Lysophospholipid Acyltransferase Research Articles

Development of an Improved Adenovirus Vector and Its Application to the Treatment of Lifestyle-Related Diseases.

The number of patients with lifestyle-related diseases such as type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has continued to increase worldwide. Therefore, development of innovative therapeutic methods targeting lifestyle-related diseases is required. Gene therapy has attracted considerable attention as an advanced medical treatment. Safe and high-performance vectors are essential for the practical application of gene therapy. Replication-incompetent adenovirus (Ad) vectors are widely used in clinical gene therapy and basic research. Here, we developed a novel Ad vector, named Ad-E4-122aT, exhibiting higher and longer-term transgene expression and lower hepatotoxicity than conventional Ad vectors. We also elucidated the mechanisms underlying Ad vector-induced hepatotoxicity during the early phase using Ad-E4-122aT. Next, we examined the therapeutic effects of the genes of interest, namely zinc finger AN1-type domain 3 (ZFAND3), lipoprotein lipase (LPL), and lysophospholipid acyltransferase 10 (LPLAT10), on lifestyle-related diseases using Ad-E4-122aT. We showed that the overexpression of ZFAND3 in the liver improved glucose tolerance and insulin resistance. Liver-specific LPL overexpression suppressed hepatic lipid accumulation and improved glucose metabolism. LPLAT10 overexpression in the liver suppressed postprandial hyperglycemia by increasing glucose-stimulated insulin secretion. Furthermore, we also focused on foods to advance research on the pathophysiology and treatment of lifestyle-related diseases. Cranberry and calamondin, which are promising functional foods, attenuated the progression of MASLD/NAFLD. Our findings will aid the development of new therapeutic methods, including gene therapy, for lifestyle-related diseases such as T2DM and MASLD/NAFLD.

Lysophospholipid acyltransferase-mediated formation of saturated glycerophospholipids maintained cell membrane integrity for hypoxic adaptation.

Adaptation to hypoxia has attracted much public interest because of its clinical significance. However, hypoxic adaptation in the body is complicated and difficult to fully explore. To explore previously unknown conserved mechanisms and key proteins involved in hypoxic adaptation in different species, we first used a yeast model for mechanistic screening. Further multi-omics analyses in multiple species including yeast, zebrafish and mice revealed that glycerophospholipid metabolism was significantly involved in hypoxic adaptation with up-regulation of lysophospholipid acyltransferase (ALE1) in yeast, a key protein for the formation of dipalmitoyl phosphatidylcholine [DPPC (16:0/16:0)], which is a saturated phosphatidylcholine. Importantly, a mammalian homolog of ALE1, lysophosphatidylcholine acyltransferase 1 (LPCAT1), enhanced DPPC levels at the cell membrane and exhibited the same protective effect in mammalian cells under hypoxic conditions. DPPC supplementation effectively attenuated growth restriction, maintained cell membrane integrity and increased the expression of epidermal growth factor receptor under hypoxic conditions, but unsaturated phosphatidylcholine did not. In agreement with these findings, DPPC treatment could also repair hypoxic injury of intestinal mucosa in mice. Taken together, ALE1/LPCAT1-mediated DPPC formation, a key pathway of glycerophospholipid metabolism, is crucial for cell viability under hypoxic conditions. Moreover, we found that ALE1 was also involved in glycolysis to maintain sufficient survival conditions for yeast. The present study offers a novel approach to understanding lipid metabolism under hypoxia and provides new insights into treating hypoxia-related diseases.

Abstract 6379: Comparison of lipid profiles in children with solid tumors receiving chemotherapy and healthy children

