Optimization of chromatographic buffer conditions for the simultaneous analysis of phosphatidylinositol and phosphatidylinositol phosphate species in canola.

The phosphatidylinositols and phosphatidylinositol phosphates are a set of closely related lipids known to influence various cellular functions. Irregular distributions of these molecules have been correlated with the development and progression of multiple diseases, including Alzheimer's, bipolar disorder, and various cancers. As a result, there is continued interest regarding the speciation of these compounds, with specific consideration on how their distribution may differ between healthy and diseased tissue. The comprehensive analysis of these compounds is challenging due to their varied and unique chemical characteristics, and current generalized lipidomics methods have proven unsuitable for phosphatidylinositol analysis and remain incapable of phosphatidylinositol phosphate analysis. Here we improved upon current methods by enabling the sensitive and simultaneous analysis of phosphatidylinositol and phosphatidylinositol phosphate species, whilst enhancing their characterization through chromatographic resolution between isomeric species. A 1 mM ammonium bicarbonate and ammonia buffer was determined optimal for this goal, enabling the identification of 148 phosphatidylinositide species, including 23 lyso-phosphatidylinositols, 51 phosphatidylinositols, 59 oxidized-phosphatidylinositols, and 15 phosphatidylinositol phosphates. As a result of this analysis, four distinct canola cultivars were differentiated based exclusively on their unique phosphatidylinositide-lipidome, indicating analyses of this type may be of use when considering the development and progression of the disease through lipidomic profiles.

Plasma Membrane Phosphatidylinositol 4-Phosphate Is Necessary for Virulence of Candida albicans.

Phosphatidylinositol lipids regulate key processes, including vesicle trafficking and cell polarity. A recent study identified novel roles for phosphatidylinositol 4-phosphate (PI4P) in the plasma membrane of the fungal pathogen Candida albicans, including polarized hyphal growth and cell wall organization. Studies in other organisms were not able to separate the roles of PI4P in the plasma membrane and Golgi, but the C. albicans plasma membrane pool of PI4P could be selectively eliminated by deleting the STT4 kinase, which creates PI4P. Interestingly, stt4Δ mutants were strongly defective in disseminated candidiasis in mice but were not defective in an oral infection. This suggested that abnormal exposure of ß-glucan in the mutant cell walls increased recruitment of innate immune cells during disseminated infection, which is not expected to impact oral infection. These results highlight novel roles of PI4P and reinforce the need to test the virulence of C. albicans mutants at different host sites.

Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection.

Mycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and ß-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited. IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus.

Structure of the ancient TRPY1 channel from Saccharomyces cerevisiae reveals mechanisms of modulation by lipids and calcium.

Transient receptor potential (TRP) channels emerged in fungi as mechanosensitive osmoregulators. The Saccharomyces cerevisiae vacuolar TRP yeast 1 (TRPY1) is the most studied TRP channel from fungi, but the structure and details of channel modulation remain elusive. Here, we describe the full-length cryoelectron microscopy structure of TRPY1 at 3.1 Å resolution in a closed state. The structure, despite containing an evolutionarily conserved and archetypical transmembrane domain, reveals distinctive structural folds for the cytosolic N and C termini, compared with other eukaryotic TRP channels. We identify an inhibitory phosphatidylinositol 3-phosphate (PI(3)P) lipid-binding site, along with two Ca2+-binding sites: a cytosolic site, implicated in channel activation and a vacuolar lumen site, implicated in inhibition. These findings, together with data from microsecond-long molecular dynamics simulations and a model of a TRPY1 open state, provide insights into the basis of TRPY1 channel modulation by lipids and Ca2+, and the molecular evolution of TRP channels.

A hybrid strategy combining solution NMR spectroscopy and isothermal titration calorimetry to characterize protein-nanodisc interaction.

NMR is a powerful tool for characterizing intermolecular interactions at atomic resolution. However, the nature of the complex interactions of membrane-binding proteins makes it difficult to elucidate the interaction mechanisms. Here, we demonstrated that structural and thermodynamic analyses using solution NMR spectroscopy and isothermal titration calorimetry (ITC) can clearly detect a specific interaction between the pleckstrin homology (PH) domain of ceramide transport protein (CERT) and phosphatidylinositol 4-monophosphate (PI4P) embedded in the lipid nanodisc, and distinguish the specific interaction from nonspecific interactions with the bulk surface of the lipid nanodisc. This NMR-ITC hybrid strategy provides detailed characterization of protein-lipid membrane interactions.

Activation Mechanisms of the VPS34 Complexes.

