-protein coupled receptor 110 (GPR110, ADGRF1) as the synaptamide receptor, mediating synaptamide-induced bioactivity in a cAMP-dependent manner.

Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis.

Threonine 34 phosphorylation by phosphoinositide-dependent protein kinase 1 facilitates dissociation of Akt from the plasma membrane.

Akt is a key mediator of cell proliferation, survival and metabolism. After translocation to the membrane and phosphorylation at T308 and S473, the activated Akt dissociates from the plasma membrane to cytoplasm, which is an important step to phosphorylate its downstream targets. In addition to its central role in regulating the kinase activity, phosphorylation of T308 in the kinase loop has been reported to be necessary for this dissociation process.

Phosphatidylserine in the brain: metabolism and function.

Phosphatidylserine (PS) is the major anionic phospholipid class particularly enriched in the inner leaflet of the plasma membrane in neural tissues. PS is synthesized from phosphatidylcholine or phosphatidylethanolamine by exchanging the base head group with serine, and this reaction is catalyzed by phosphatidylserine synthase 1 and phosphatidylserine synthase 2 located in the endoplasmic reticulum. Activation of Akt, Raf-1 and protein kinase C signaling, which supports neuronal survival and differentiation, requires interaction of these proteins with PS localized in the cytoplasmic leaflet of the plasma membrane.

Effective identification of Akt interacting proteins by two-step chemical crosslinking, co-immunoprecipitation and mass spectrometry.

Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis.

Integration of phosphoproteomic, chemical, and biological strategies for the functional analysis of targeted protein phosphorylation.

Reversible phosphorylation, tightly controlled by protein kinases and phosphatases, plays a central role in mediating biological processes, such as protein-protein interactions, subcellular translocation, and activation of cellular enzymes. MS-based phosphoproteomics has now allowed the detection and quantification of tens of thousands of phosphorylation sites from a typical biological sample in a single experiment, which has posed new challenges in functional analysis of each and every phosphorylation site on specific signaling phosphoproteins of interest. In this article, we review recent advances in the functional analysis of targeted phosphorylation carried out by various chemical and biological approaches in combination with the MS-based phosphoproteomics.

Effects of ethanol on conformational changes of Akt studied by chemical cross-linking, mass spectrometry, and (18)O labeling.

Although PI3K/Akt signaling that regulates neuronal survival has been implicated in the deleterious effects of ethanol on the central nervous system, underlying molecular mechanisms have not been fully elucidated. Akt-membrane interaction is a prerequisite step for Akt activation since it induces interdomain conformational changes to an open conformer that allows Akt phosphorylation by upstream kinases. In this study, we investigated the effect of ethanol on Akt activation by quantitatively probing Akt conformation using chemical cross-linking, (18)O labeling and mass spectrometry.

Post-translational modification by β-lysylation is required for activity of Escherichia coli elongation factor P (EF-P).

Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical β-lysyl-lysine.

Effects of docosahexaenoic acid on mouse brain synaptic plasma membrane proteome analyzed by mass spectrometry and (16)O/(18)O labeling.

Docosahexenoic acid (DHA, 22:6n-3) plays an important role in development of proper brain function in mammals. We have previously reported that DHA promotes synaptogenesis and synaptic function in hippocampal neurons while DHA-depletion in the brain due to n-3 fatty acid deficiency produces opposite effects. To gain insight into underlying molecular mechanisms, we investigated whether the brain DHA status affects the synaptic plasma membrane (SPM) proteome by using nanoLC-ESI-MS/MS and (16)O/(18)O labeling.

Phosphatidylserine is a critical modulator for Akt activation.

Akt activation relies on the binding of Akt to phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) in the membrane. Here, we demonstrate that Akt activation requires not only PIP(3) but also membrane phosphatidylserine (PS). The extent of insulin-like growth factor-induced Akt activation and downstream signaling as well as cell survival under serum starvation conditions positively correlates with plasma membrane PS levels in living cells.

N-Docosahexaenoylethanolamide promotes development of hippocampal neurons.

DHA (docosahexaenoic acid, C22:6,n-3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression.

Conformational changes in Akt1 activation probed by amide hydrogen/deuterium exchange and nano-electrospray ionization mass spectrometry.

Amide hydrogen exchange coupled to nano-electrospray ionization mass spectrometry (nano-ESI-MS) has been used to identify and characterize localized conformational changes of Akt upon activation. Active or inactive Akt was incubated in D(2)O buffer, digested with pepsin, and analyzed by nano-ESI-MS to determine the deuterium incorporation. The hydrogen/deuterium (H/D) exchange profiles revealed that Akt undergoes considerable conformational changes in the core structures of all three individual domains after activation.

Probing Akt-inhibitor interaction by chemical cross-linking and mass spectrometry.

The serine/threonine kinase Akt is a critical enzyme that regulates cell survival. As high Akt activity has been shown to contribute to the pathogenesis of various human malignancies, inhibition of Akt activation is a promising therapeutic strategy for cancers. We have previously demonstrated that changes in Akt interdomain arrangements from a closed to open conformation occur upon Akt-membrane interaction, which in turn allows Akt phosphorylation/activation.

Simultaneous determination of salsolinol enantiomers and dopamine in human plasma and cerebrospinal fluid by chemical derivatization coupled to chiral liquid chromatography/electrospray ionization-tandem mass spectrometry.

A sensitive, specific, and robust method to simultaneously determine enantiomeric salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, SAL), a potential biomarker implicated in alcohol-related neurotoxicity in a stereoselective manner, and its precursor dopamine (DA) has been developed using simple chemical derivatization and chiral separation coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS). SAL enantiomers and DA were converted to stable pentafluorobenzyl (PFB) derivatives directly from aqueous media. Bulky PFB groups introduced into the SAL structure enabled baseline separation of SAL stereoisomers on a chiral column without cumbersome chiral derivatization to unstable SAL diastereomers.

Multiple pathways involved in the biosynthesis of anandamide.

Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways.

A biosynthetic pathway for anandamide.

The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released "on demand" by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE-PLD).

Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry.

Akt, a serine/threonine kinase, plays a critical role in cell survival. Upon growth factor receptor stimulation, cytosolic Akt is recruited to the plasma membrane by phospholipid binding and activated through phosphorylation at Thr(308) and Ser(473). Although crystal structures for the parts of Akt have been reported, neither the three-dimensional structure of the whole molecule nor sequential conformational changes during activation have been demonstrated.

Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry.

Mass spectrometry with chemical cross-linking was used to probe the conformational changes of HSA (human serum albumin) in solution on interaction with monounsaturated OA (oleic acid) or polyunsaturated AA (arachidonic acid) or DHA (docosahexaenoic acid). Fatty acid-free or -bound HSA was modified with lysine-specific cross-linkers and digested with trypsin. Cross-linked peptides were analysed by nano-electrospray ionization MS to localize the sites of cross-linking.

Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry.

Serum albumin is the principal transporter of fatty acids that are otherwise insoluble in circulating plasma. While the crystal structure of human serum albumin (HSA) as well as its binding with fatty acids has been characterized, the three dimensional structure of bovine serum albumin (BSA) has not been determined although both albumins share 76% sequence homology. In this study we used mass spectrometry coupled with chemical cross-linking, to probe the tertiary structure of BSA.