Phosphatidylinositol Cycle Research Articles

Diacylglycerol kinase-ε is S-palmitoylated on cysteine in the cytoplasmic end of its N-terminal transmembrane fragment

Diacylglycerol kinase-ε (DGKε) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol, which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGKε is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity and was also found crucial for lipid metabolism. DGKε dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and possibly other human diseases. Despite the DGKε significance, data on its regulation by cotranslational and/or post-translational modifications are scarce. Here, we report that DGKε is S-palmitoylated at Cys38/40 (mouse/human DGKε) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKε was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry and with acyl-biotin and acyl-polyethylene glycol exchange assays. The S-acyltransferases zDHHC7 (zinc finger DHHC domain containing) and zDHHC17 and the zDHHC6/16 tandem were found to catalyze DGKε S-palmitoylation, which also increased the DGKε abundance. Mouse DGKε-Myc ectopically expressed in human embryonic kidney 293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated, whereas hyperpalmitoylation of wild-type DGKε reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. In addition, the substitution of neighboring Pro31 with Ala also diminished the activity of DGKε. Taken together, our data indicate that S-palmitoylation can fine-tune DGKε activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.

Human Diacylglycerol Kinase ε N-Terminal Segment Regulates the Phosphatidylinositol Cycle, Controlling the Rate but Not the Acyl Chain Composition of Its Lipid Intermediates.

Diacylglycerol kinase ε (DGKε), an enzyme of the phosphatidylinositol (PI) cycle, bears a highly conserved hydrophobic N-terminal segment, which was proposed to anchor the enzyme into the membrane. However, the importance of this segment to the DGKε function remains to be determined. To address this question, it is here reported an in silico and in vitro combined research strategy. Capitalizing on the AlphaFold 2.0 predicted structure of human DGKε, it is shown that its hydrophobic N-terminal segment anchors it into the membrane via a transmembrane α-helix. Coarse-grained based elastic network model studies showed that a conformational change in the hydrophobic N-terminal segment determines the proximity between the active site of DGKε and the membrane-water interface, likely regulating its kinase activity. In vitro studies with a purified DGKε construct lacking the hydrophobic N-terminal segment (His-SUMO*-Δ50-DGKε) corroborated the role of the N-terminus in regulating DGKε enzymatic properties. The comparison between the enzymatic properties of DGKε and His-SUMO*-Δ50-DGKε showed that the conserved N-terminal segment markedly inhibits the enzyme activity and its sensitivity to membrane intrinsic negative curvature, while also playing a role in the modulation of the enzyme by phosphatidylserine. On the other hand, this segment did not strongly affect its diacylglycerol acyl chain specificity, the modulation of the enzyme by membrane morphological changes, or the activation by phosphatidic acid-rich lipid domains. Hence, these results suggest that the conservation of the hydrophobic N-terminal segment of DGKε throughout evolution guaranteed not only membrane anchorage but also an efficient and elegant manner to regulate the rate of the PI cycle.

Anterior cingulate cortex neuro-metabolic changes underlying lithium-induced euthymia in bipolar depression: A longitudinal 1H-MRS study

