Seipin accumulates and traps diacylglycerols and triglycerides in its ring-like structure.

Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein-TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis.

A single point mutation in the TRPC3 lipid-recognition window generates supersensitivity to benzimidazole channel activators.

Mutation of a single residue within the recently identified lipid (diacylglycerol) recognition window of TRPC3 (G652A) was found to abolish channel activation via endogenous lipid mediators while retaining sensitivity to the non-lipid activator GSK1702934A (abb. GSK). The mechanism of this change in chemical sensing by TRPC3 was analysed by whole-cell and single channel electrophysiology as well as Ca2+ imaging. Currents initiated by GSK or the structural (benzimidazole) analog BI-2 were significantly larger in cells expressing the G652A mutant as compared to wild type (WT) channels. Whole cell patch-clamp experiments revealed that enhanced sensitivity to benzimidazoles was not due to augmented potency but reflected enhanced efficacy of benzimidazoles. Single channel analysis demonstrated that neither unitary conductance nor I-V characteristics were altered by the G652A mutation, precluding altered pore architecture as the basis of enhanced efficacy. These experiments uncovered a distinct gating pattern of BI-2-activated G652A mutant channels, featuring a unique, long-lived open state. Moreover, G652A mutant channels lacked PLC/diacylglycerol mediated cross-desensitization for GSK activation as typically observed for TRPC3. Lack of desensitization in G652A channels enabled large GSK/BI-2-induced Ca2+ signals in conditions that fully desensitized TRPC3 WT channels. We demonstrate that the lipid-recognition window of TRPC3 determines both sensitivity to lipid mediators and chemical gating by benzimidazoles. TRPC3 mutations within this lipid interaction site are suggested as a basis for chemogenetic targeting of TRPC3-signaling.

A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study.

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.

The glycerol-3-phosphate acyltransferase PLAT2 functions in the generation of DHA-rich glycerolipids in Aurantiochytrium limacinum F26-b.

Thraustochytrids possess docosahexaenoic acid (DHA, 22:6n-3) as acyl chain(s) of triacylglycerol (TG) and phosphatidylcholine (PC), some of which contain multiple DHAs. However, little is known about how these DHA-rich glycerolipids are produced in thraustochytrids. In this study, we identified PLAT2 in Aurantiochytrium limacinum F26-b as a glycerol-3-phosphate (G3P) acyltransferase (GPAT) by heterologous expression of the gene in budding yeast. Subsequently, we found that GPAT activity was reduced by disruption of the PLAT2 gene in A. limacinum, resulting in a decrease in DHA-containing lysophosphatidic acid (LPA 22:6). Conversely, overexpression of PLAT2 increased both GPAT activity and LPA 22:6. These results indicate that PLAT2 is a GPAT that transfers DHA to G3P in vivo as well as in vitro. Overexpression of the PLAT2 gene increased the production of a two DHA-containing diacylglycerol (DG 44:12), followed by an increase in the three DHA-containing TG (TG 66:18), two-DHA-containing TG (TG 60:12), and two DHA-containing PC (PC 44:12). However, overexpression of PLAT2 did not increase DHA-free DG (DG32:0), which was preferentially converted to three 16:0-containing TG (TG 48:0) but not two 16:0-containing PC (PC 32:0). Collectively, we revealed that DHA-rich glycerolipids are produced from a precursor, LPA 22:6, which is generated by incorporating DHA to G3P by PLAT2 in the A. limacinum.

Of local translation control and lipid signaling in neurons.

