The leukotriene B4 receptors BLT1 and BLT2 as potential therapeutic targets.

Leukotriene B4 (LTB4 ) was recognized as an arachidonate-derived chemotactic factor for inflammatory cells and an important drug target even before the molecular identification of its receptors. We cloned the high- and low-affinity LTB4 receptors, BLT1 and BLT2, respectively, and examined their functions by generating and studying gene-targeted mice. BLT1 is involved in the pathogenesis of various inflammatory and immune diseases, including asthma, psoriasis, contact dermatitis, allergic conjunctivitis, age-related macular degeneration, and immune complex-mediated glomerulonephritis. Meanwhile, BLT2 is a high-affinity receptor for 12-hydroxyheptadecatrienoic acid, which is involved in the maintenance of dermal and intestinal barrier function, and the acceleration of skin and corneal wound healing. Thus, BLT1 antagonists and BLT2 agonists are promising candidates in the treatment of inflammatory diseases.

Cell-trafficking impairment in disease-associated LPA6 missense mutants and a potential pharmacoperone therapy for autosomal recessive woolly hair/hypotrichosis.

In human autosomal recessive woolly hair/hypotrichosis (ARWH/HT), many mutations have been identified in a gene encoding LPA6, a G protein-coupled receptor (GPCR) for lysophosphatidic acid (LPA). However, information regarding the effects of such mutations on receptor function is limited. In this study, we examined functional impacts of selected amino acid changes in LPA6 identified in ARWH/HT patients. In our exogenous expression experiments, all mutants except S3T failed to respond to LPA, indicating that they are loss-of-function mutants. Among the nine mutants, five (D63V, G146R, N246D, L277P and C278Y) displayed impaired expression at the cell surface because of endoplasmic reticulum (ER) retention, indicating that these mutants are trafficking-defective, as reported in other disease-associated GPCRs. Notably, alkyl-OMPT, a potent synthetic agonist for LPA6 restored the defective cell surface expression of two of the ER-retained mutants, D63V and N246D, possibly by its chaperoning function that allows them to escape intracellular retention as well as proteasomal degradation. Furthermore, the alkyl-OMPT-rescued N246D mutant was shown be functional. Our findings encourage future application of pharmacoperone therapy for ARWH/HT patients with specific LPA6 mutations.

Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.

LPCAT3/LPLAT12 deficiency in the liver ameliorates acetaminophen-induced acute liver injury.

Acetaminophen (APAP) is a double-edged sword, mainly depending on the dosage. A moderate dose of APAP is effective for fever and pain relief; however, an overdose induces acute liver injury. The mechanism underlying APAP-induced acute liver failure is unclear, and its treatment is limited. A recent report has shown that several oxidized phospholipids are associated with APAP-induced acute liver failure. Lysophosphatidylcholine acyltransferase 3 (Lpcat3, Lplat12), which is highly expressed in the liver, preferentially catalyzes the incorporation of arachidonate into lysophospholipids (PLs). In the present study, we investigated the roles of Lpcat3 on APAP-induced acute liver injury using liver-specific Lpcat3-knockout mice. Hepatic Lpcat3 deficiency reduced the degree of APAP-induced necrosis of hepatocytes around Zone 3 and ameliorated the elevation of hepatic injury serum marker levels, and prolonged survival. Lipidomic analysis showed that the accumulation of oxidized and hydroperoxidized phospholipids was suppressed in Lpcat3-knockout mice. The amelioration of APAP-induced acute liver injury was due not only to the reduction in the lipid synthesis of arachidonic acid PLs because of Lpcat3 deficiency, but also to the promotion of the APAP detoxification pathway by facilitating the conjugation of glutathione and N-acetyl-p-benzoquinone imine. Our findings suggest that Lpcat3 is a potential therapeutic target for treating APAP-induced acute liver injury.

Lysophospholipid Acyltransferase 9 Promotes Emphysema Formation via Platelet-activating Factor.