The diagnosis and treatment of bipolar depression (BDep) poses complex clinical challenges for psychiatry. Proton magnetic resonance spectroscopy (1H-MRS) is a useful imaging tool for investigating in vivo levels of brain neuro-metabolites, critical to understanding the process of mood dysregulation in Bipolar Disorder. Few studies have evaluated longitudinal clinical outcomes in BDep associated with 1H-MRS metabolic changes. This study aimed to longitudinally assess brain 1H-MRS metabolites in the anterior cingulate cortex (ACC) correlated with improvement in depression (from BDep to euthymia) after lithium treatment in BDep patients versus matched healthy controls (HC). Twenty-eight medication-free BDep patients and 28 HC, matched for age and gender, were included in this study. All subjects were submitted to a 3-Tesla brain 1H-MRS scan in the ACC using a single-voxel (8cm3) PRESS sequence at baseline. At follow-up (6 weeks), 14 BDep patients repeated the exam in euthymia. Patients with current BDep had higher baseline Myo-inositol/Cr (mI/Cr) and Choline/Cr (Cho/Cr) compared to HC. After six weeks, mI/Cr or Cho/Cr levels in subjects that achieved euthymia no longer differed to levels in HC, while high Cho/Cr levels persisted in non-responders . Elevated ACC mI/Cr and Cho/Cr in BDep might indicate increased abnormal membrane phospholipid metabolism and phosphatidylinositol (PI) cycle activity. Return of mI/Cr and Cho/Cr to normal levels after lithium-induced euthymia suggests a critical regulatory effect of lithium targeting the PI cycle involved in mood regulation.

Towards a Comprehensive Understanding of Bipolar Disorder: In Vivo MRS Investigation of the Phosphatidylinositol Cycle


 
 
 
 At present, both the neuropathophysiology of bipolar disorder as well as the mechanism(s) through which current mood-stabilizing agents provide symptom relief are unknown. Through the recent utilization of magnetic resonance spectroscopy (MRS), progress has been made; with optimistic interest being focused on the phosphatidylinositol (PI) cycle as a likely neuropathophysiological factor in bipolar disorder. The present manuscript reviews this interesting and promising area, placing emphasis on the magnetic resonance spectroscopy investigations of PI- cycle function in bipolar disorder reported to date. While relatively few well-designed MRS studies have investigated PI-cycle function in bipolar disorder, current evidence does lend support to PI-cycle involvement in bipolar neuropathophysiology as a means through which mood-stabilizing agents act; pointing to PI-cycle dysfunction as an important neuropathophysiological factor in bipolar disorder. However, there still remains a dearth of information about this interesting hypothesis. In addition to lithium, more data is needed regarding the effects on PI-cycle function of the other commonly prescribed mood-stabilizing agents. As well, studies investigating the temporal relationship between medication effect on PI-cycle functioning and symptom improvement are warranted. In tandem with clinical MRS investigation of bipolar disorder, further preclinical study of the neurochemical effects of mood-stabilizing agents is needed. Finally, greater methodological rigor needs to be implemented during the design phase of any future MRS investigation into bipolar disorder, with particular attention aimed at recruiting a large homogenous sample of bipolar patients.
 
 
 

A model of the PI cycle reveals the regulating roles of lipid-binding proteins and pitfalls of using mosaic biological data

The phosphatidylinositol (PI) cycle is central to eukaryotic cell signaling. Its complexity, due to the number of reactions and lipid and inositol phosphate intermediates involved makes it difficult to analyze experimentally. Computational modelling approaches are seen as a way forward to elucidate complex biological regulatory mechanisms when this cannot be achieved solely through experimental approaches. Whilst mathematical modelling is well established in informing biological systems, many models are often informed by data sourced from multiple unrelated cell types (mosaic data) or from purified enzyme data. In this work, we develop a model of the PI cycle informed by experimental and omics data taken from a single cell type, namely platelets. We were able to make a number of predictions regarding the regulation of PI cycle enzymes, the importance of the number of receptors required for successful GPCR signaling and the importance of lipid- and protein-binding proteins in regulating second messenger outputs. We then consider how pathway behavior differs, when fully informed by data for HeLa cells and show that model predictions remain consistent. However, when informed by mosaic experimental data model predictions greatly vary illustrating the risks of using mosaic datasets from unrelated cell types.

Integrated regulation of the phosphatidylinositol cycle and phosphoinositide-driven lipid transport at ER-PM contact sites.

