Inositol Phospholipid Research Articles

Towards a thorough understanding of mammalian glycosylphosphatidylinositol-anchored protein biosynthesis.

Glycosylphosphatidylinositols (GPIs) are glycolipids found ubiquitously in eukaryotes. They consist of a glycan and an inositol phospholipid, and act as membrane anchors of many cell-surface proteins by covalently linking to their C-termini. GPIs also exist as unlinked, free glycolipids on the cell surface. In human cells, at least 160 proteins with various functions are GPI-anchored proteins. Because the attachment of GPI is required for the cell-surface expression of GPI-anchored proteins, a thorough knowledge of the molecular basis of mammalian GPI-anchored protein biosynthesis is important for understanding the basic biochemistry and biology of GPI-anchored proteins and their medical significance. In this paper, I review our previous knowledge of the biosynthesis of mammalian GPI-anchored proteins and then examine new findings made since 2020.

Adaptive Changes in Group 2 Metabotropic Glutamate Receptors Underlie the Deficit in Recognition Memory Induced by Methamphetamine in Mice.

Cognitive dysfunction is associated with methamphetamine use disorder (MUD). Here, we used genetic and pharmacological approaches to examine the involvement of either Group 2 metabotropic glutamate (mGlu2) or mGlu3 receptors in memory deficit induced by methamphetamine in mice. Methamphetamine treatment (1 mg/kg, i.p., once a day for 5 d followed by 7 d of withdrawal) caused an impaired performance in the novel object recognition test in wild-type mice, but not in mGlu2-/- or mGlu3-/- mice. Memory deficit in wild-type mice challenged with methamphetamine was corrected by systemic treatment with selectively negative allosteric modulators of mGlu2 or mGlu3 receptors (compounds VU6001966 and VU0650786, respectively). Methamphetamine treatment in wild-type mice caused large increases in levels of mGlu2/3 receptors, the Type 3 activator of G-protein signaling (AGS3), Rab3A, and the vesicular glutamate transporter, vGlut1, in the prefrontal cortex (PFC). Methamphetamine did not alter mGlu2/3-mediated inhibition of cAMP formation but abolished the ability of postsynaptic mGlu3 receptors to boost mGlu5 receptor-mediated inositol phospholipid hydrolysis in PFC slices. Remarkably, activation of presynaptic mGlu2/3 receptors did not inhibit but rather amplified depolarization-induced [3H]-D-aspartate release in synaptosomes prepared from the PFC of methamphetamine-treated mice. These findings demonstrate that exposure to methamphetamine causes changes in the expression and function of mGlu2 and mGlu3 receptors, which might alter excitatory synaptic transmission in the PFC and raise the attractive possibility that selective inhibitors of mGlu2 or mGlu3 receptors (or both) may be used to improve cognitive dysfunction in individuals affected by MUD.

Abstract 1946 The long road paved by inositol phospholipids

Abstract 1946 The long road paved by inositol phospholipids

Sir Michael John Berridge. 22 October 1938—13 February 2020

Sir Michael Berridge was a giant in the fields of physiology and biochemistry who, by his discovery in 1983 of inositol 1,4,5-trisphosphate (IP 3 ) as the ubiquitous intracellular messenger releasing Ca 2+ from intracellular stores, revolutionized our concepts of signal transduction mechanisms. Mike, as he was universally known, discovered one of the most important regulatory mechanisms in animal cells, involved in the control of virtually all bodily functions, including secretion, contraction and memory. Mike, working at the Department of Zoology at the University of Cambridge, personally identified IP 3 as one of the two initial products of hormone- or neurotransmitter-elicited inositol phospholipid breakdown and then, in collaboration with the research group led by Irene Schulz at the Max Planck Institute for Biophysics in Frankfurt, demonstrated directly the Ca 2+ -releasing effect of IP 3 in pancreatic acinar cells. Subsequently, in collaborations with research groups in Switzerland and the USA, Mike confirmed the Ca 2 + -releasing action of IP 3 in insulin-secreting cells, hepatocytes and photoreceptors. The IP 3 –Ca 2 + -releasing pathway became accepted amazingly quickly as one of the key elements of cellular signal transduction mechanisms and is now featured in virtually all textbooks of physiology and biochemistry. Mike was also a great synthesizer, who created an entirely new field, namely calcium signalling. Through his numerous, highly cited review articles, and his insightful keynote lectures at the most important biomedical congresses, he dominated this large and increasingly important research field for more than 30 years following his momentous 1983 discovery.