Cigarette smoking is known to be the leading cause of chronic obstructive pulmonary disease (COPD). However, the detailed mechanisms have not been elucidated. PAF (platelet-activating factor), a potent inflammatory mediator, is involved in the pathogenesis of various respiratory diseases such as bronchial asthma and COPD. We focused on LPLAT9 (lysophospholipid acyltransferase 9), a biosynthetic enzyme of PAF, in the pathogenesis of COPD. LPLAT9 gene expression was observed in excised COPD lungs and single-cell RNA sequencing data of alveolar macrophages (AMs). LPLAT9 was predominant and upregulated in AMs, particularly monocyte-derived AMs, in patients with COPD. To identify the function of LPLAT9/PAF in AMs in the pathogenesis of COPD, we exposed systemic LPLAT9-knockout (LPALT9-/-) mice to cigarette smoke (CS). CS increased the number of AMs, especially the monocyte-derived fraction, which secreted MMP12 (matrix metalloprotease 12). Also, CS augmented LPLAT9 phosphorylation/activation on macrophages and, subsequently, PAF synthesis in the lung. The LPLAT9-/- mouse lung showed reduced PAF production after CS exposure. Intratracheal PAF administration accumulated AMs by increasing MCP1 (monocyte chemoattractant protein-1). After CS exposure, AM accumulation and subsequent pulmonary emphysema, a primary pathologic change of COPD, were reduced in LPALT9-/- mice compared with LPLAT9+/+ mice. Notably, these phenotypes were again worsened by LPLAT9+/+ bone marrow transplantation in LPALT9-/- mice. Thus, CS-induced LPLAT9 activation in monocyte-derived AMs aggravated pulmonary emphysema via PAF-induced further accumulation of AMs. These results suggest that PAF synthesized by LPLAT9 has an important role in the pathogenesis of COPD.

LPGAT1/LPLAT7 regulates acyl chain profiles at the sn-1 position of phospholipids in murine skeletal muscles.

Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle.

Stepwise phosphorylation of BLT1 defines complex assemblies with ß-arrestin serving distinct functions.

G protein-coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B4 (LTB4 ), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr308 and Ser310 . Ser310 phosphorylation occurs at a lower LTB4 concentration than Thr308 , leading to a shift in ligand affinity from a high-to-low state. However, the implications of BLT1 phosphorylation in signal transduction processes or the underlying mechanisms have remained unclear. Here, we identify the sequential BLT1-engaged conformations of ß-arrestin and subsequent alterations in signal transduction. Stimulation of the high-affinity BLT1 with LTB4 induces phosphorylation at Ser310 via the ERK1/2-GRK pathway, resulting in a ß-arrestin-bound low-affinity state. This configuration, referred to as the "low-LTB4 -induced complex," necessitates the finger loop region and the phosphoinositide-binding motif of ß-arrestins to interact with BLT1 and deactivates the ERK1/2 signaling. Under high LTB4 concentrations, the low-affinity BLT1 again binds to the ligand and triggers the generation of the low-LTB4 -induced complex into a different form termed "high-LTB4 -induced complex." This change is propelled by The308 -phosphorylation-dependent basal phosphorylation by PKCs. Within the high-LTB4 -induced complex, ß-arrestin adapts a unique configuration that involves additional N domain interaction to the low-affinity BLT1 and stimulates the PI3K/AKT pathway. We propose that the stepwise phosphorylation of BLT1 defines the formation of complex assemblies, wherein ß-arrestins perform distinct functions.

Constitutively active GPR43 is crucial for proper leukocyte differentiation.

The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.

Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells.

Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.

Dietary omega-3 fatty acid does not improve male infertility caused by lysophospholipid acyltransferase 3 (LPLAT3/AGPAT3) deficiency.

Docosahexaenoic acid (DHA), an omega-3 fatty acid, usually presents as a constituent of phospholipids in the cellular membrane. Lysophospholipid acyltransferase 3 (LPLAT3; AGPAT3) is the primary enzyme that incorporates DHA into phospholipids. LPLAT3-KO mice show male infertility and visual dysfunction accompanied by decreased phospholipids (PLs) containing DHA (PL-DHA) in the testis and retina, respectively. In this study, we evaluated the effect of diets consisting mainly of triacylglycerol-bound DHA (fish oil) and PL-bound DHA (salmon roe oil) on the amount of PL-DHA in a broad range of tissues and on reproductive functions. Both diets elevated phosphatidylcholines (PCs)-containing DHA in most tissues of wild type (WT) mice. Although LPLAT3-KO mice acquired a minimal amount of PC-DHA in the testes and sperm by eating either of the diets, reproductive function did not improve. The present study suggests that DHA-rich diets do not restore sufficient PL-DHA to improve male infertility in LPLAT3-KO mice. Alternatively, PL-DHA can be biosynthesized by LPLAT3 but not by external supplementation, which may be necessary for normal reproductive function.

Prophylactic endoscopic gallbladder stent placement for cholecystitis after covered metal stent placement for distal biliary obstruction (with video).