Among the structural phospholipids that form the bulk of eukaryotic cell membranes, phosphatidylinositol (PtdIns) is unique in that it also serves as the common precursor for low-abundance regulatory lipids, collectively referred to as polyphosphoinositides (PPIn). The metabolic turnover of PPIn species has received immense attention because of the essential functions of these lipids as universal regulators of membrane biology and their dysregulation in numerous human pathologies. The diverse functions of PPIn lipids occur, in part, by orchestrating the spatial organization and conformational dynamics of peripheral or integral membrane proteins within defined subcellular compartments. The emerging role of stable contact sites between adjacent membranes as specialized platforms for the coordinate control of ion exchange, cytoskeletal dynamics, and lipid transport has also revealed important new roles for PPIn species. In this review, we highlight the importance of membrane contact sites formed between the endoplasmic reticulum (ER) and plasma membrane (PM) for the integrated regulation of PPIn metabolism within the PM. Special emphasis will be placed on non-vesicular lipid transport during control of the PtdIns biosynthetic cycle as well as toward balancing the turnover of the signaling PPIn species that define PM identity.

Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids

Diacylglycerol kinase ε (DGKε) is a membrane-bound enzyme that catalyzes the ATP-dependent phosphorylation of diacylglycerol to form phosphatidic acid (PA) in the phosphatidylinositol cycle. DGKε lacks a putative regulatory domain and has recently been reported to be regulated by highly curved membranes. To further study the effect of other membrane properties as a regulatory mechanism of DGKε, our work reports the effect of negatively charged phospholipids on DGKε activity and substrate acyl chain specificity. These studies were conducted using purified DGKε and detergent-free phospholipid aggregates, which present a more suitable model system to access the impact of membrane physical properties on membrane-active enzymes. The structural properties of the different model membranes were studied by means of differential scanning calorimetry and 31P-NMR. It is shown that the enzyme is inhibited by a variety of negatively charged phospholipids. However, PA, which is a negatively charged phospholipid and the product of DGKε catalyzed reaction, showed a varied regulatory effect on the enzyme from being an activator to an inhibitor. The type of feedback regulation of DGKε by PA depends on the particular PA molecular species as well as the physical properties of the membrane that the enzyme binds to. In the presence of highly packed PA-rich domains, the enzyme is activated. However, its acyl chain specificity is only observed in liposomes containing 1,2-dioleoyl PA in the presence of Ca2+. It is proposed that to endow the enzyme with its substrate acyl chain specificity, a highly dehydrated (hydrophobic) membrane interface is needed. The presence of an overlap of mechanisms to regulate DGKε ensures proper phosphatidylinositol cycle function regardless of the trigged stimulus and represents a sophisticated and specialized manner of membrane-enzyme regulation.

Membrane Physical Properties Regulate the Rate and Acyl Chain Specificity of One of the Steps of the Phosphatidylinositol Cycle