Fine-tuned regulation of new proteins synthesis is key to the fast adaptation of cells to their changing environment and their response to external cues. Protein synthesis regulation is particularly refined and important in the case of highly polarized cells like neurons where translation occurs in the subcellular dendritic compartment to produce long-lasting changes that enable the formation, strengthening and weakening of inter-neuronal connection, constituting synaptic plasticity. The changes in local synaptic proteome of neurons underlie several aspects of synaptic plasticity and new protein synthesis is necessary for long-term memory formation. Details of how neuronal translation is locally controlled only start to be unraveled. A generally accepted view is that mRNAs are transported in a repressed state and are translated locally upon externally cued triggering signaling cascades that derepress or activate translation machinery at specific sites. Some important yet poorly considered intermediates in these cascades of events are signaling lipids such as diacylglycerol and its balancing partner phosphatidic acid. A link between these signaling lipids and the most common inherited cause of intellectual disability, Fragile X syndrome, is emphasizing the important role of these secondary messages in synaptically controlled translation.

Diacylglycerol kinases: Relationship to other lipid kinases.

Lipid kinases regulate a wide variety of cellular functions and have emerged as one the most promising targets for drug design. Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PtdOH). Despite the critical role in lipid biosynthesis, both DAG and PtdOH have been shown as bioactive lipids mediating a number of signaling pathways. Although there is increasing recognition of their role in signaling systems, our understanding of the key enzyme which regulate the balance of these two lipid messages remain limited. Solved structures provide a wealth of information for understanding the function and regulation of these enzymes. Solving the structures of mammalian DGKs by traditional NMR and X-ray crystallography approaches have been challenging and so far, there are still no three-dimensional structures of these DGKs. Despite this, some insights may be gained by examining the similarities and differences between prokaryotic DGKs and other mammalian lipid kinases. This review focuses on summarizing and comparing the structure of prokaryotic and mammalian DGKs as well as two other lipid kinases: sphingosine kinase and phosphatidylinositol-3-kinase. How these known lipid kinases structures relate to mammalian DGKs will also be discussed.

Thylakoid membrane lipid sulfoquinovosyl-diacylglycerol (SQDG) is required for full functioning of photosystem II in Thermosynechococcus elongatus.

Sulfoquinovosyl-diacylglycerol (SQDG) is one of the four lipids present in the thylakoid membranes. Depletion of SQDG causes different degrees of effects on photosynthetic growth and activities in different organisms. Four SQDG molecules bind to each monomer of photosystem II (PSII), but their role in PSII function has not been characterized in detail, and no PSII structure without SQDG has been reported. We analyzed the activities of PSII from an SQDG-deficient mutant of the cyanobacterium Thermosynechococcus elongatus by various spectroscopic methods, which showed that depletion of SQDG partially impaired the PSII activity by impairing secondary quinone (QB) exchange at the acceptor site. We further solved the crystal structure of the PSII dimer from the SQDG deletion mutant at 2.1 Å resolution and found that all of the four SQDG-binding sites were occupied by other lipids, most likely PG molecules. Replacement of SQDG at a site near the head of QB provides a possible explanation for the QB impairment. The replacement of two SQDGs located at the monomer-monomer interface by other lipids decreased the stability of the PSII dimer, resulting in an increase in the amount of PSII monomer in the mutant. The present results thus suggest that although SQDG binding in all of the PSII-binding sites is necessary to fully maintain the activity and stability of PSII, replacement of SQDG by other lipids can partially compensate for their functions.

DIACYLGLYCEROL ACYLTRANSFERASE and DIACYLGLYCEROL KINASE Modulate Triacylglycerol and Phosphatidic Acid Production in the Plant Response to Freezing Stress.