BACKGROUND AND AIMS: Acute cholecystitis is occasionally observed after biliary drainage using a covered self-expandable metal stent (CSEMS) for distal biliary obstruction (DBO). Gallbladder drainage before CSEMS placement may reduce cholecystitis. This study aimed to examine the preventive effect of endoscopic gallbladder stent placement (EGBS) on cholecystitis with CSEMSs. METHODS: We retrospectively analyzed patients with DBO who underwent CSEMS placement across the orifice of the cystic duct between November 2014 and October 2021 and were negative for cholecystitis on biliary drainage. Prophylactic EGBS was attempted before CSEMS placement. The incidence of cholecystitis was compared between patients with and without EGBS. RESULTS: In total, 286 patients (128 men; median age, 75 years) were included in this study. EGBS was attempted in 32 patients before CSEMS placement, and technical success was achieved in 24 patients (75%). Adverse events were noted in 3 patients (9.4%; penetration of cystic duct in 1 and acute pancreatitis in 2). The cumulative incidence of cholecystitis was significantly lower in patients with EGBS than in those without EGBS (1 [4.2%] vs 56 [21.4%], P = .045). In multivariable analysis, EGBS was a significant protective factor against cholecystitis (hazard ratio, .11; 95% confidence interval, .01-.79; P = .028). CONCLUSIONS: Although the transpapillary approach to the gallbladder is not easy for patients with DBO, EGBS is effective in preventing cholecystitis associated with CSEMS placement.

Efficacy of pancreatic duct stenting to prevent postendoscopic retrograde cholangiopancreatography pancreatitis after covered self-expandable metal stent deployment.

OBJECTIVES: Although covered self-expandable metal stents (CSEMSs) are associated with the risk of postendoscopic retrograde cholangiopancreatography (ERCP) pancreatitis due to pancreatic duct (PD) orifice obstruction, they are often used for biliary drainage treatment in malignant biliary obstruction (MBO). This study aimed to investigate the efficacy of PD stenting in preventing post-ERCP pancreatitis after CSEMS implantation. METHODS: This retrospective cohort study analyzed 554 patients with transpapillary CSEMS for MBO. Patients with noninitial deployment, benign disease, CSEMS deployment above the papilla, surgically altered anatomy, uncovered self-expandable metal stents, multiple thin self-expandable metal stents, and unavailable procedure videos were excluded. Logistic regression analysis estimated the association between PD stenting and post-ERCP pancreatitis incidence. We adjusted for age, sex, pancreatitis history, prophylactic rectal nonsteroidal anti-inflammatory drug use, naïve papilla, MBO etiology, and prolonged biliary cannulation time. RESULTS: Among 554 patients, 67 (12.1%) experienced post-ERCP pancreatitis. Post-ERCP pancreatitis was recorded in 13.7% of patients in the non-PD stenting and 4.3% in the PD stenting groups. Pancreatic duct stenting was associated with lower risks of post-ERCP pancreatitis (odds ratio [OR] 0.28; 95% confidence interval [CI] 0.099-0.79; P = 0.028). In multivariable analysis, the association between PD stenting and lower post-ERCP pancreatitis incidence was consistent (OR 0.19; 95% CI 0.062-0.58; P = 0.0034). CONCLUSIONS: Pancreatic duct stenting could reduce the risk of post-ERCP pancreatitis after CSEMSs.

Identification and characterization of LPLAT7 as an sn-1-specific lysophospholipid acyltransferase.

The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs). Here, we used this method to identify the enzymes capable of incorporating fatty acids into the sn-1 position of lyso-PLs (sn-1 lysophospholipid acyltransferase [LPLAT]). Screenings using siRNA knockdown and recombinant proteins for 14 LPLATs identified LPLAT7/lysophosphatidylglycerol acyltransferase 1 (LPGAT1) as a candidate. In vitro, we found LPLAT7 mainly incorporated several fatty acids into the sn-1 position of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), with weak activities toward other lyso-PLs. Interestingly, however, only C18:0-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were specifically reduced in the LPLAT7-mutant cells and tissues from knockout mice, with a concomitant increase in the level of C16:0- and C18:1-containing PC and PE. Consistent with this, the incorporation of deuterium-labeled C18:0 into PLs dramatically decreased in the mutant cells, while deuterium-labeled C16:0 and C18:1 showed the opposite dynamic. Identifying LPLAT7 as an sn-1 LPLAT facilitates understanding the biological significance of sn-1 fatty acid remodeling of PLs. We also propose to use the new nomenclature, LPLAT7, for LPGAT1 since the newly assigned enzymatic activities are quite different from the LPGAT1s previously reported.

Mapping membrane lipids in the developing and adult mouse retina under physiological and pathological conditions using mass spectrometry.