The plethora of biological functions regulated by phosphatidylinositol (PI) and its phosphorylated forms makes this lipid class one of the most important in cell physiology. The major metabolic pathway for the synthesis of this lipid class is the PI‐cycle. One of the steps of the PI‐cycle is the ATP‐dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), a reaction catalyzed by the epsilon isoform of diacylglycerol kinases (DGKɛ). The enzyme when assayed with mixed‐micelles exhibits a high degree of acyl chain specificity for its DAG substrate, with 1‐stearoyl‐2‐arachidonoyl glycerol (SAG) being the preferred substrate. These acyl chains are the same as those found in the lipid intermediates of the PI‐cycle, which was proposed to be in part due to the action of DGKɛ. Opposite to other metabolic cycles, steps of the PI‐cycle spans two different locations (membranes) within a cell, the plasma membrane (PM) and endoplasmic reticulum (ER), the same membranes where DGKɛ has been reported to be found. However, it is currently unknown whether DGKɛ catalyze its reaction within the PI‐cycle in the PM and/or ER. Since DGKɛ lacks a putative regulatory domain and these two membranes have several different physical and chemical properties, we hypothesized that either DGKɛ is constitutively active or it is regulated by the properties of the membrane it's bound to. To address this question we use an assay with purified DGKɛ and liposomes. This strategy allowed us to systematically vary the membrane physical and chemical properties while avoiding complications coming from other levels of regulation. In the present work we show that the rate of DGKɛ ‐ catalyzed reaction and its substrate acyl chain specificity are regulated by membrane physical properties. In a locally flat membrane (in relation to the size of the enzyme and its membrane binding platform) the enzyme works at very low basal levels without discriminating the nature of the substrate acyl chains. In these membrane structures the majority of biological negatively charged phospholipids showed an inhibitory effect on the enzyme. However, its product (PA), which is also negatively charged, exhibited the contrary effect leading to an enhancement in enzyme activity and acyl chain specificity. This result suggested a positive feedback mechanism, contrary to what is currently believed. Likewise, the presence of highly curved membranes, specifically those membrane structures bearing negative Gaussian (saddle‐point like) curvature greatly enhanced the rate of reaction as well as the enzyme substrate acyl chain specificity, which was also dependent on the degree of curvature. To sum up, our results indicate that DGKɛ is not likely to catalyze its reaction within the PI‐cycle in stable regions of either the ER or the PM, but rather at the junction between these two organelles where highly curved membrane structures have been reported and/or at PA‐enriched domains.Support or Funding InformationThis work was supported by the Canadian Natural Sciences and Engineering Research Council grant RGPIN‐2018‐05585.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of membrane shape in regulating the phosphatidylinositol cycle at contact sites.

The extensive number of metabolic processes regulated by phosphatidylinositol (PI) phospholipids highlights their physiological importance. The major metabolic pathway for their biosynthesis in cells is the PI-cycle. Contrary to most metabolic cycles, reactions of the PI-cycle occur in two different locations; those are, the plasma membrane (PM) and endoplasmic reticulum (ER). Lipid movement between the two organelles is, therefore, a requirement of the cyclical process. Moreover, in mammals the PI-cycle yield PI molecular species enriched in specific acyl chains, namely 1-stearoyl-2-arachidonoyl acyl chains. Hence, to ensure cycle efficiency and specificity it should take place in specialized regions of PM and ER rather than being randomly distributed among those membranes. Along these lines, ER-PM contact sites have emerged as the location where a number of proteins related to the PI-cycle have been reported to localize. Of importance to this review is the presence of the epsilon isoform of diacylglycerol kinase (DGKε) at ER-PM contact sites. In the PI-cycle DGKε is in part responsible for the acyl chain enrichment of the PI molecular species. However, it has recently been shown that the enzyme can only engage in the PI-cycle upon membrane morphological changes. In this review we will discuss the PI-cycle at ER-PM contact sites and how the generation of membrane negative Gaussian curvature nearby those regions could regulate the cycle. We will focus our discussion on the hypothesis that actin polymerization provides the mechanical force needed to change membrane shape nearby ER-PM contact sites, which will transiently trigger DGKε and, therefore, link enzymatic catalysis and lipid transfer in the PI-cycle.

Lysophospholipid acyltransferases and leukotriene biosynthesis: intersection of the Lands cycle and the arachidonate PI cycle

Leukotrienes (LTs) are autacoids derived from the precursor arachidonic acid (AA) via the action of five-lipoxygenase (5-LO). When inflammatory cells are activated, 5-LO translocates to the nuclear membrane to initiate oxygenation of AA released by cytosolic phospholipase A2 (cPLA2) into leukotriene A4 (LTA4). LTA4 can also be exported from an activated donor cell into an acceptor cell by the process of transcellular biosynthesis. When thimerosal is added to cells, the level of free AA increases by inhibition of lysophospholipid acyltransferases of the Lands pathway of phospholipid remodeling. Another arachidonate phospholipid cycle involves phosphatidylinositol (PI) in the plasma membrane that undoubtedly intersects with the Lands pathway of phospholipid remodeling. The highest abundance of PI occurs between the ER and the plasma membrane and is probably a result of the importance of the PI signaling cascade in cellular biochemistry. Because transport proteins mediate the rapid intracellular movement of phospholipids, largely as result of physical membrane contact, 5-LO-dependent production of LTA4 could be mediated by the disappearance of free AA from the nuclear membrane, transfer to the ER for Lands cycle reesterification into PI, and population of PI(18:0/20:4) for cell membrane signaling.