Abstract Background: Lipid metabolism plays an important role in the regulation of synaptic function and plasticity.Cancer chemotherapy (chemo) can disturb the lipid profile, potentially linked with neuropsychological toxicities. This study aimed to compare the lipid profiles between children with cancer receiving chemo and healthy children. Methods: A prospective, observational study was conducted in 21 children with solid tumors enrolled from Children’s Healthcare of Atlanta (CHOA) and 16 controls. Children with solid tumors were consented precycle 2 chemo (T1) and followed at the end of chemo (T2). Controls completed one timepoint of data collection. At T1 (n=21) and T2 (n=13), fecal samples were collected for lipidomics analysis. Fecal lipids were processed using a high-resolution, untargeted lipidomics LC-MS approach. MetaboAnalyst and linear mixed models were used to analyze the data with false discovery rate correction (FDR 0.2). Results: There were no significant differences in age, sex, race, and body mass index between the groups. In total 241 lipid features were identified with high confidence and analyzed. A comparison between children with solid tumors at T2 (n=12) vs. controls (n=16) detected 8 differential lipids, which can be clustered into 4 classes, including 4 triglycerides (TG), 2 phosphatidylcholine (PC), 1 sphingomyelin (SM), and 1 lysophosphatidylcholine (LPC). Additionally, 24 differential lipids were detected for children with solid tumors from T1 to T2 (n=12), including 6 classes: 9 SM, 7 PC, 3 diacylglycerols (DG), 2 TG, 2 monoacylglycerols (MG), and 1 phosphatidylethanolamine (PE). Compared with controls, children with solid tumors at T2 had higher levels of SM (e.g., SM(d36:1)) and lower levels of TG (e.g., TG(18:0/18:0/20:0)), LPC (e.g., LPC(20:4)), and PC (e.g., PC(33:4). Compared with children at T1, those at T2 had higher levels of SM (e.g., SM(d36:1)), PE (e.g., PE(16:0p/20:4), PC (e.g., PC(30:0), and PC(36:1)), and lower levels of TG (e.g., TG(18:0/18:0/20:0) and TG(18:0/16:0/18:0)), LPC (e.g., LPC(20:4)), DG (e.g., DG(18:0/16:0) and DG(18:0/18:0)), and MG (e.g., MG(18:0)). Conclusion: Children with cancer chemo had alterations in fecal lipid profiles impacting the metabolism of PC, SM, TG, PE, MG, and DG. The increased PC and the dysregulation of LPC in comparison with controls suggest the decreased activity of phospholipase A2 (PLA2), primarily reported in inflammatory disease. Altered activity of lysophospholipid acyltransferase (LPCAT), diacylglyceride acyltransferase (DGAT), and phosphatidylethanolamine methyl transferase (PEMT) could also explain changes in PC and glycerolipids. Combined, these suggest dysregulation of the Kennedy or Lands pathways towards PC synthesis. Future work is needed to study whether the disturbed lipid metabolisms were associated with treatment toxicities and outcomes such as cognitive dysfunction based on literature. Citation Format: Jinbing Bai, Madelyn Houser, Melissa Martin, Christie Powell, Kristal Maner-Smith, Kathryn S. Sutton, Thomas Olson, Deborah W. Bruner. Comparison of lipid profiles in children with solid tumors receiving chemotherapy and healthy children [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6379.

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. Platelet-activating factor (PAF), a potent inflammatory mediator, is involved in the pathogenesis of various respiratory diseases, such as bronchial asthma or COPD. We focused on lysophospholipid acyltransferase 9 (LPLAT9), 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 macrophage (AM). LPLAT9 was predominant and upregulated in AM, particularly monocyte-derived AM, in patients with COPD. To identify the function of LPLAT9/PAF in AM on the pathogenesis of COPD, we exposed cigarette smoke (CS) to systemic LPLAT9 knockout (LPALT9-/-) mice. CS increased the number of AM, especially monocyte-derived fraction, which secreted matrix metalloprotease 12 (MMP12). Also, CS augmented LPLAT9 phosphorylation/activation on macrophage and, subsequently, PAF synthesis in mice lung. LPLAT9-/- mice lung reduced PAF production after CS exposure. Intratracheal PAF administration accumulated AM by increasing monocyte chemoattractant protein-1 (MCP1). After CS exposure, AM accumulation and subsequent pulmonary emphysema, a primary pathologic change of COPD, were reduced in LPALT9-/- mice than in LPLAT9+/+ mice. Notably, these phenotypes got worsened again by LPLAT9+/+ bone marrow transplantation in LPALT9-/- mice. Thus, CS-induced LPLAT9 activation in monocyte-derived AM aggravated pulmonary emphysema via PAF-induced further AM accumulation. These results suggest that PAF synthesized by LPLAT9 has an important role in the pathogenesis of COPD.

Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Efficiency of phospholipid remodeling via acyl‐CoA:lysophospholipid acyltransferases action is modified by acyl‐CoAs

AbstractThis study investigated the impact of different acyl‐CoAs on remodeling efficiency of PC and PE via the action of acyl‐CoA:lysophospholipid acyltransferases (LPLAT). Microsomal fractions from yeast ΔALE1 mutant transformed with an empty plasmid pYES2 or plasmid carrying AtLPCAT or ALE1 encoding genes were used in the experiments. In the preliminary assays, presence of 18:1‐CoA in the reaction mixture increased remodeling efficiency of [14C]PC integrated with microsomal fraction of yeast overexpressing AtLPCAT compared to assays without exogenous acyl‐CoA. In further experiments, the effect of five different acyl‐CoAs (16:0‐CoA, 18:0‐CoA, 18:1‐CoA, 18:2‐CoA, and 18:3‐CoA) on remodeling efficiency of yeast microsomal PC and PE via the action of three LPLATs (yeast SLC1 and ALE1 and Arabidopsis LPCAT2) were tested. It has been shown that acyl‐CoAs used in the experiments had different effect on the remodeling intensity of PC and PE via action of the tested acyltransferases. Moreover, each acyl‐CoA used had a different effect on the tested acyltransferases. Additionally, the assays with DTNB (inhibitor of the LPLAT backward reaction) showed that remodeling of PC and PE by LPLATs can also occur through reactions other than the backward reaction carried out by these enzymes, and that acyl‐CoA present in the reaction mixture affected these processes.

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.

Mapping a mutation causing pale yellow petals in Brassica rapa.

Petal color is an important trait for both ornamental purposes and also for attracting pollinators. Here, we report a mutation of Brassica rapa R-o-18 with pale yellow petals that we retrieved from an EMS population and named whiter shade of pale ( wsp ). Phenotypic segregation ratio of an F2 mapping population indicates the phenotype is controlled by a single recessive gene. Mapping data from the whole genome sequencing coupled with allele frequency analysis suggests the mutation is located in a ~2 Mbp interval on chromosome 2. The interval contains a putative esterase/lipase/thioesterase protein previously demonstrated to account for floral color in B. rapa . We demonstrate that wsp carries a G to A missense mutation causing an aspartate to asparagine substitution within the putative lysophospholipid acyltransferase domain.

Dual Role of ACBD6 in the Acylation Remodeling of Lipids and Proteins.

The transfer of acyl chains to proteins and lipids from acyl-CoA donor molecules is achieved by the actions of diverse enzymes and proteins, including the acyl-CoA binding domain-containing protein ACBD6. N-myristoyl-transferase (NMT) enzymes catalyze the covalent attachment of a 14-carbon acyl chain from the relatively rare myristoyl-CoA to the N-terminal glycine residue of myr-proteins. The interaction of the ankyrin-repeat domain of ACBD6 with NMT produces an active enzymatic complex for the use of myristoyl-CoA protected from competitive inhibition by acyl donor competitors. The absence of the ACBD6/NMT complex in ACBD6.KO cells increased the sensitivity of the cells to competitors and significantly reduced myristoylation of proteins. Protein palmitoylation was not altered in those cells. The specific defect in myristoyl-transferase activity of the ACBD6.KO cells provided further evidence of the essential functional role of the interaction of ACBD6 with the NMT enzymes. Acyl-CoAs bound to the acyl-CoA binding domain of ACBD6 are acyl donors for the lysophospholipid acyl-transferase enzymes (LPLAT), which acylate single acyl-chain lipids, such as the bioactive molecules LPA and LPC. Whereas the formation of acyl-CoAs was not altered in ACBD6.KO cells, lipid acylation processes were significantly reduced. The defect in PC formation from LPC by the LPCAT enzymes resulted in reduced lipid droplets content. The diversity of the processes affected by ACBD6 highlight its dual function as a carrier and a regulator of acyl-CoA dependent reactions. The unique role of ACBD6 represents an essential common feature of (acyl-CoA)-dependent modification pathways controlling the lipid and protein composition of human cell membranes.