Gut microbiota analyses of cutaneous T-cell lymphoma patients undergoing narrowband ultraviolet B therapy reveal alterations associated with disease treatment.

Recent studies have shown a close relationship between cutaneous T-cell lymphoma (CTCL) and its microbiome. CTCL disease progression is associated with gut dysbiosis and alterations in bacterial taxa parallel those observed in immunologically similar atopic dermatitis. Moreover, the microbial profile of lesional skin may predict response to narrowband ultraviolet B (nbUVB), a common skin-directed therapy. However, the relationship between the gut microbiome, an immunologically vital niche, and nbUVB remains unexplored in CTCL. Herein, we performed 16S rRNA sequencing and PICRUSt2 predictive metagenomics on DNA extracted from stool swabs of 13 CTCL patients treated with nbUVB, 8 non-treated patients, and 13 healthy controls. Disease response was assessed with modified Severity Weighted Assessment Tool (mSWAT); of nbUVB-treated patients, 6 improved (decreased mSWAT), 2 remained stable, and 5 worsened (increased mSWAT). Protective commensal bacteria including Lactobacillaceae and Erysipelatoclostridiaceae were significantly less abundant in CTCL patients compared to controls. With treatment, the CTCL gut microbiome exhibited decreased phylogenetic diversity and lower relative abundance of pro-inflammatory Sutterellaceae. Sutterellaceae was also significantly more abundant in patients who worsened, and Eggerthellaceae and Erysipelotrichaceae trended higher in patients who improved. Finally, PICRUSt2 functional predictions based on shifts in abundance of bacterial sequences repeatedly identified alterations in inositol degradation, which plays a key role in host immunomodulation, including inositol phospholipid signaling relevant to T-cell survival and proliferation. Our results bolster the paradigm of gut dysbiosis in CTCL and its functional implications in disease pathogenesis, and further delineate bacterial taxa associated with nbUVB response and with nbUVB treatment itself.

Toxoplasma membrane inositol phospholipid binding protein TgREMIND is essential for secretory organelle function and host infection

Apicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.

Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: A neutron reflectometry, AFM and QCM-D structural study

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.

Comparative transcriptome analysis of grafting to improve chilling tolerance of cucumber.

Grafting with pumpkin as rootstock could improve chilling tolerance of cucumber; however, the underlying mechanism of grafting-induced chilling tolerance remains unclear. Here, we analyzed the difference of physiological and transcriptional level between own-rooted (Cs/Cs) and hetero-grafted (Cs/Cm) cucumber seedlings under chilling stress. The results showed that grafting with pumpkin significantly alleviated the chilling injury as evidenced by slightly symptoms, lower contents of electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2-) and higher relative water content in Cs/Cm seedlings compared with Cs/Cs seedlings under chilling stress. RNA-seq data showed that grafting induced more DGEs at 8 °C/5 °C compared with 25 °C/18 °C. In accordance with the increase of the activities of antioxidant enzymes (SOD, POD, CAT, APX), grafting upregulated the expression of the regulated redox-related genes such as GST, SOD, and APX. Moreover, grafting increased the expression of genes participated in central carbon metabolism to promote the conversion and decomposition of sugar, which provided more energy for the growth of Cs/Cm seedlings under chilling stress. In addition, grafting regulated the genes involved in the intracellular signal transduction pathways such as calcium signal (CAML, CML, and CDPK) and inositol phospholipid signal (PLC), as well as changed the gene expression of plant hormone signal transduction pathways (ARF, GAI, ABF, and PYR/PYL). These results provide a physiological and transcriptional basis for the molecular mechanism of grafting-induced chilling tolerance of cucumber seedlings.

Advances in MDS/AML and inositide signalling.