Membrane curvature allosterically regulates the phosphatidylinositol cycle, controlling its rate and acyl-chain composition of its lipid intermediates

Signaling events at membranes are often mediated by membrane lipid composition or membrane physical properties. These membrane properties could act either by favoring the membrane binding of downstream effectors or by modulating their activity. Several proteins can sense/generate membrane physical curvature (i.e. shape). However, the modulation of the activity of enzymes by a membrane's shape has not yet been reported. Here, using a cell-free assay with purified diacylglycerol kinase ϵ (DGKϵ) and liposomes, we studied the activity and acyl-chain specificity of an enzyme of the phosphatidylinositol (PI) cycle, DGKϵ. By systematically varying the model membrane lipid composition and physical properties, we found that DGKϵ has low activity and lacks acyl-chain specificity in locally flat membranes, regardless of the lipid composition. On the other hand, these enzyme properties were greatly enhanced in membrane structures with a negative Gaussian curvature. We also found that this is not a consequence of preferential binding of the enzyme to those structures, but rather is due to a curvature-mediated allosteric regulation of DGKϵ activity and acyl-chain specificity. Moreover, in a fine-tuned interplay between the enzyme and the membrane, DGKϵ favored the formation of structures with greater Gaussian curvature. DGKϵ does not bear a regulatory domain, and these findings reveal the importance of membrane curvature in regulating DGKϵ activity and acyl-chain specificity. Hence, this study highlights that a hierarchic coupling of membrane physical property and lipid composition synergistically regulates membrane signaling events. We propose that this regulatory mechanism of membrane-associated enzyme activity is likely more common than is currently appreciated.

Complex polymorphisms in endocytosis genes suggest alpha-cyclodextrin as a treatment for breast cancer.

Most breast cancer deaths are caused by metastasis and treatment options beyond radiation and cytotoxic drugs, which have severe side effects, and hormonal treatments, which are or become ineffective for many patients, are urgently needed. This study reanalyzed existing data from three genome-wide association studies (GWAS) using a novel computational biostatistics approach (muGWAS), which had been validated in studies of 600–2000 subjects in epilepsy and autism. MuGWAS jointly analyzes several neighboring single nucleotide polymorphisms while incorporating knowledge about genetics of heritable diseases into the statistical method and about GWAS into the rules for determining adaptive genome-wide significance. Results from three independent GWAS of 1000–2000 subjects each, which were made available under the National Institute of Health’s “Up For A Challenge” (U4C) project, not only confirmed cell-cycle control and receptor/AKT signaling, but, for the first time in breast cancer GWAS, also consistently identified many genes involved in endo-/exocytosis (EEC), most of which had already been observed in functional and expression studies of breast cancer. In particular, the findings include genes that translocate (ATP8A1, ATP8B1, ANO4, ABCA1) and metabolize (AGPAT3, AGPAT4, DGKQ, LPPR1) phospholipids entering the phosphatidylinositol cycle, which controls EEC. These novel findings suggest scavenging phospholipids as a novel intervention to control local spread of cancer, packaging of exosomes (which prepare distant microenvironment for organ-specific metastases), and endocytosis of β1 integrins (which are required for spread of metastatic phenotype and mesenchymal migration of tumor cells). Beta-cyclodextrins (βCD) have already been shown to be effective in in vitro and animal studies of breast cancer, but exhibits cholesterol-related ototoxicity. The smaller alpha-cyclodextrins (αCD) also scavenges phospholipids, but cannot fit cholesterol. An in-vitro study presented here confirms hydroxypropyl (HP)-αCD to be twice as effective as HPβCD against migration of human cells of both receptor negative and estrogen-receptor positive breast cancer. If the previous successful animal studies with βCDs are replicated with the safer and more effective αCDs, clinical trials of adjuvant treatment with αCDs are warranted. Ultimately, all breast cancer are expected to benefit from treatment with HPαCD, but women with triple-negative breast cancer (TNBC) will benefit most, because they have fewer treatment options and their cancer advances more aggressively.