Plants accumulate the lipids phosphatidic acid (PA), diacylglycerol (DAG), and triacylglycerol (TAG) during cold stress, but how plants balance the levels of these lipids to mediate cold responses remains unknown. The enzymes ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) and DIACYLGLYCEROL KINASE (DGK) catalyze the conversion of DAG to TAG and PA, respectively. Here, we show that DGAT1, DGK2, DGK3, and DGK5 contribute to the response to cold in Arabidopsis (Arabidopsis thaliana). With or without cold acclimation, the dgat1 mutants exhibited higher sensitivity upon freezing exposure compared with the wild type. Under cold conditions, the dgat1 mutants showed reduced expression of C-REPEAT/DRE BINDING FACTOR2 and its regulons, which are essential for the acquisition of cold tolerance. Lipid profiling revealed that freezing significantly increased the levels of PA and DAG while decreasing TAG in the rosettes of dgat1 mutant plants. During freezing stress, the accumulation of PA in dgat1 plants stimulated NADPH oxidase activity and enhanced RbohD-dependent hydrogen peroxide production compared with the wild type. Moreover, the cold-inducible transcripts of DGK2, DGK3, and DGK5 were significantly more up-regulated in the dgat1 mutants than in the wild type during cold stress. Consistent with this observation, dgk2, dgk3, and dgk5 knockout mutants showed improved tolerance and attenuated PA production in response to freezing temperatures. Our findings demonstrate that the conversion of DAG to TAG by DGAT1 is critical for plant freezing tolerance, acting by balancing TAG and PA production in Arabidopsis.

Where do substrates of diacylglycerol kinases come from? Diacylglycerol kinases utilize diacylglycerol species supplied from phosphatidylinositol turnover-independent pathways.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprises ten isozymes (α-κ) and regulates a wide variety of physiological and pathological events, such as cancer, type II diabetes, neuronal disorders and immune responses. DG and PA consist of various molecular species that have different acyl chains at the sn-1 and sn-2 positions, and consequently, mammalian cells contain at least 50 structurally distinct DG/PA species. Because DGK is one of the components of phosphatidylinositol (PI) turnover, the generally accepted dogma is that all DGK isozymes utilize 18:0/20:4-DG derived from PI turnover. We recently established a specific liquid chromatography-mass spectrometry method to analyze which PA species were generated by DGK isozymes in a cell stimulation-dependent manner. Interestingly, we determined that DGKδ, which is closely related to the pathogenesis of type II diabetes, preferentially utilized 14:0/16:0-, 14:0/16:1-, 16:0/16:0-, 16:0/16:1-, 16:0/18:0- and 16:0/18:1-DG species (X:Y = the total number of carbon atoms: the total number of double bonds) supplied from the phosphatidylcholine-specific phospholipase C pathway, but not 18:0/20:4-DG, in high glucose-stimulated C2C12 myoblasts. Moreover, DGKα mainly consumed 14:0/16:0-, 16:0/18:1-, 18:0/18:1- and 18:1/18:1-DG species during cell proliferation in AKI melanoma cells. Furthermore, we found that 16:0/16:0-PA was specifically produced by DGKζ in Neuro-2a cells during retinoic acid- and serum starvation-induced neuronal differentiation. These results indicate that DGK isozymes utilize a variety of DG molecular species derived from PI turnover-independent pathways as substrates in different stimuli and cells. DGK isozymes phosphorylate various DG species to generate various PA species. It was revealed that the modes of activation of conventional and novel protein kinase isoforms by DG molecular species varied considerably. However, PA species-selective binding proteins have not been found to date. Therefore, we next attempted to identify PA species-selective binding proteins from the mouse brain and identified α-synuclein, which has causal links to Parkinson's disease. Intriguingly, we determined that among phospholipids, including several PA species (16:0/16:0-PA, 16:0/18:1-PA, 18:1/18:1-PA, 18:0/18:0-PA and 18:0/20:4-PA); 18:1/18:1-PA was the most strongly bound PA to α-synuclein. Moreover, 18:1/18:1-PA strongly enhanced secondary structural changes from the random coil form to the α-helix form and generated a multimeric and proteinase K-resistant α-synuclein protein. In contrast with the dogma described above, our recent studies strongly suggest that PI turnover-derived DG species and also various DG species derived from PI turnover-independent pathways are utilized by DGK isozymes. DG species supplied from distinct pathways may be utilized by DGK isozymes based on different stimuli present in different types of cells, and individual PA molecular species would have specific targets and exert their own physiological functions.

Mechanism by which arylamine N-acetyltransferase 1 ablation causes insulin resistance in mice.