Phosphatidylinositol-3-phosphate (PtdIns(3)P) is essential for cell survival, and its intracellular synthesis is spatially and temporally regulated. It has major roles in two distinctive cellular pathways, namely, the autophagy and endocytic pathways. PtdIns(3)P is synthesized from phosphatidylinositol (PtdIns) by PIK3C3C/VPS34 in mammals or Vps34 in yeast. Pathway-specific VPS34/Vps34 activity is the consequence of the enzyme being incorporated into two mutually exclusive complexes: complex I for autophagy, composed of VPS34/Vps34-Vps15/Vps15-Beclin 1/Vps30-ATG14L/Atg14 (mammals/yeast), and complex II for endocytic pathways, in which ATG14L/Atg14 is replaced with UVRAG/Vps38 (mammals/yeast). Because of its involvement in autophagy, defects in which are closely associated with human diseases such as cancer and neurodegenerative diseases, developing highly selective drugs that target specific VPS34/Vps34 complexes is an essential goal in the autophagy field. Recent studies on the activation mechanisms of VPS34/Vps34 complexes have revealed that a variety of factors, including conformational changes, lipid physicochemical parameters, upstream regulators, and downstream effectors, greatly influence the activity of these complexes. This review summarizes and highlights each of these influences as well as clarifying key questions remaining in the field and outlining future perspectives.

Simulations of Kindlin-2 PIP binding domains reveal protonation-dependent membrane binding modes.

Kindlin-2, a member of the Kindlin family of peripheral membrane proteins, is important for integrin activation and stabilization of epidermal growth factor receptor. It associates with the cytoplasmic face of the plasma membrane via dedicated phosphatidylinositol phosphate binding domains located in the N-terminal F0 and Pleckstrin Homology domains. These domains have binding affinity for phosphatidylinositol 4,5-bisphosphate and, to a greater degree, phosphatidylinositol 3,4,5-trisphosphate. The biological significance of the differential binding of these phosphatidylinositol phosphates to Kindlin-2 and the mechanism by which they activate Kindlin-2 are not well understood. Recently, ssNMR identified the predominant protonation states of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate near physiological pH in the presence of anionic lipids. Here, we perform atomistic simulation of the bound state of the Pleckstrin Homology and F0 domains of Kindlin-2 at membranes containing phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate with differing protonation states. This computational approach demonstrates that these two phosphatidylinositol phosphates differently modulate Kindlin-2 subdomain binding in a protonation-state-dependent manner. We speculate these variations in binding mode provide a mechanism for intracellular pH and Ca2+ influx to control the membrane binding behavior and activity of Kindlin-2.

Monitoring the cellular metabolism of a membrane-permeant photo-caged phosphatidylinositol 3,4,5-trisphosphate derivative.

To deliver charged lipid derivatives to the cell interior, bioactivatable and photo-activatable protecting groups are frequently used. The intracellular metabolism of the protecting groups, as well as the lipid itself, are key factors that determine biological activity. Here we followed the cellular metabolism of cell-permeant photo-activatable ("caged") and non-caged membrane-permeant analogs of dioctanoyl phosphatidylinositol 3,4,5-trisphosphate (diC8PIP3) carrying biodegradable protecting groups by mass spectrometry. After successful cell entry, the photo-activatable group can be removed on demand by a light pulse. Hence, UV irradiation acts as a switch to expose the cellular metabolism to a bolus of active compound. To investigate lipid metabolites and to capture a more complete metabolome, we adapted standard extraction methods and employed multi-reaction monitoring mass spectrometry (MRM-MS). This required a previously developed permethylation method that stabilized metabolites and enhanced volatility of the phosphoinositide metabolites. The mass spectrometric analysis allowed for the monitoring of the intracellular removal of photo-activatable caging as well as biodegradable protecting groups from the membrane-permeant phosphoinositides along with cellular turnover, namely by dephosphorylation. We found that phosphate masking groups, namely acetoxymethyl esters, were rapidly removed by endogenous enzymes while butyrates masking hydroxy groups showed a longer lifetime, giving rise to trapped intermediates. We further identified key intermediate metabolites and demonstrated the beneficial effect of caging groups and their removal on the formation of favorable metabolites. Surprisingly, caging and protecting groups were found to influence each other's stability.

Selective increment of phosphatidylserine on the autophagic body membrane in the yeast vacuole.

In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.

In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate.

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

Analysis of Phosphoinositides from Complex Plant Samples by Solid-Phase Adsorption Chromatography and Subsequent Quantification via Thin-Layer and Gas Chromatography.