Lipopolysaccharide Upregulates Palmitoylated Enzymes of the Phosphatidylinositol Cycle: An Insight from Proteomic Studies

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria that induces strong proinflammatory reactions of mammals. These processes are triggered upon sequential binding of LPS to CD14, a GPI-linked plasma membrane raft protein, and to the TLR4/MD2 receptor complex. We have found earlier that upon LPS binding, CD14 triggers generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a lipid controlling subsequent proinflammatory cytokine production. Here we show that stimulation of RAW264 macrophage-like cells with LPS induces global changes of the level of fatty-acylated, most likely palmitoylated, proteins. Among the acylated proteins that were up-regulated in those conditions were several enzymes of the phosphatidylinositol cycle. Global profiling of acylated proteins was performed by metabolic labeling of RAW264 cells with 17ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labeled proteins using biotin-azide/streptavidin and their identification with mass spectrometry. This proteomic approach revealed that 154 fatty-acylated proteins were up-regulated, 186 downregulated, and 306 not affected in cells stimulated with 100 ng/ml LPS for 60 min. The acylated proteins affected by LPS were involved in diverse biological functions, as found by Ingenuity Pathway Analysis. Detailed studies of 17ODYA-labeled and immunoprecipitated proteins revealed that LPS induces S-palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) β, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P2 precursor. Silencing of PI4KIIβ and PI4KIIα inhibited LPS-induced expression and production of proinflammatory cytokines, especially in the TRIF-dependent signaling pathway of TLR4. Reciprocally, this LPS-induced signaling pathway was significantly enhanced after overexpression of PI4KIIβ or PI4KIIα; this was dependent on palmitoylation of the kinases. However, the S-palmitoylation of PI4KIIα, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers S-palmitoylation and activation of PI4KIIβ, which generates PI(4)P involved in signaling pathways controlling production of proinflammatory cytokines.

Expression, Purification, and Properties of a Human Arachidonoyl-Specific Isoform of Diacylglycerol Kinase.

Diacylglycerol kinase ε (DGKε) catalyzes the phosphorylation of diacylglycerol, producing phosphatidic acid. DGKε demonstrates exquisite specificity for the acyl chains of diacylglycerol. This contributes to the enrichment of particular acyl chains within the lipids of the phosphatidylinositol cycle. Phosphatidylinositol is highly enriched with 1-stearoyl-2-arachidonoyl, which is important for maintaining cellular health. Dysregulation of DGKε perturbs lipid signaling and biosynthesis, which has been linked to epilepsy, Huntington's disease, and heart disease. Recessive loss-of-function mutations in the DGKε gene cause atypical hemolytic uremic syndrome. Because DGKε has never been purified, little is known about its molecular properties. We expressed human DGKε and a truncated version lacking the first 40 residues (DGKεΔ40) and purified both proteins to near homogeneity using nickel affinity chromatography. Kinase activity measurements showed that both purified constructs retained their acyl chain specificity for diacylglycerol with an activity level comparable to that of N-terminally FLAG epitope-tagged forms of these proteins expressed in COS7 cells. Both constructs lost activity upon being stored, particularly upon freezing and thawing, which was minimized by the addition of glycerol. Circular dichroism revealed that DGKε and DGKεΔ40 both contain significant amounts of α-helical and β structure and exhibit biphasic thermal denaturations. The loss of secondary structure upon heating was irreversible for both constructs, with relatively little effect of added dioleoylphosphatidylcholine. The addition of 50% glycerol stabilized both constructs and facilitated refolding of their secondary structures after heating. This is the first successful purification and characterization of DGKε's enzymatic and conformational properties. The purification of DGKε permits detailed analyses of this unique enzyme and will improve our understanding of DGKε-related diseases.