A single-nucleotide polymorphism in the human arylamine N-acetyltransferase 2 (Nat2) gene has recently been identified as associated with insulin resistance in humans. To understand the cellular and molecular mechanisms by which alterations in Nat2 activity might cause insulin resistance, we examined murine ortholog Nat1 knockout (KO) mice. Nat1 KO mice manifested whole-body insulin resistance, which could be attributed to reduced muscle, liver, and adipose tissue insulin sensitivity. Hepatic and muscle insulin resistance were associated with marked increases in both liver and muscle triglyceride (TAG) and diacylglycerol (DAG) content, which was associated with increased PKCε activation in liver and increased PKCθ activation in skeletal muscle. Nat1 KO mice also displayed reduced whole-body energy expenditure and reduced mitochondrial oxygen consumption in white adipose tissue, brown adipose tissue, and hepatocytes. Taken together, these studies demonstrate that Nat1 deletion promotes reduced mitochondrial activity and is associated with ectopic lipid-induced insulin resistance. These results provide a potential genetic link among mitochondrial dysfunction with increased ectopic lipid deposition, insulin resistance, and type 2 diabetes.

Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of chronic kidney disease. Here, we identified recessive mutations in the gene encoding the actin-binding protein advillin (AVIL) in 3 unrelated families with SRNS. While all AVIL mutations resulted in a marked loss of its actin-bundling ability, truncation of AVIL also disrupted colocalization with F-actin, thereby leading to impaired actin binding and severing. Additionally, AVIL colocalized and interacted with the phospholipase enzyme PLCE1 and with the ARP2/3 actin-modulating complex. Knockdown of AVIL in human podocytes reduced actin stress fibers at the cell periphery, prevented recruitment of PLCE1 to the ARP3-rich lamellipodia, blocked EGF-induced generation of diacylglycerol (DAG) by PLCE1, and attenuated the podocyte migration rate (PMR). These effects were reversed by overexpression of WT AVIL but not by overexpression of any of the 3 patient-derived AVIL mutants. The PMR was increased by overexpression of WT Avil or PLCE1, or by EGF stimulation; however, this increased PMR was ameliorated by inhibition of the ARP2/3 complex, indicating that ARP-dependent lamellipodia formation occurs downstream of AVIL and PLCE1 function. Together, these results delineate a comprehensive pathogenic axis of SRNS that integrates loss of AVIL function with alterations in the action of PLCE1, an established SRNS protein.

Steroidogenic acute regulatory protein (StAR) overexpression attenuates HFD-induced hepatic steatosis and insulin resistance.

Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology. Intracellular lipid accumulation is the first step in the development and progression of NAFLD. Steroidogenic acute regulatory protein (StAR) plays an important role in the synthesis of bile acid and intracellular lipid homeostasis and cholesterol metabolism. We hypothesize that StAR is involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. The hypothesis was identified using free fatty acid (FFA)-overloaded NAFLD in vitro model and high-fat diet (HFD)-induced NAFLD mouse model transfected by recombinant adenovirus encoding StAR (StAR). StAR expression was also examined in pathology samples of patients with fatty liver by immunohistochemical staining. We found that the expression level of StAR was reduced in the livers obtained from fatty liver patients and NAFLD mice. Additionally, StAR overexpression decreased the levels of hepatic lipids and maintained the hepatic glucose homeostasis due to the activation of farnesoid x receptor (FXR). StAR overexpression attenuated the impairment of insulin signaling in fatty liver. This protective role of StAR was owing to a reduction of intracellular diacylglycerol levels and the phosphorylation of PKCε. Furthermore, FXR inactivation reversed the observed beneficial effects of StAR. The present study revealed that StAR overexpression can reduce hepatic lipid accumulation, regulate glucose metabolism and attenuate insulin resistance through a mechanism involving the activation of FXR. Our study suggests that StAR may be a potential therapeutic target for NAFLD.

