Expanding functions of the phosphatidylinositol/phosphatidate lipid transporter, PITPNC1 in physiology and in pathology.

PITPNC1 was the last of the PITPs to be identified and has been characterized as a binding protein for phosphatidylinositol and phosphatidate. In mammals, PITPNC1 is expressed as two splice variants whilst in zebrafish is expressed from two separate genes. The two splice variants have different expression profiles with the long splice variant having a prominent role in the brain. Several physiological functions have been identified including neuronal and metabolic functions. PITPNC1 also plays a significant role in cancer and has been identified as a risk factor in type 2 diabetes. Here, we review our current understanding of PITPNC1 in cell physiology and pathology.

Fatty acid synthesis promotes inflammasome activation through NLRP3 palmitoylation.

Despite its significance, the role of lipid metabolism in NLRP3 inflammasome remains elusive. Here, we reveal a critical role for fatty acid synthase (FASN) in NLRP3 inflammasome activation. We demonstrate that pharmacological or genetic depletion of FASN dampens NLRP3 activation in primary mouse and human macrophages and in mice. This disruption in NLRP3 activation is contingent upon FASN activity. Accordingly, abolishing cellular palmitoylation, a post-translational modification in which the FASN product palmitate is reversibly conjugated to cysteine residues of target proteins, blunts inflammasome signaling. Correspondingly, an acyl-biotin exchange assay corroborated NLRP3 palmitoylation. Mechanistically, Toll-like receptor (TLR) ligation introduces palmitoylation at NLRP3 Cys898, permitting NLRP3 translocation to dispersed trans-Golgi network (dTGN) vesicles, the site of inflammasome assembly, upon NLRP3 activation. Accordingly, the NLRP3 Cys898 mutant exhibits reduced palmitoylation, limited translocation to the dTGN compartment, and diminished inflammasome activation. These results underscore mechanistic insights through which lipid metabolism licenses NLRP3 inflammasome assembly and activation.

The expanding roles of PI4P and PI(4,5)P2 at the plasma membrane: Role of phosphatidylinositol transfer proteins.

Phosphoinositides are phosphorylated derivatives of phosphatidylinositol, a phospholipid that is synthesised at the endoplasmic reticulum. The plasma membrane contains the enzymes to phosphorylate phosphatidylinositol and is therefore rich in the phosphorylated derivatives, PI4P and PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P2 levels.

Individual phosphatidylinositol transfer proteins have distinct functions that do not involve lipid transfer activity.

Platelets use signal transduction pathways facilitated by class I phosphatidylinositol transfer proteins (PITPs). The 2 mammalian class I PITPs, PITPα and PITPß, are single PITP domain soluble proteins that are encoded by different genes and share 77% sequence identity, although their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominantly expressed murine PITP isoform, had no effect on hemostasis but impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we found that mice lacking the less expressed PITPß in their platelets exhibited a similar phenotype. However, in contrast to PITPα-null platelet lysates, which have impaired lipid transfer activity, PITPß-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs led to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our study also demonstrated that PITP isoforms are required to maintain endogenous phosphoinositide PtdInsP2 levels and agonist-stimulated second messenger formation. The data shown here demonstrate that the 2 isoforms are functionally overlapping and that a single isoform is able to maintain the homeostasis of platelets. However, both class I PITP isoforms contribute to phosphoinositide signaling in platelets through distinct biochemical mechanisms or different subcellular domains.

Yeast Sec14-like lipid transfer proteins Pdr16 and Pdr17 bind and transfer the ergosterol precursor lanosterol in addition to phosphatidylinositol.

Yeast Sec14-like phosphatidylinositol transfer proteins (PITPs) contain a hydrophobic cavity capable of accepting a single molecule of phosphatidylinositol (PI) or another molecule in a mutually exclusive manner. We report here that two yeast Sec14 family PITPs, Pdr16p (Sfh3p) and Pdr17p (Sfh4p), possess high-affinity binding and transfer towards lanosterol. To our knowledge, this is the first identification of lanosterol transfer proteins. In addition, a pdr16Δpdr17Δ double mutant had a significantly increased level of cellular lanosterol compared with the corresponding wild-type. Based on the lipid profiles of wild-type and pdr16Δpdr17Δ cells grown in aerobic and anaerobic conditions, we suggest that PI-lanosterol transfer proteins are important predominantly for the optimal functioning of the post-lanosterol part of sterol biosynthesis.