Aberrant signaling pathways regulating proliferation and differentiation of hematopoietic stem cells (HSCs) can contribute to disease pathogenesis and neoplastic growth. Phosphoinositides (PIs) are inositol phospholipids that are implicated in the regulation of critical signaling pathways: aberrant regulation of Phospholipase C (PLC) beta1, PLCgamma1 and the PI3K/Akt/mTOR pathway play essential roles in the pathogenesis of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML).

Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.

Phosphoinositide metabolism is critically involved in human cancer cell migration and metastatic growth. The formation of lamellipodia at the leading edge of migrating cells is regulated by metabolism of the inositol phospholipid PI(4,5)P2 into PI(3,4,5)P3. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane and regulates guanine nucleotide exchange factor Rac-mediated actin filament remodeling. Here, we investigated if VIPR2, a receptor for vasoactive intestinal peptide (VIP), has a potential role in regulating cell migration via this pathway. We found that silencing of VIPR2 in MDA-MB-231 and MCF-7 human breast cancer cells inhibited VIP-induced cell migration. In contrast, stable expression of exogenous VIPR2 promoted VIP-induced tumor cell migration, an effect that was inhibited by the addition of a PI3-kinase (PI3K)γ inhibitor or a VIPR2-selective antagonist. VIPR2 stably-expressing cells exhibited increased PI3K activity. Membrane localization of PI(3,4,5)P3 was significantly attenuated by VIPR2-silencing. VIPR2-silencing in MDA-MB-231 cells suppressed lamellipodium extension; in VIPR2-overexpressing cells, VIPR2 accumulated in the cell membrane on lamellipodia and co-localized with WAVE2. Conversely, VIPR2-silencing reduced WAVE2 level on the cell membrane and inhibited the interaction between WAVE2, actin-related protein 3, and actin. These findings suggest that VIP–VIPR2 signaling controls cancer migration by regulating WAVE2-mediated actin nucleation and elongation for lamellipodium formation through the synthesis of PI(3,4,5)P3.

2-O-Acetyl-3,4,5,6-tetra-O-benzyl-d-myo-inosityl diphenylphosphate: A new useful intermediate to inositol phosphate and phospholipids.

Inositol phosphates and inositol phospholipids are ubiquitous in biochemistry and play a central role in cell signaling and regulation events. For this reason, their synthesis has attracted widespread interest. This paper describes the preparation of a new optically active inositol phosphate derivative, 2‐O‐acetyl‐3,4,5,6‐tetra‐O‐benzyl‐d‐myo‐inosityl diphenylphosphate (6), and its characterization by spectroscopic methods. Compound (6) represents a useful intermediate for the preparation of inositol phosphate and phospholipids, in particular of glycerophosphoinositol (GPI), a natural anti‐inflammatory agent.

Control of the Cellular Lipid Landscape by Inositol Lipids

Cellular organization of eukaryotic cells relies upon compartmentalization of their signaling nodes on special membrane platforms forming the various organelles. Unique lipid composition of the different organelle membranes not only defines their identity but also is critical for the proper assembly and functioning of the protein signaling complexes associated with them. Inositol phospholipids (PPIns), a class of regulatory lipids, play a critical role in defining membrane identity and forming membrane microdomains with unique signaling properties. Recent developments in lipid membrane biology revealed that phosphatidylinositol (PI) 4‐phosphate (PI4P) gradients and the PI 4‐kinases that form them drive non‐vesicular transport of several structural lipids against their concentration gradients at membrane contact sites (MCSs). These processes clearly depend on the delivery of PI from its site of synthesis in the ER to the membranes where PI4Ks convert them to PI4P. Therefore, our recent efforts have been focused on the question of how PI synthesis and PI transport systems provide the means of proper PI delivery to their other membrane destinations. In this presentation we will review our recent data on the generation and use of molecular tools to visualize and manipulate PI metabolism and delivery and demonstrate, how they can help us better understand the central role of PPIns in defining the overall lipid landscape of eukaryotic cells.