The determination of phosphoinositide molecular species in plant material is challenging because of their low abundance concurrent with a very high abundance of other membrane lipids, such as plastidial glycolipids. Phosphoinositides harbor an inositol headgroup which carries one or more phosphate groups at different positions of the inositol, linked to diacylglycerol via a phosphodiester. Thus, a further analytical challenge is to distinguish the different inositol-phosphate headgroups as well as the fatty acids of the diacylglycerol backbone. The method presented in this chapter expands on previous protocols for phosphoinositide analysis by employing chromatographic enrichment of phospholipids and their separation from other, more abundant lipid classes, before analysis. Lipids extracted from plant material are first separated by solid-phase adsorption chromatography into fractions containing neutral lipids, glycolipids, or phospholipids. Lipids from the phospholipid fraction are then separated by thin-layer chromatography (TLC) according to their characteristic head groups, and the individual phosphatidylinositol-monophosphates and phosphatidylinositol-bisphosphates are isolated. Finally, the fatty acids associated with each isolated phosphatidylinositol-monophosphate or phosphatidylinositol-bisphosphate are analyzed in a quantitative fashion using gas chromatography (GC). The analysis of phosphoinositides by this combination of methods provides a cost-efficient and reliable alternative to lipidomics approaches requiring more extensive instrumentation.

The chemistry and biology of phosphatidylinositol 4-phosphate at the plasma membrane.

Phosphoinositides are an important class of anionic, low abundance signaling lipids distributed throughout intracellular membranes. The plasma membrane contains three phosphoinositides: PI(4)P, PI(4,5)P2, and PI(3,4,5)P3. Of these, PI(4)P has remained the most mysterious, despite its characterization in this membrane more than a half-century ago. Fortunately, recent methodological innovations at the chemistry-biology interface have spurred a renaissance of interest in PI(4)P. Here, we describe these new toolsets and how they have revealed novel functions for the plasma membrane PI(4)P pool. We examine high-resolution structural characterization of the plasma membrane PI 4-kinase complex that produces PI(4)P, tools for modulating PI(4)P levels including isoform-selective PI 4-kinase inhibitors, and fluorescent probes for visualizing PI(4)P. Collectively, these chemical and biochemical approaches have revealed insights into how cells regulate synthesis of PI(4)P and its downstream metabolites as well as new roles for plasma membrane PI(4)P in non-vesicular lipid transport, membrane homeostasis and trafficking, and cell signaling pathways.

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation.

The TSC complex is a critical negative regulator of the small GTPase Rheb and mTORC1 in cellular stress signaling. The TSC2 subunit contains a catalytic GTPase activating protein domain and interacts with multiple regulators, while the precise function of TSC1 is unknown. Here we provide a structural characterization of TSC1 and define three domains: a C-terminal coiled-coil that interacts with TSC2, a central helical domain that mediates TSC1 oligomerization, and an N-terminal HEAT repeat domain that interacts with membrane phosphatidylinositol phosphates (PIPs). TSC1 architecture, oligomerization, and membrane binding are conserved in fungi and humans. We show that lysosomal recruitment of the TSC complex and subsequent inactivation of mTORC1 upon starvation depend on the marker lipid PI3,5P2, demonstrating a role for lysosomal PIPs in regulating TSC complex and mTORC1 activity via TSC1. Our study thus identifies a vital role of TSC1 in TSC complex function and mTORC1 signaling.

Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.

Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.

Dynamic remodeling of host membranes by self-organizing bacterial effectors.

During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.

Probing Protein-Membrane Interactions and Dynamics Using Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Cellular membranes are a central hub for initiation and execution of many signaling processes. Integral to these processes being accomplished appropriately is the highly controlled recruitment and assembly of proteins at membrane surfaces. The study of the molecular mechanisms that mediate protein-membrane interactions can be facilitated by utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS is a robust analytical technique that allows for the measurement of the exchange rate of backbone amide hydrogens with solvent to make inferences about protein structure and conformation. This chapter discusses the use of HDX-MS as a tool to study the conformational changes that occur within peripheral membrane proteins upon association with membrane. Particular reference will be made to the analysis of the protein kinase Akt and its activation upon binding phosphatidylinositol (3,4,5) tris-phosphate (PIP3)-containing membranes to illustrate specific methodological principles.

Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Simultaneous Detection of Phosphoinositide Lipids by Radioactive Metabolic Labeling.

Phosphoinositide (PPI) lipids are a crucial class of low-abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-3H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.

Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry.

Phosphoinositides (PIs), the seven phosphorylated derivatives of phosphatidylinositol, are recognized as key molecules in the control of multiple molecular events in eukaryotic cells. Within cells, PIs are low-abundance lipids making their detection and quantification challenging. While many methods that allow radiolabeling and quantification of PIs in the context of cultured cells are available, these are not useful in the context of in vivo animal models where cell and developmental processes are best studied. In this chapter, we describe radionuclide-free, mass spectrometry-based methods for the detection and quantification of PIs from Drosophila tissues in vivo. The use of these methods should facilitate the discovery of novel modes by which PIs regulate cellular and developmental processes in complex metazoans.

Profiling of Phosphoinositide Molecular Species in Resting or Activated Human or Mouse Platelets by a LC-MS Method.

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP2, and PIP3, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.