NLRP3 Inflammasome Priming and Activation Are Regulated by a Phosphatidylinositol-Dependent Mechanism.

Imbalance in lipid homeostasis is associated with discrepancies in immune signaling and is tightly linked to metabolic disorders. The diverse ways in which lipids impact immune signaling, however, remain ambiguous. The phospholipid phosphatidylinositol (PI), which is implicated in numerous immune disorders, is chiefly defined by its phosphorylation status. By contrast, the significance of the two fatty acid chains attached to the PI remains unknown. In this study, by using a mass spectrometry-based assay, we demonstrate a role for PI acyl group chains in regulating both the priming and activation steps of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in mouse macrophages. In response to NLRP3 stimuli, cells deficient in ABC transporter ATP Binding Cassette Subfamily B Member 1 (ABCB1), which effluxes lipid derivatives, revealed defective inflammasome activation. Mechanistically, Abcb1 deficiency shifted the total PI configuration exhibiting a reduced ratio of short-chain to long-chain PI acyl lipids. Consequently, Abcb1 deficiency initiated the rapid degradation of Toll/IL-1R domain-containing adaptor protein, the TLR adaptor protein that binds PI (4,5)-bisphosphate, resulting in defective TLR-dependent signaling, and thus NLRP3 expression. Moreover, this accompanied increased NLRP3 phosphorylation at the Ser291 position and contributed to blunted inflammasome activation. Exogenously supplementing wild-type cells with linoleic acid (LA), but not arachidonic acid, reconfigured PI acyl chains. Accordingly, LA supplementation increased Toll/IL-1R domain-containing adaptor protein degradation, elevated NLRP3 phosphorylation, and abrogated inflammasome activation. Furthermore, NLRP3 Ser291 phosphorylation was dependent on PGE2-induced protein kinase A signaling because pharmacological inhibition of this pathway in LA-enriched cells dephosphorylated NLRP3. Altogether, our study reveals, to our knowledge, a novel metabolic-inflammatory circuit that contributes to calibrating immune responses.

Mammalian lipids: structure, synthesis and function.

Lipids are essential constituents of cellular membranes. Once regarded merely as structural components, lipids have taken centre stage with the discovery of their roles in cell signalling and in the generation of bioactive metabolites. Lipids regulate many physiological functions of cells and alterations in membrane lipid metabolism are associated with major diseases including cancer, Type II diabetes, cardiovascular disease and immune disorders. Understanding lipid diversity, their synthesis and metabolism to generate signalling molecules will provide insight into the fundamental function of the cell. This review summarises the biosynthesis of the lipids of the mammalian cell; phospholipids, sphingolipids and cholesterol and how lipid diversity is achieved. The fatty acids (FAs) are the main building blocks of lipids and contribute to the diversity. Lipid synthesis is intimately connected to their transport within cells; the contribution by proteins that transport lipids, lipid transport proteins will be described. Cellular lipids are metabolised by phospholipases, lipid kinases and phosphatases to make new bioactive metabolites. These transient bioactive metabolites allow cells to respond to the external environment to maintain cellular health. The function of individual metabolites is also highlighted. Bioactive metabolites can be second messengers, or released to the external medium to regulate other cells. Alternatively, bioactive lipids also provide a platform for reversible recruitment of proteins to membranes using their lipid-binding domains. The wide range of physiological processes in which a specific involvement of lipids has been identified explains the need for lipid diversity present in mammalian cells.

Courier service for phosphatidylinositol: PITPs deliver on demand.

Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPß and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.

Phospholipase C families: Common themes and versatility in physiology and pathology.

Phosphoinositide-specific phospholipase Cs (PLCs) are expressed in all mammalian cells and play critical roles in signal transduction. To obtain a comprehensive understanding of these enzymes in physiology and pathology, a detailed structural, biochemical, cell biological and genetic information is required. In this review, we cover all these aspects to summarize current knowledge of the entire superfamily. The families of PLCs have expanded from 13 enzymes to 16 with the identification of the atypical PLCs in the human genome. Recent structural insights highlight the common themes that cover not only the substrate catalysis but also the mechanisms of activation. This involves the release of autoinhibitory interactions that, in the absence of stimulation, maintain classical PLC enzymes in their inactive forms. Studies of individual PLCs provide a rich repertoire of PLC function in different physiologies. Furthermore, the genetic studies discovered numerous mutated and rare variants of PLC enzymes and their link to human disease development, greatly expanding our understanding of their roles in diverse pathologies. Notably, substantial evidence now supports involvement of different PLC isoforms in the development of specific cancer types, immune disorders and neurodegeneration. These advances will stimulate the generation of new drugs that target PLC enzymes, and will therefore open new possibilities for treatment of a number of diseases where current therapies remain ineffective.

Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane.

Phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) has become a major focus in biochemistry, cell biology and physiology owing to its diverse functions at the plasma membrane. As a result, the functions of PI(4,5)P2 can be explored in two separate and distinct roles - as a substrate for phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) and as a primary messenger, each having unique properties. Thus PI(4,5)P2 makes contributions in both signal transduction and cellular processes including actin cytoskeleton dynamics, membrane dynamics and ion channel regulation. Signalling through plasma membrane G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immune receptors all use PI(4,5)P2 as a substrate to make second messengers. Activation of PI3K generates PI(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate), a lipid that recruits a plethora of proteins with pleckstrin homology (PH) domains to the plasma membrane to regulate multiple aspects of cellular function. In contrast, PLC activation results in the hydrolysis of PI(4,5)P2 to generate the second messengers, diacylglycerol (DAG), an activator of protein kinase C and inositol(1,4,5)trisphosphate (IP3/I(1,4,5)P3) which facilitates an increase in intracellular Ca2+. Decreases in PI(4,5)P2 by PLC also impact on functions that are dependent on the intact lipid and therefore endocytosis, actin dynamics and ion channel regulation are subject to control. Spatial organisation of PI(4,5)P2 in nanodomains at the membrane allows for these multiple processes to occur concurrently.

CDP-Diacylglycerol Synthases (CDS): Gateway to Phosphatidylinositol and Cardiolipin Synthesis.

Cytidine diphosphate diacylglycerol (CDP-DAG) is a key intermediate in the synthesis of phosphatidylinositol (PI) and cardiolipin (CL). Both PI and CL have highly specialized roles in cells. PI can be phosphorylated and these phosphorylated derivatives play major roles in signal transduction, membrane traffic, and maintenance of the actin cytoskeletal network. CL is the signature lipid of mitochondria and has a plethora of functions including maintenance of cristae morphology, mitochondrial fission, and fusion and for electron transport chain super complex formation. Both lipids are synthesized in different organelles although they share the common intermediate, CDP-DAG. CDP-DAG is synthesized from phosphatidic acid (PA) and CTP by enzymes that display CDP-DAG synthase activities. Two families of enzymes, CDS and TAMM41, which bear no sequence or structural relationship, have now been identified. TAMM41 is a peripheral membrane protein localized in the inner mitochondrial membrane required for CL synthesis. CDS enzymes are ancient integral membrane proteins found in all three domains of life. In mammals, they provide CDP-DAG for PI synthesis and for phosphatidylglycerol (PG) and CL synthesis in prokaryotes. CDS enzymes are critical for maintaining phosphoinositide levels during phospholipase C (PLC) signaling. Hydrolysis of PI (4,5) bisphosphate by PLC requires the resynthesis of PI and CDS enzymes catalyze the rate-limiting step in the process. In mammals, the protein products of two CDS genes (CDS1 and CDS2) localize to the ER and it is suggested that CDS2 is the major CDS for this process. Expression of CDS enzymes are regulated by transcription factors and CDS enzymes may also contribute to CL synthesis in mitochondria. Studies of CDS enzymes in protozoa reveal spatial segregation of CDS enzymes from the rest of the machinery required for both PI and CL synthesis identifying a key gap in our understanding of how CDP-DAG can cross the different membrane compartments in protozoa and in mammals.

Phosphatidylinositol synthesis at the endoplasmic reticulum.