Phospholipase Cγ1 (PLCγ1) Controls Osteoclast Numbers via Colony-stimulating Factor 1 (CSF-1)-dependent Diacylglycerol/ß-Catenin/CyclinD1 Pathway.

Phospholipases Cγ (PLCγ) 1 and 2 are a class of highly homologous enzymes modulating a variety of cellular pathways through production of inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Our previous studies demonstrated the importance of PLCγ2 in osteoclast (OC) differentiation by modulating inositol 1,4,5-trisphosphate-mediated calcium oscillations and the up-regulation of the transcription factor NFATc1. Surprisingly, despite being expressed throughout osteoclastogenesis, PLCγ1 did not compensate for PLCγ2 deficiency. Because both isoforms are activated during osteoclastogenesis, it is plausible that PLCγ1 modulates OC development independently of PLCγ2. Here, we utilized PLCγ1-specific shRNAs to delete PLCγ1 in OC precursors derived from wild type (WT) mice. Differently from PLCγ2, we found that PLCγ1 shRNA significantly suppresses OC differentiation by limiting colony-stimulating factor 1 (CSF-1)-dependent proliferation and ß-catenin/cyclinD1 levels. Confirming the specificity toward CSF-1 signaling, PLCγ1 is recruited to the CSF-1 receptor following exposure to the cytokine. To understand how PLCγ1 controls cell proliferation, we turned to its downstream effector, DAG. By utilizing cells lacking the DAG kinase ζ, which have increased DAG levels, we demonstrate that DAG modulates CSF-1-dependent proliferation and ß-catenin/cyclinD1 levels in OC precursors. Most importantly, the proliferation and osteoclastogenesis defects observed in the absence of PLCγ1 are normalized in PLCγ1/DAG kinase ζ double null cells. Taken together, our study shows that PLCγ1 controls OC numbers via a CSF-1-dependent DAG/ß-catenin/cyclinD1 pathway.

Diacylglycerol kinase ζ limits the polarized recruitment of diacylglycerol-enriched organelles to the immune synapse in T cells.

The antigen-induced formation of an immune synapse (IS) between T cells and antigen-presenting cells results in the rapid generation of the lipid second messenger diacylglycerol (DAG) in T cells. Diacylglycerol kinase ζ (DGKζ) converts DAG into phosphatidic acid (PA). Cytotoxic T lymphocytes (CTLs) from mice deficient in DGKζ have enhanced antiviral and antitumor activities, indicating that the amount of DAG controls the effectiveness of the T cell response. We characterized the second C1 domain of protein kinase Cθ (PKCθ), a DAG-binding protein that is specifically recruited to the IS, as a biological sensor to observe the generation of a DAG gradient during IS formation. In experiments with transgenic mouse CTLs expressing the OT-I T cell receptor (TCR), we showed that both strong and weak interactions between antigen and the TCR led to the rapid generation of DAG, whereas only strong interactions induced the movement of DAG-enriched organelles toward the IS. In DGKζ-deficient CTLs, antigen stimulation led to the enhanced accumulation of DAG-containing organelles at the IS; however, impaired activation of the PA effector PKCζ resulted in lack of reorientation of the microtubule-organizing center toward the IS, a process needed for effective T cell activation. Together, these data suggest that the activation of DGKζ downstream of antigen recognition provides a mechanism that ensures the activation of PA-dependent signaling as a direct result of the strength of TCR-dependent DAG mobilization.

Residue Asn277 affects the stability and substrate specificity of the SMG1 lipase from Malassezia globosa.

Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273-278 and the 3104 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.

Diacylglycerol, phosphatidic acid, and their metabolic enzymes in synaptic vesicle recycling.