DGK-θ: Structure, Enzymology, and Physiological Roles.

Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PtdOH). The recognition of the importance of these enzymes has been increasing ever since it was determined that they played a role in the phosphatidylinositol (PtdIns) cycle and a number of excellent reviews have already been written [(see van Blitterswijk and Houssa, 2000; Kanoh et al., 2002; Mérida et al., 2008; Tu-Sekine and Raben, 2009, 2011; Shulga et al., 2011; Tu-Sekine et al., 2013) among others]. We now know there are ten mammalian DGKs that are organized into five classes. DGK-θ is the lone member of the Type V class of DGKs and remains as one of the least studied. This review focuses on our current understanding of the structure, enzymology, regulation, and physiological roles of this DGK and suggests some future areas of research to understand this DGK isoform.

Features of the Phosphatidylinositol Cycle and its Role in Signal Transduction.

The phosphatidylinositol cycle (PI-cycle) has a central role in cell signaling. It is the major pathway for the synthesis of phosphatidylinositol and its phosphorylated forms. In addition, some lipid intermediates of the PI-cycle, including diacylglycerol and phosphatidic acid, are also important lipid signaling agents. The PI-cycle has some features that are important for the understanding of its role in the cell. As a cycle, the intermediates will be regenerated. The PI-cycle requires a large amount of metabolic energy. There are different steps of the cycle that occur in two different membranes, the plasma membrane and the endoplasmic reticulum. In order to complete the PI-cycle lipid must be transferred between the two membranes. The role of the Nir proteins in the process has recently been elucidated. The lipid intermediates of the PI-cycle are normally highly enriched with 1-stearoyl-2-arachidonoyl molecular species in mammals. This enrichment will be retained as long as the intermediates are segregated from other lipids of the cell. However, there is a significant fraction (>15%) of lipids in the PI-cycle of normal cells that have other acyl chains. Phosphatidylinositol largely devoid of arachidonoyl chains are found in cancer cells. Phosphatidylinositol species with less unsaturation will not be as readily converted to phosphatidylinositol-3,4,5-trisphosphate, the lipid required for the activation of Akt with resulting effects on cell proliferation. Thus, the cyclical nature of the PI-cycle, its dependence on acyl chain composition and its requirement for lipid transfer between two membranes, explain many of the biological properties of this cycle.

Nuclear Phosphatidylinositol Signaling: Focus on Phosphatidylinositol Phosphate Kinases and Phospholipases C

Phosphatidylinositol (PI) metabolism represents the core of a network of signaling pathways which modulate many cellular functions including cell proliferation, cell differentiation, apoptosis, and membrane trafficking. An array of kinases, phosphatases, and lipases acts on PI creating an important number of second messengers involved in different cellular processes. Although, commonly, PI signaling was described to take place at the plasma membrane, many evidences indicated the existence of a PI cycle residing in the nuclear compartment of eukaryotic cells. The discovery of this mechanism shed new light on many nuclear functions, such as gene transcription, DNA modifications, and RNA expression. As these two PI cycles take place independently of one another, understanding how nuclear lipid signaling functions and modulates nuclear output is fundamental in the study of many cellular processes. J. Cell. Physiol. 231: 1645-1655, 2016. © 2015 Wiley Periodicals, Inc.