Phosphatidylinositol (PI) is a minor phospholipid with a characteristic fatty acid profile; it is highly enriched in stearic acid at the sn-1 position and arachidonic acid at the sn-2 position. PI is phosphorylated into seven specific derivatives, and individual species are involved in a vast array of cellular functions including signalling, membrane traffic, ion channel regulation and actin dynamics. De novo PI synthesis takes place at the endoplasmic reticulum where phosphatidic acid (PA) is converted to PI in two enzymatic steps. PA is also produced at the plasma membrane during phospholipase C signalling, where hydrolysis of phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) leads to the production of diacylglycerol which is rapidly phosphorylated to PA. This PA is transferred to the ER to be also recycled back to PI. For the synthesis of PI, CDP-diacylglycerol synthase (CDS) converts PA to the intermediate, CDP-DG, which is then used by PI synthase to make PI. The de novo synthesised PI undergoes remodelling to acquire its characteristic fatty acid profile, which is altered in p53-mutated cancer cells. In mammals, there are two CDS enzymes at the ER, CDS1 and CDS2. In this review, we summarise the de novo synthesis of PI at the ER and the enzymes involved in its subsequent remodelling to acquire its characteristic acyl chains. We discuss how CDS, the rate limiting enzymes in PI synthesis are regulated by different mechanisms. During phospholipase C signalling, the CDS1 enzyme is specifically upregulated by cFos via protein kinase C.

Yeast phosphatidylinositol transfer protein Pdr17 does not require high affinity phosphatidylinositol binding for its cellular function.

Yeast phosphatidylinositol transfer protein (PITP) Pdr17 is an essential component of the complex required for decarboxylation of phosphatidylserine (PS) to phosphatidylethanolamine (PE) at a non-mitochondrial location. According to current understanding, this process involves the transfer of PS from the endoplasmic reticulum to the Golgi/endosomes. We generated a Pdr17E237A, K269A mutant protein to better understand the mechanism by which Pdr17p participates in the processes connected to the decarboxylation of PS to PE. We show that the Pdr17E237A, K269A mutant protein is not capable of binding phosphatidylinositol (PI) using permeabilized human cells, but still retains the ability to transfer PI between two membrane compartments in vitro. We provide data together with molecular models showing that the mutations E237A and K269A changed only the lipid binding cavity of Pdr17p and not its surface properties. In contrast to Pdr16p, a close homologue, the ability of Pdr17p to bind PI is not required for its major cellular function in the inter-membrane transfer of PS. We hypothesize that these two closely related yeast PITPs, Pdr16p and Pdr17p, have evolved from a common ancestor. Pdr16p fulfills those role(s) in which the ability to bind and transfer PI is required, while Pdr17p appears to have adapted to a different role which does not require the high affinity binding of PI, although the protein retains the capacity to transfer PI. Our results indicate that PITPs function in complex ways in vivo and underscore the need to consider multiple PITP parameters when studying these proteins in vitro.

Sustained phospholipase C stimulation of H9c2 cardiomyoblasts by vasopressin induces an increase in CDP-diacylglycerol synthase 1 (CDS1) through protein kinase C and cFos.

Chronic stimulation (24 h) with vasopressin leads to hypertrophy in H9c2 cardiomyoblasts and this is accompanied by continuous activation of phospholipase C. Consequently, vasopressin stimulation leads to a depletion of phosphatidylinositol levels. The substrate for phospholipase C is phosphatidylinositol (4, 5) bisphosphate (PIP2) and resynthesis of phosphatidylinositol and its subsequent phosphorylation maintains the supply of PIP2. The resynthesis of PI requires the conversion of phosphatidic acid to CDP-diacylglycerol catalysed by CDP-diacylglycerol synthase (CDS) enzymes. To examine whether the resynthesis of PI is regulated by vasopressin stimulation, we focussed on the CDS enzymes. Three CDS enzymes are present in mammalian cells: CDS1 and CDS2 are integral membrane proteins localised at the endoplasmic reticulum and TAMM41 is a peripheral protein localised in the mitochondria. Vasopressin selectively stimulates an increase CDS1 mRNA that is dependent on protein kinase C, and can be inhibited by the AP-1 inhibitor, T-5224. Vasopressin also stimulates an increase in cFos protein which is inhibited by a protein kinase C inhibitor. We conclude that vasopressin stimulates CDS1 mRNA through phospholipase C, protein kinase C and cFos and provides a potential mechanism for maintenance of phosphatidylinositol levels during long-term phospholipase C signalling.

Pitpnc1a Regulates Zebrafish Sleep and Wake Behavior through Modulation of Insulin-like Growth Factor Signaling.