Membrane phospholipids play pivotal roles in various cellular processes, and their levels are tightly regulated. In the retina, phospholipids had been scrutinized because of their distinct composition and requirement in visual transduction. However, how lipid composition changes during retinal development remains unclear. Here, we used liquid chromatography-mass spectrometry (LC-MS) to assess the dynamic changes in the levels of two main glycerophospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in the developing mouse retina under physiological and pathological conditions. The total levels of PC and PE increased during retinal development, and individual lipid species exhibited distinct level changes. The amount of very-long-chain PC and PE increased dramatically in the late stages of retinal development. The mRNA levels of Elovl2 and Elovl4, genes encoding enzymes essential for the synthesis of very-long-chain polyunsaturated fatty acids, increased in developing photoreceptors. Cell sorting based on CD73 expression followed by LC-MS revealed distinct changes in PC and PE levels in CD73-positive rod photoreceptors and CD73-negative retinal cells. Finally, using the NaIO3-induced photoreceptor degeneration model, we identified photoreceptor-specific changes in PC and PE levels from 1 day after NaIO3 administration, before the outer segment of photoreceptors displayed morphological impairment. In conclusion, our findings provide insight into the dynamic changes in PC and PE levels in the developing and adult mouse retina under physiological and pathological conditions. Furthermore, we provide evidence that cell sorting followed by LC-MS is a promising approach for investigating the relevance of lipid homeostasis in the function of different retinal cell types.

Mutagenesis and homology modeling reveal a predicted pocket of lysophosphatidylcholine acyltransferase 2 to catch Acyl-CoA.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions and promotes several pathological events, including anaphylaxis and neuropathic pain. PAF is biosynthesized by two types of lyso-PAF acetyltransferases: lysophosphatidylcholine acyltransferase 1 (LPCAT1) and LPCAT2, which are constitutive and inducible forms of lyso-PAF acetyltransferase, respectively. Because LPCAT2 mainly produces PAF under inflammatory stimuli, understanding the structure of LPCAT2 is important for developing specific drugs against PAF-related inflammatory diseases. Although the structure of LPCAT2 has not been determined, the crystal structure was reported for Thermotoga maritima PlsC, an enzyme in the same gene family as LPCAT2. Here, we identified residues in mouse LPCAT2 essential for its enzymatic activity and a potential acyl-coenzyme A (CoA)-binding pocket, based on homology modeling of mouse LPCAT2 with PlsC. We also found that Ala115 of mouse LPCAT2 was important for acyl-CoA selectivity. In conclusion, these results predict the three-dimensional (3D) structure of mouse LPCAT2. Our findings have implications for the future development of new drugs against PAF-related diseases.

Paraganglioma of the urinary bladder initially diagnosed as gastrointestinal stromal tumor requiring combined resection of the rectum: a case report.

BACKGROUND: Paraganglioma of the urinary bladder (Pub) is rare and presents with clinical symptoms caused by catecholamine production and release. The typical symptoms of Pub are hypertension, macroscopic hematuria, and a hypertensive crisis during micturition. The average size of detected Pubs is approximately 3 cm. Herein, we report a case of a large Pub in which the symptoms were masked by oral medication, precise preoperative diagnosis was difficult, and intraoperative confirmation of tumoral adhesion to the rectum resulted in hypertensive attacks during surgery. CASE PRESENTATION: A 64-year-old Japanese male with a history of hypertension and arrhythmia controlled with oral medication presented with a large tumor in the pelvic region, detected on examination for weight loss, with no clinical symptoms. Computed tomography and magnetic resonance imaging revealed a tumor measuring 77 mm in diameter in the posterior wall of the urinary bladder. The border with the rectum was unclear, and the tumor showed heterogeneous enhancement in the solid part with an enhancing hypodense lesion. Cystoscopy revealed compression of the bladder trigone by external masses; however, no tumor was visible in the lumen. Endoscopic ultrasonography-guided fine-needle aspiration revealed CD34-positive spindle-shaped cells in the fibrous tissue, suggestive of a mesenchymal neoplasm. The tumor was suspected to be a gastrointestinal stromal tumor, and surgery was performed. After laparotomy, we suspected that the tumor had invaded the rectum, and total cystectomy and anterior resection of the rectum were performed. Histologically, the tumor cells had granular or clear amphophilic cytoplasm with an oval nucleus and nests of cells delimited by connective tissue and vascular septations. Immunohistochemically, the tumor was positive for chromogranin A, CD56, and synaptophysin, and a diagnosis of paraganglioma of the urinary bladder was confirmed. There was no tumor recurrence at the 7-month follow-up. CONCLUSION: This case highlights the importance of careful examination of pelvic tumors, including endocrine testing, for detecting paraganglioma of the urinary bladder in patients with a history of hypertension or arrhythmia.