An Integrated Pharmacological, Structural, and Genetic Analysis of Extracellular Versus Intracellular ROS Production in Neutrophils

The neutrophil NADPH oxidase produces both intracellular and extracellular reactive oxygen species (ROS). Although oxidase activity is essential for microbial killing, and ROS can act as signaling molecules in the inflammatory process, excessive extracellular ROS directly contributes to inflammatory tissue damage, as well as to cancer progression and immune dysregulation in the tumor microenvironment. How specific signaling pathways contribute to ROS localization is unclear. Here we used a systems pharmacology approach to identify the specific Class I PI3-K isoform p110β, and PLD1, but not PLD2, as critical regulators of extracellular, but not intracellular ROS production in primary neutrophils. Combined crystallographic and molecular dynamics analysis of the PX domain of the oxidase component p47phox, which binds the lipid products of PI 3-K and PLD, was used to clarify the membrane-binding mechanism and guide the design of mutant mice whose p47phox is unable to bind 3-phosphorylated inositol phospholipids. Neutrophils from these K43A mutant animals were specifically deficient in extracellular, but not intracellular, ROS production, and showed increased dependency on signaling through the remaining PLD1 arm. These findings identify the PX domain of p47phox as a critical integrator of PLD1 and p110β signaling for extracellular ROS production, and as a potential therapeutic target for modulating tissue damage and extracellular signaling during inflammation.

Genomic profiling and expression analysis of the diacylglycerol kinase gene family in heterologous hexaploid wheat

The inositol phospholipid signaling system mediates plant growth, development, and responses to adverse conditions. Diacylglycerol kinase (DGK) is one of the key enzymes in the phosphoinositide-cycle (PI-cycle), which catalyzes the phosphorylation of diacylglycerol (DAG) to form phosphatidic acid (PA). To date, comprehensive genomic and functional analyses of DGKs have not been reported in wheat. In this study, 24 DGK gene family members from the wheat genome (TaDGKs) were identified and analyzed. Each putative protein was found to consist of a DGK catalytic domain and an accessory domain. The analyses of phylogenetic and gene structure analyses revealed that each TaDGK gene could be grouped into clusters I, II, or III. In each phylogenetic subgroup, the TaDGKs demonstrated high conservation of functional domains, for example, of gene structure and amino acid sequences. Four coding sequences were then cloned from Chinese Spring wheat. Expression analysis of these four genes revealed that each had a unique spatial and developmental expression pattern, indicating their functional diversification across wheat growth and development processes. Additionally, TaDGKs were also prominently up-regulated under salt and drought stresses, suggesting their possible roles in dealing with adverse environmental conditions. Further cis-regulatory elements analysis elucidated transcriptional regulation and potential biological functions. These results provide valuable information for understanding the putative functions of DGKs in wheat and support deeper functional analysis of this pivotal gene family. The 24 TaDGKs identified and analyzed in this study provide a strong foundation for further exploration of the biological function and regulatory mechanisms of TaDGKs in response to environmental stimuli.

Targeting SHIP1 and SHIP2 in Cancer

Simple SummaryPhosphoinositol signaling pathways and their dysregulation have been shown to have a fundamental role in health and disease, respectively. The SH2-containing 5′ inositol phosphatases, SHIP1 and SHIP2, are regulators of the PI3K/AKT pathway that have crucial roles in cancer progression. This review aims to summarize the role of SHIP1 and SHIP2 in cancer signaling and the immune response to cancer, the discovery and use of SHIP inhibitors and agonists as possible cancer therapeutics.Membrane-anchored and soluble inositol phospholipid species are critical mediators of intracellular cell signaling cascades. Alterations in their normal production or degradation are implicated in the pathology of a number of disorders including cancer and pro-inflammatory conditions. The SH2-containing 5′ inositol phosphatases, SHIP1 and SHIP2, play a fundamental role in these processes by depleting PI(3,4,5)P3, but also by producing PI(3,4)P2 at the inner leaflet of the plasma membrane. With the intent of targeting SHIP1 or SHIP2 selectively, or both paralogs simultaneously, small molecule inhibitors and agonists have been developed and tested in vitro and in vivo over the last decade in various disease models. These studies have shown promising results in various pre-clinical models of disease including cancer and tumor immunotherapy. In this review the potential use of SHIP inhibitors in cancer is discussed with particular attention to the molecular structure, binding site and efficacy of these SHIP inhibitors.

Inositol Phospholipid Interactions with Inward Rectifier Potassium Channels and Their Role in Disease

Ion channels are frequently modulated by specific protein-lipid interactions in the phospholipid bilayer. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised in biophysical studies. Here, we provide an extensive application of a coarse-grained molecular dynamics free-energy perturbation (CG-MD-FEP) methodology to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step protocol, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid and in the presence of anionic phospholipid such as phosphatidylserine (PS). By perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K has increased PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. This is in good agreement with electrophysiological studies showing the E179K mutant exhibits a reduction in neomycin sensitivity, which implies that PIP2 binds more tightly to E179K mutant channels. We conclude that our application provides a valuable method to compare affinities between lipid species, and for annotating amino acid side chains within lipid-binding pockets.

Release of Anti-Inflammatory Palmitoleic Acid and Its Positional Isomers by Mouse Peritoneal Macrophages.

Positional isomers of hexadecenoic acid are considered as fatty acids with anti-inflammatory properties. The best known of them, palmitoleic acid (cis-9-hexadecenoic acid, 16:1n-7), has been identified as a lipokine with important beneficial actions in metabolic diseases. Hypogeic acid (cis-7-hexadecenoic acid, 16:1n-9) has been regarded as a possible biomarker of foamy cell formation during atherosclerosis. Notwithstanding the importance of these isomers as possible regulators of inflammatory responses, very little is known about the regulation of their levels and distribution and mobilization among the different lipid pools within the cell. In this work, we describe that the bulk of hexadecenoic fatty acids found in mouse peritoneal macrophages is esterified in a unique phosphatidylcholine species, which contains palmitic acid at the sn-1 position, and hexadecenoic acid at the sn-2 position. This species markedly decreases when the macrophages are activated with inflammatory stimuli, in parallel with net mobilization of free hexadecenoic acid. Using pharmacological inhibitors and specific gene-silencing approaches, we demonstrate that hexadecenoic acids are selectively released by calcium-independent group VIA phospholipase A2 under activation conditions. While most of the released hexadecenoic acid accumulates in free fatty acid form, a significant part is also transferred to other phospholipids to form hexadecenoate-containing inositol phospholipids, which are known to possess growth-factor-like-properties, and are also used to form fatty acid esters of hydroxy fatty acids, compounds with known anti-diabetic and anti-inflammatory properties. Collectively, these data unveil new pathways and mechanisms for the utilization of palmitoleic acid and its isomers during inflammatory conditions, and raise the intriguing possibility that part of the anti-inflammatory activity of these fatty acids may be due to conversion to other lipid mediators.

Evaluating inositol phospholipid interactions with inward rectifier potassium channels and characterising their role in disease

Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues.

Emerging roles of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate as regulators of multiple steps in autophagy.

Inositol phospholipids are low-abundance regulatory lipids that orchestrate diverse cellular functions in eukaryotic organisms. Recent studies have uncovered involvement of the lipids in multiple steps in autophagy. The late endosome-lysosome compartment plays critical roles in cellular nutrient sensing and in the control of both the initiation of autophagy and the late stage of eventual degradation of cytosolic materials destined for elimination. It is particularly notable that inositol lipids are involved in almost all steps of the autophagic process. In this review, we summarize how inositol lipids regulate and contribute to autophagy through the endomembrane compartments, primarily focusing on PI4P and PI(4,5)P2.

Phosphoinositide Lipids in Ocular Tissues.

Inositol phospholipids play an important role in cell physiology. The inositol head groups are reversibly phosphorylated to produce seven distinct phosphorylated inositides, commonly referred to as phosphoinositides (PIs). These seven PIs are dynamically interconverted from one PI to another by the action of PI kinases and PI phosphatases. The PI signals regulate a wide variety of cellular functions, including organelle distinction, vesicular transport, cytoskeletal organization, nuclear events, regulation of ion channels, cell signaling, and host–pathogen interactions. Most of the studies of PIs in ocular tissues are based on the PI enzymes and PI phosphatases. In this study, we examined the PI levels in the cornea, retinal pigment epithelium (RPE), and retina using PI-binding protein as probes. We have examined the lipids PI(3)P, PI(4)P, PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3, and each is present in the cornea, RPE, and retina. Alterations in the levels of these PIs in mouse models of retinal disease and corneal infections have been reported, and the results of our study will help in the management of anomalous phosphoinositide metabolism in ocular tissues.