The lipid transporters of the phosphatidylinositol transfer protein (PITP) family dictate phosphoinositide compartmentalization, and specific phosphoinositides play crucial roles in signaling cascades, membrane traffic, ion channel regulation, and actin dynamics. Although PITPs are enriched in the brain, their physiological functions in neuronal signaling pathways in vivo remain ill defined. We describe a CRISPR/Cas9-generated zebrafish mutant in a brain-specific, conserved class II PITP member, pitpnc1a. Zebrafish pitpnc1a mutants are healthy but display widespread aberrant neuronal activity and increased wakefulness across the day-night cycle. The loss of Pitpnc1a increases insulin-like growth factor (IGF) signaling in the brain, and inhibition of IGF pathways is sufficient to rescue both neuronal and behavioral hyperactivity in pitpnc1a mutants. We propose that Pitpnc1a-expressing neurons alter behavior via modification of neuro-modulatory IGF that acts on downstream wake-promoting circuits.

Phospholipid transport protein function at organelle contact sites.

Phospholipids are synthesized at the endoplasmic reticulum (ER), the largest membrane bound organelle that forms membrane contact sites (MCS) with almost every other organelle. MCS are locations at which the membrane es of two organelles are closely positioned to provide a microenvironment where proteins in one membrane can interact with the opposite membrane. Thus, MCS provide an ideal location at which lipid transfer proteins (LTPs) can achieve the efficient transfer of individual classes of lipids from the ER to other organelles via non-vesicular transport. Here we provide an overview of emerging findings on the localization and biochemical activity of LTPs at MCS between the ER and other cellular membranes. The localization of LTPs at MCS offers an elegant cell biological solution to tune local lipid composition to ongoing cell physiology.

Mitochondrial CDP-diacylglycerol synthase activity is due to the peripheral protein, TAMM41 and not due to the integral membrane protein, CDP-diacylglycerol synthase 1.

CDP diacylglycerol synthase (CDS) catalyses the conversion of phosphatidic acid (PA) to CDP-diacylglycerol, an essential intermediate in the synthesis of phosphatidylglycerol, cardiolipin and phosphatidylinositol (PI). CDS activity has been identified in mitochondria and endoplasmic reticulum of mammalian cells apparently encoded by two highly-related genes, CDS1 and CDS2. Cardiolipin is exclusively synthesised in mitochondria and recent studies in cardiomyocytes suggest that the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1α and ß) serve as transcriptional regulators of mitochondrial biogenesis and up-regulate the transcription of the CDS1 gene. Here we have examined whether CDS1 is responsible for the mitochondrial CDS activity. We report that differentiation of H9c2 cells with retinoic acid towards cardiomyocytes is accompanied by increased expression of mitochondrial proteins, oxygen consumption, and expression of the PA/PI binding protein, PITPNC1, and CDS1 immunoreactivity. Both CDS1 immunoreactivity and CDS activity were found in mitochondria of H9c2 cells as well as in rat heart, liver and brain mitochondria. However, the CDS1 immunoreactivity was traced to a peripheral p55 cross-reactive mitochondrial protein and the mitochondrial CDS activity was due to a peripheral mitochondrial protein, TAMM41, not an integral membrane protein as expected for CDS1. TAMM41 is the mammalian equivalent of the recently identified yeast protein, Tam41. Knockdown of TAMM41 resulted in decreased mitochondrial CDS activity, decreased cardiolipin levels and a decrease in oxygen consumption. We conclude that the CDS activity present in mitochondria is mainly due to TAMM41, which is required for normal mitochondrial function.

Phosphatidylinositol transfer protein-α in platelets is inconsequential for thrombosis yet is utilized for tumor metastasis.

Platelets are increasingly recognized for their contributions to tumor metastasis. Here, we show that the phosphoinositide signaling modulated by phosphatidylinositol transfer protein type α (PITPα), a protein which shuttles phosphatidylinositol between organelles, is essential for platelet-mediated tumor metastasis. PITPα-deficient platelets have reduced intracellular pools of phosphoinositides and an 80% reduction in IP3 generation upon platelet activation. Unexpectedly, mice lacking platelet PITPα form thrombi normally at sites of intravascular injuries. However, following intravenous injection of tumor cells, mice lacking PITPα develop fewer lung metastases due to a reduction of fibrin formation surrounding the tumor cells, rendering the metastases susceptible to mucosal immunity. These findings demonstrate that platelet PITPα-mediated phosphoinositide signaling is inconsequential for in vivo hemostasis, yet is critical for in vivo dissemination. Moreover, this demonstrates that signaling pathways within platelets may be segregated into pathways that are essential for thrombosis formation and pathways that are important for non-hemostatic functions.