Molecular effects of lithium are partially mimicked by inositol-monophosphatase (IMPA)1 knockout mice in a brain region-dependent manner

We have previously shown that homozygote knockout (KO) of inositol-monophosphatase1 (IMPA1) results in lithium (Li)-like behavior. We now aimed to find out whether Li-treated mice and IMPA1 KO mice exhibit neurochemical similarity at the gene- and protein-expression level. Hippocampal and frontal cortex B-cell lymphoma (Bcl-2), Bcl-2-associated X protein (BAX), P53, Perodoxin2 (PRDX2), myristoylated alanine-rich C kinase substrate (MARCKS) and neuropeptide Y (NPY) mRNA levels, and hippocampal, frontal cortex and hypothalamic cytokine levels, all previously reported to be affected by lithium treatment, were measured in three groups of mice: wildtype (WT) on regular-food (RF), WT on Li-supplemented food (Li-treated) and IMPA1-KOs.Hippocampal and frontal cortex Bcl-2 and MARCKS were the only genes commonly affected (downregulated) by Li and IMPA1 KO; Bcl-2 - by 28% and 19%, respectively; MARCKS – by about 20% in both regions.The effect of Li and of IMPA1 KO on cytokine levels differed among the three brain areas studied. Only in the hippocampus both interventions exerted similar effects. Frontal cortex cytokine levels were unaffected neither by Li nor by IMPA1 KO.Similar changes in Bcl-2 and MARCKS but not in PRDX2 and NPY following both Li-treatment and IMPA1 KO suggest a mechanism different than inositol-monophosphatase1 inhibition for Li׳s effect on the latter genes. The cytokine levels results suggest that the mechanism mediating Li׳s effect on the inflammatory system differs among brain regions. Only in the hippocampus the results favor the involvement of the phosphatidylinositol (PI) cycle.

Lithium carbonate teratogenic effects in chick cardiomyocyte micromass system and mouse embryonic stem cell derived cardiomyocyte – Possible protective role of myo-inositol

The drug lithium carbonate (Li2CO3) use during pregnancy increases the possibility of cardiovascular anomalies. The earlier studies confirm its phosphatidylinositol cycle (PI) inhibition and Wnt pathways mimicking properties, which might contribute to its teratogenic effects. In this study the toxic effects of Li2CO3 in chick embryonic cardiomyocyte micromass system (MM) and embryonic stem cell derived cardiomyocyte (ESDC) were evaluated, with possible protective role of myo-inositol. In MM system the Li2CO3 did not alter the toxicity estimation endpoints, whereas in ESDC system the cardiomyocytes contractile activity stopped at 1500μM and above with significant increase in total cellular protein contents. In ESDC system when myo-inositol was added along with Li2CO3 to continue PI cycle, the contractile activity was recovered with decreased protein content. The lithium toxic effects depend on the role of PI cycle at particular stage of cardiogenesis, while relation between myo-inositol and reduced cellular protein contents remains unknown.

DGKE Variants Cause a Glomerular Microangiopathy That Mimics Membranoproliferative GN

Renal microangiopathies and membranoproliferative GN (MPGN) can manifest similar clinical presentations and histology, suggesting the possibility of a common underlying mechanism in some cases. Here, we performed homozygosity mapping and whole exome sequencing in a Turkish consanguineous family and identified DGKE gene variants as the cause of a membranoproliferative-like glomerular microangiopathy. Furthermore, we identified two additional DGKE variants in a cohort of 142 unrelated patients diagnosed with membranoproliferative GN. This gene encodes the diacylglycerol kinase DGKε, which is an intracellular lipid kinase that phosphorylates diacylglycerol to phosphatidic acid. Immunofluorescence confocal microscopy demonstrated that mouse and rat Dgkε colocalizes with the podocyte marker WT1 but not with the endothelial marker CD31. Patch-clamp experiments in human embryonic kidney (HEK293) cells showed that DGKε variants affect the intracellular concentration of diacylglycerol. Taken together, these results not only identify a genetic cause of a glomerular microangiopathy but also suggest that the phosphatidylinositol cycle, which requires DGKE, is critical to the normal function of podocytes.