Multiplex fatty acid imaging inside cells by Raman microscopy.

Visualizing intracellular fatty acids (including free and esterified form) is very useful for understanding how and where such molecules are incorporated, stored, and metabolized within cells. However, techniques of imaging multiple intracellular fatty acids have been limited by their small size, making it difficult to label and track without changing their biological and biophysical characteristics. Here, we present a new method for simultaneously visualizing up to five atomically labeled intracellular fatty acid species. For this, we utilized the distinctive Raman spectra depending on the labeling patterns and created a new, extensible opensource software to perform by-pixel analysis of extracting original spectra from mixed ones. Our multiplex imaging method revealed that fatty acids with more double bonds tend to concentrate more efficiently at lipid droplets. This novel approach contributes to reveal not only the spatial dynamics of fatty acids, but also of any other metabolites inside cells.

Roles of polyunsaturated fatty acids, from mediators to membranes.

PUFAs, such as AA and DHA, are recognized as important biomolecules, but understanding their precise roles and modes of action remains challenging. PUFAs are precursors for a plethora of signaling lipids, for which knowledge about synthetic pathways and receptors has accumulated. However, due to their extreme diversity and the ambiguity concerning the identity of their cognate receptors, the roles of PUFA-derived signaling lipids require more investigation. In addition, PUFA functions cannot be explained just as lipid mediator precursors because they are also critical for the regulation of membrane biophysical properties. The presence of PUFAs in membrane lipids also affects the functions of transmembrane proteins and peripheral membrane proteins. Although the roles of PUFAs as membrane lipid building blocks were difficult to analyze, the discovery of lysophospholipid acyltransferases (LPLATs), which are critical for their incorporation, advanced our understanding. Recent studies unveiled how LPLATs affect PUFA levels in membrane lipids, and their genetic manipulation became an excellent strategy to study the roles of PUFA-containing lipids. In this review, we will provide an overview of metabolic pathways regulating PUFAs as lipid mediator precursors and membrane components and update recent progress about their functions. Some issues to be solved for future research will also be discussed.

Lysophosphatidylethanolamine acyltransferase 2 (LPEAT2) incorporates DHA into phospholipids and has possible functions for fatty acid-induced cell death.

Glycerophospholipids, one of the main constituents of biological membranes, are synthesized from glycerol-3-phosphate through the de novo pathway, and are reconstituted through the remodeling pathway. Lysophosphatidylethanolamine acyltransferase 2 (LPEAT2), one of the enzymes that play a role in the remodeling pathway, has been previously reported to have LPEAT, lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities with 16:0-CoA, 18:0-CoA, and 18:1-CoA as donors. In this study, we found that LPEAT2 is active with 22:6-CoA. Knockdown studies using Neuro 2A cells showed that LPEAT2 has endogenous LPEAT activity with 22:6-CoA, and that LPEAT2 has functions for modulating 22:6/20:4 ratios of phospholipids. In addition, we demonstrated that Neuro 2A cells overexpressing LPEAT2 underwent cell death with necrotic morphology when differentiated into neuron-like cells, with supplementation with 22:6 (DHA). These results suggest that LPEAT2 plays a role in inducing cell death DHA-dependently. This study will lead to better understand how DHA levels are regulated in phospholipids, especially in the brain where LPEAT2 is highly expressed. Our study also provides insight to understand the mechanism of cell death induced by DHA.

Na+-mimicking ligands stabilize the inactive state of leukotriene B4 receptor BLT1.

Most G-protein-coupled receptors (GPCRs) are stabilized in common in the inactive state by the formation of the sodium ion-centered water cluster with the conserved Asp2.50 inside the seven-transmembrane domain. We determined the crystal structure of the leukotriene B4 (LTB4) receptor BLT1 bound with BIIL260, a chemical bearing a benzamidine moiety. Surprisingly, the amidine group occupies the sodium ion and water locations, interacts with D662.50, and mimics the entire sodium ion-centered water cluster. Thus, BLT1 is fixed in the inactive state, and the transmembrane helices cannot change their conformations to form the active state. Moreover, the benzamidine molecule alone serves as a negative allosteric modulator for BLT1. As the residues involved in the benzamidine binding are widely conserved among GPCRs, the unprecedented inverse-agonist mechanism by the benzamidine moiety could be adapted to other GPCRs. Consequently, the present structure will enable the rational development of inverse agonists specific for each GPCR.