Comparative transcriptomic and lipidomic analyses indicate that cold stress enhanced the production of the long C18-C22 polyunsaturated fatty acids in Aurantiochytrium sp.

Aurantiochytrium sp. belonging to Thraustochytrids are known for their capacity to produce long-chain polyunsaturated fatty acids (PUFAs). However, effects of cold stress accompanied with staged-temperature control on the fatty acid metabolism in Aurantiochytrium sp. were rarely studied. In this study, cold stress (15°C, 5°C) was applied for Aurantiochytrium sp., with the physiological responses (morphology, growth, fatty acid profiling) and gene expression related FA synthesis, lipid metabolism, and regulatory processes was observed. Results showed that there is a significant change for the lipid types under 5°C (251 species) and 15°C (97 species) treatment. The 5°C treatment was benefit for the C18–C22 PUFAs with the yield of docosahexaenoic acid (DHA) increased to 1.25 times. After incubation at 15°C, the accumulation of eicosadienoic acid (EA) (20:2) was increased to 2.00-fold. Based on transcriptomic and qPCR analysis, an increase in genes involved in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways was observed under low-temperature treatment. With upregulation of 3-ketoacyl-CoA synthase (2.44-fold), ketoreductase (2.50-fold), and dTDP-glucose 4,6-Dehydratase (rfbB) (2.31-fold) involved in PKS pathway, the accumulation of DHA was enhanced under 5°C. While, FAS and fatty elongase 3 (ELO) involved in the FAS pathway were upregulated (1.55-fold and 2.45-fold, respectively) to accumulate PUFAs at 15°C. Additionally, glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT), phosphatidic acid phosphatase (PAP), phosphatidylserine synthase (PSS), and phosphatidylserine decarboxylase (PSD) involved in glycerophospholipid biosynthesis were upregulated at 5°C increasing the accumulation of phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). However, glycolysis and the TCA cycle were inhibited under 5°C. This study provides a contribution to the application of two-staged temperature control in the Aurantiochytrium sp. fermentation for producing cold stress-enhancing PUFAs, in order to better understand the function of the key genes for future genetic engineering.

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.

Abnormal male reproduction and embryonic development induced by downregulation of a phospholipid fatty acid-introducing enzyme Lpgat1 in zebrafish

Phospholipids in the membrane consist of diverse pairs of fatty acids bound to a glycerol backbone. The biological significance of the diversity, however, remains mostly unclear. Part of this diversity is due to lysophospholipid acyltransferases (LPLATs), which introduce a fatty acid into lysophospholipids. The human genome has 14 LPLATs and most of them are highly conserved in vertebrates. Here, we analyzed the function of one of these enzymes, lysophosphatidylglycerol acyltransferase 1 (Lpgat1), in zebrafish. We found that the reproduction of heterozygous (lpgat1+/−) male mutants was abnormal. Crosses between heterozygous males and wild-type females produced many eggs with no obvious cleavage, whereas eggs produced by crosses between heterozygous females and wild-type males cleaved normally. Consistent with this, spermatozoa from heterozygous males had reduced motility and abnormal morphology. We also found that the occurrence of lpgat1 homozygous (lpgat1−/−) mutants was far lower than expected. In addition, downregulation of lpgat1 by morpholino antisense oligonucleotides resulted in severe developmental defects. Lipidomic analysis revealed that selective phospholipid species with stearic acid and docosahexaenoic acid were reduced in homozygous larvae and spermatozoa from heterozygotes. These results suggest that the specific phospholipid molecular species produced by Lpgat1 have an essential role in sperm fertilization and in embryonic development.

Comparative effects of dietary soybean oil and fish oil on the growth performance, fatty acid composition and lipid metabolic signaling of grass carp, Ctenopharyngodon idella

Lipid was an essential constituent of aquatic feeds, among which fish oil (FO) was the most excellent lipid source for its nutritive values. However, high price and limited production restricted its application, thus leading to the consideration of developing other alternative lipid sources, and soybean oil (SO) instead of FO was applied as the main lipid source in the commercial feed of grass carp. In the present study, two typical lipids sources FO and SO were used to prepare grass carp feed, and their comparative effects on the growth performance, nutrient composition and metabolic signaling were investigated. The growth performance of two groups exhibited no significant difference, while crude lipid was much higher in the muscle of SO group. Additionally, muscle fatty acid composition of both groups were highly correlated with that of diets, and the proportion of muscle n-3 polyunsaturated fatty acids was notably higher in the FO group, especially that of C20:5n-3 (EPA) and C22:6n-3 (DHA). Comparative digital gene expression profiles (DGE) was conducted and 1261 differentially expressed genes (DEGs) had been identified, then gene ontology (GO) enrichment analysis revealed the enriched functional subterms were primarily focused on cellular metabolic processes and regulation. KEGG pathway analysis elucidated the main metabolism related pathways, and DEGs involved in fatty acid elongation, fatty acid metabolism, and glycerolipid metabolism pathways were listed, including elongation of very long chain fatty acids protein 7 (el7), lysophospholipid acyltransferase 4 (lpcat4), long-chain acyl-CoA synthetase (acs), phosphatidate phosphatase LPIN (lpin), cathepsin L, and glucose-6-phosphate isomerase (gpi) et al. In conclusion, the nutritional composition rather than growth performance of grass carp was affected by different lipid sources, and the underlying regulatory signaling was predicted, which would provide theoretical basis for fish diet formulation.

In Vitro Growth Conditions Boost Plant Lipid Remodelling and Influence Their Composition.

Acyl-lipids are vital components for all life functions of plants. They are widely studied using often in vitro conditions to determine inter alia the impact of genetic modifications and the description of biochemical and physiological functions of enzymes responsible for acyl-lipid metabolism. What is currently lacking is knowledge of if these results also hold in real environments—in in vivo conditions. Our study focused on the comparative analysis of both in vitro and in vivo growth conditions and their impact on the acyl-lipid metabolism of Camelina sativa leaves. The results indicate that in vitro conditions significantly decreased the lipid contents and influenced their composition. In in vitro conditions, galactolipid and trienoic acid (16:3 and 18:3) contents significantly declined, indicating the impairment of the prokaryotic pathway. Discrepancies also exist in the case of acyl-CoA:lysophospholipid acyltransferases (LPLATs). Their activity increased about 2–7 times in in vitro conditions compared to in vivo. In vitro conditions also substantially changed LPLATs’ preferences towards acyl-CoA. Additionally, the acyl editing process was three times more efficient in in vitro leaves. The provided evidence suggests that the results of acyl-lipid research from in vitro conditions may not completely reflect and be directly applicable in real growth environments.

Diatoms and Plants Acyl-CoA:lysophosphatidylcholine Acyltransferases (LPCATs) Exhibit Diverse Substrate Specificity and Biochemical Properties.

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).

Aspariginase-Triggered Hyperglycerimedia: An Analysis on Germline Regulatory Gene Variations in Adult Acute Lymphoblastic Leukemia Patients

Asparaginase (ASP) is considered as a backbone of successful treatment of acute lymphoblastic leukemia (ALL). Cell growth and division of malignant lymphoblasts are highly dependent on asparagine and glutamine. ASP depletes them from extracellular fluid which leads to apoptosis of lymphoblasts. Aside from the aimed antileukemic properties, asparaginase exposes patients to multiple adverse effects including severe hypertriglyceridemia which potentially hampers efficient leukemia therapy. Molecular background of ASP-induced hypertriglyceridemia and its regulators are not well known. We aimed to investigate constitutional genetic factors as biomarkers for hypertriglyceridemia following ASP therapy.We analyzed clinical and laboratory data recorded in The Finnish Hematology Registry (FHR) from 52 adult ALL patients treated at the Helsinki University Hospital, Department of Hematology in 2014-2016. Study subjects were selected for further analyses if germline whole exome sequencing (WES) had been performed, patients had received ASP and data on basic chemistry and lipids before and after ASP dosing were available. Sixteen patients fulfilled these criteria.The patients' germline DNA was extracted from a skin biopsy taken in conjunction with a bone marrow sampling. Roche MedExome kit was used to target the coding regions of the genome and sequencing was conducted using an Illumina HiSeq sequencer. The reads were processed and aligned to the GRCh37 human reference genome. SNVs and indels in the exome sequencing data were analyzed using an in-house developed analysis tool. Shared variants in patients with similar serum triglyceride level changes after ASP treatment were analyzed and variants in genes associated with lipid metabolism were selected as candidate target genes.Three patient groups were identified based on to the maximum triglyceride changes pre- and post ASP infusion (both clinical and exome data available, n=7, 5, and 4; respectively). Mean changes in the Groups 1-3 were 1.7, 8.4, and 72,7 mmol/L, respectively. Differences in patients' BMI or corticosteroids use did not explain the distribution. Hence, a germline determinant was hypothesized and a candidate gene analysis (xx known genes affecting lipid and ASP metabolisms) performed from patients' germline WES.Mean changes in the Groups 1-3 were 1.7, 8.4, and 72,7 mmol/L, respectively. Differences in patients' BMI or usage of corticosteroids did not explain the distribution. Hence, a germline determinant was hypothesized and candidate gene search performed on patients' germline WES.Four of the candidate variants in ATF5, NKX1-1, THEM4, and LPCAT2 were enriched in patients with ASP-induced hyperTG. None of the variants suggested here have been previously reported to be associated with ASP-induced hypertriglyceridemia, but another SNP in ATF5 has been linked to adverse outcome in ASP-treated patients. ATF5, a transcription factor, regulates asparagine synthetase biosynthesis and is also involved in the cell cycle and apoptosis regulation, in the cellular stress response and the ceramide pathway. NKX1-1 is a transcription factor and an essential regulator of organ development, and it is also probably associated with energy homeostasis. THEM4 is an acyl-CoA thioesterase and is probably involved in mitochondrial fatty acid metabolism as well as in AKT1 signaling and apoptosis. LPCAT2 is a member of the lysophospholipid acyltransferase family involved in the synthesis of phospholipids, but also in the biosynthesis of platelet-activating factor (PAF) in inflammatory conditions.Here, we present four candidate genes and variations in them possibly linking to ASP-induced hypertriglyceridemia in adult ALL patients. Prospective studies exploring the impact of these findings are called for.Figure 1. Patient groups (3) identified according to the maximum triglyceride changes pre (green dot) - and post (red dot) ASP infusion. [Display omitted] DisclosuresPorkka:Pfizer: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

Editing of phosphatidic acid and phosphatidylethanolamine by acyl-CoA: lysophospholipid acyltransferases in developing Camelina sativa seeds

Main conclusionsThe main source of polyunsaturated acyl-CoA in cytoplasmic acyl-CoA pool of Camelina sativa seeds are fatty acids derived from phosphatidylcholine followed by phosphatidic acid. Contribution of phosphatidylethanolamine is negligible.While phosphatidylethanolamine (PE) is the second most abundant phospholipid, phosphatidic acid (PA) only constitutes a small fraction of C. sativa seeds’ polar lipids. In spite of this, the relative contribution of PA in providing fatty acids for the synthesis of acyl-CoA, supplying cytosolic acyl-CoA pool seems to be much higher than the contribution of PE. Our data indicate that up to 5% of fatty acids present in mature C. sativa seeds are first esterified with PA, in comparison to 2% first esterified with PE, before being transferred into acyl-CoA pool via backward reactions of either acyl-CoA:lysophosphatidic acid acyltransferases (CsLPAATs) or acyl-CoA:lysophoshatidylethanolamine acyltransferases (CsLPEATs). Those acyl-CoAs are later reused for lipid biosynthesis or remodelling. In the forward reactions both aforementioned acyltransferases display the highest activity at 30 °C. The spectrum of optimal pH differs for both enzymes with CsLPAATs most active between pH 7.5–9.0 and CsLPEATs between pH 9.0 to 10.0. Whereas addition of magnesium ions stimulates CsLPAATs, calcium and potassium ions inhibit them in concentrations of 0.05–2.0 mM. All three types of ions inhibit CsLPEATs activity. Both tested acyltransferases present the highest preferences towards 16:0-CoA and unsaturated 18-carbon acyl-CoAs in forward reactions. However, CsLPAATs preferentially utilise 18:1-CoA and CsLPEATs preferentially utilise 18:2-CoA while catalysing fatty acid remodelling of PA and PE, respectively.

ABHD12 and LPCAT3 Interplay Regulates a Lyso-phosphatidylserine-C20:4 Phosphatidylserine Lipid Network Implicated in Neurological Disease.

PHARC (polyneuropathy, hearing loss, cerebellar ataxia, retinitis pigmentosa, and cataract) is a human neurological disorder caused by deleterious mutations in the ABHD12 gene, which encodes an integral membrane lyso-phosphatidylserine (lyso-PS) lipase. Pharmacological or genetic disruption of ABHD12 leads to higher levels of lyso-PS lipids in human cells and the central nervous system (CNS) of mice. ABHD12 loss also causes rapid rewiring of PS content, resulting in selective increases in the level of arachidonoyl (C20:4) PS and decreases in the levels of other PS species. The biochemical basis for ABHD12-dependent PS remodeling and its pathophysiological significance remain unknown. Here, we show that genetic deletion of the lysophospholipid acyltransferase LPCAT3 blocks accumulation of brain C20:4 PS in mice lacking ABHD12 and concurrently produces hyper-increases in the level of lyso-PS in these animals. These lipid changes correlate with exacerbated auditory dysfunction and brain microgliosis in mice lacking both ABHD12 and LPCAT3. Taken together, our findings reveal that ABHD12 and LPCAT3 coordinately regulate lyso-PS and C20:4 PS content in the CNS and point to lyso-PS lipids as the likely bioactive metabolites contributing to PHARC-related neuropathologies.

Biosynthetic Enzymes of Membrane Glycerophospholipid Diversity as Therapeutic Targets for Drug Development.

Biophysical properties of membranes are dependent on their glycerophospholipid compositions. Lysophospholipid acyltransferases (LPLATs) selectively incorporate fatty chains into lysophospholipids to affect the fatty acid composition of membrane glycerophospholipids. Lysophosphatidic acid acyltransferases (LPAATs) of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family incorporate fatty chains into phosphatidic acid during the de novo glycerophospholipid synthesis in the Kennedy pathway. Other LPLATs of both the AGPAT and the membrane bound O-acyltransferase (MBOAT) families further modify the fatty chain compositions of membrane glycerophospholipids in the remodeling pathway known as the Lands' cycle. The LPLATs functioning in these pathways possess unique characteristics in terms of their biochemical activities, regulation of expressions, and functions in various biological contexts. Essential physiological functions for LPLATs have been revealed in studies using gene-deficient mice, and important roles for several enzymes are also indicated in human diseases where their mutation or dysregulation causes or contributes to the pathological condition. Now several LPLATs are emerging as attractive therapeutic targets, and further understanding of the mechanisms underlying their physiological and pathological roles will aid in the development of novel therapies to treat several diseases that involve altered glycerophospholipid metabolism.

Introduction: Druggable Lipid Signaling Pathways.

Lipids are essential for life. They store energy, constitute cellular membranes, serve as signaling molecules, and modify proteins. In the long history of lipid research, many drugs targeting lipid receptors and enzymes that are responsible for lipid metabolism and function have been developed and applied to a variety of diseases. For example, non-steroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed medications for fever, pain, and inflammation. The NSAIDs block prostaglandin production by inhibiting cyclooxygenases. A recent innovative breakthrough in drug discovery for the lipid biology field was the development of the sphingosine 1-phosphate receptor modulators (fingolimod, siponimodand ozanimod) for the treatment of multiple sclerosis, which were approved by the United States Food and Drug Administration in 2010, 2019 and 2020,respectively. This review series of "Druggable Lipid Signaling Pathways" provides 9 outstanding reviews that summarize the currently available drugs that target lipid signaling pathways and also outlines future directions for drug discovery. The review chapters include lipid signaling pathways (prostanoids, leukotrienes, epoxy fatty acids, sphingolipids, lysophospholipids, endocannabinoids, and phosphoinositides) and lipid signaling proteins (lysophospholipid acyltransferases, phosphoinositide 3-kinase,and G protein-coupled receptors (GPCRs)). Drugs targeting lipid signaling pathways promise to be life changing magic for the future of human health and well-being.