The synaptic vesicle (SV) cycle includes exocytosis of vesicles loaded with a neurotransmitter such as glutamate, coordinated recovery of SVs by endocytosis, refilling of vesicles, and subsequent release of the refilled vesicles from the presynaptic bouton. SV exocytosis is tightly linked with endocytosis, and variations in the number of vesicles, and/or defects in the refilling of SVs, will affect the amount of neurotransmitter available for release (Sudhof, 2004). There is increasing interest in the roles synaptic vesicle lipids and lipid metabolizing enzymes play in this recycling. Initial emphasis was placed on the role of polyphosphoinositides in SV cycling as outlined in a number of reviews (Lim and Wenk, 2009; Martin, 2012; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Other lipids are now recognized to also play critical roles. For example, PLD1 (Humeau et al., 2001; Rohrbough and Broadie, 2005) and some DGKs (Miller et al., 1999; Nurrish et al., 1999) play roles in neurotransmission which is consistent with the critical roles for phosphatidic acid (PtdOH) and diacylglycerol (DAG) in the regulation of SV exo/endocytosis (Cremona et al., 1999; Exton, 1994; Huttner and Schmidt, 2000; Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). PLD generates phosphatidic acid by catalyzing the hydrolysis of phosphatidylcholine (PtdCho) and in some systems this PtdOH is de-phosphorylated to generate DAG. In contrast, DGK catalyzes the phosphorylation of DAG thereby converting it into PtdOH. While both enzymes are poised to regulate the levels of DAG and PtdOH, therefore, they both lead to the generation of PtdOH and could have opposite effects on DAG levels. This is particularly important for SV cycling as PtdOH and DAG are both needed for evoked exocytosis (Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Two lipids and their involved metabolic enzymes, two sphingolipids have also been implicated in exocytosis: sphingosine (Camoletto et al., 2009; Chan et al., 2012; Chan and Sieburth, 2012; Darios et al., 2009; Kanno et al., 2010; Rohrbough et al., 2004) and sphingosine-1-phosphate (Chan, Hu, 2012; Chan and Sieburth, 2012; Kanno et al., 2010). Finally a number of reports have focused on the somewhat less well studies roles of sphingolipids and cholesterol in SV cycling. In this report, we review the recent understanding of the roles PLDs, DGKs, and DAG lipases, as well as sphingolipids and cholesterol play in synaptic vesicle cycling.

Yeast Pah1p phosphatidate phosphatase is regulated by proteasome-mediated degradation.

Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.

Loss of ß-carotene 15,15'-oxygenase in developing mouse tissues alters esterification of retinol, cholesterol and diacylglycerols.

We provide novel insights into the function(s) of ß-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving ß-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue.

Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.

Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

Diacylglycerol kinases terminate diacylglycerol signaling during the respiratory burst leading to heterogeneous phagosomal NADPH oxidase activation.

It is commonly assumed that all phagosomes have identical molecular composition. This assumption has remained largely unchallenged due to a paucity of methods to distinguish individual phagosomes. We devised an assay that extends the utility of nitro blue tetrazolium for detection and quantification of NAPDH oxidase (NOX) activity in individual phagosomes. Implementation of this assay revealed that in murine macrophages there is heterogeneity in the ability of individual phagosomes to generate superoxide, both between and within cells. To elucidate the molecular basis of the variability in NOX activation, we employed genetically encoded fluorescent biosensors to evaluate the uniformity in the distribution of phospholipid mediators of the oxidative response. Despite variability in superoxide generation, the distribution of phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, and phosphatidic acid was nearly identical in all phagosomes. In contrast, diacylglycerol (DAG) was not generated uniformly across the phagosomal population, varying in a manner that directly mirrored superoxide production. Modulation of DAG levels suggested that NOX activation is precluded when phagosomes fail to reach a critical DAG concentration. In particular, forced expression of diacylglycerol kinase ß abrogated DAG accumulation at the phagosome, leading to impaired respiratory burst. Conversely, pharmacological inhibition of DAG kinases or expression of an inactive diacylglycerol kinase ß mutant increased the proportion of DAG-positive phagosomes, concomitantly potentiating phagosomal NOX activity. Our data suggest that diacylglycerol kinases limit the extent of NADPH oxidase activation, curtailing the production of potentially harmful reactive oxygen species. The resulting heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms.