Catalytic mechanism of trans-2-enoyl-CoA reductases in the fatty acid elongation cycle and its cooperative action with fatty acid elongases.

The fatty acid (FA) elongation cycle produces very-long-chain FAs with ≥C21, which have unique physiological functions. Trans-2-enoyl-CoA reductases (yeast, Tsc13; mammals, TECR) catalyze the reduction reactions in the fourth step of the FA elongation cycle and in the sphingosine degradation pathway. However, their catalytic residues and coordinated action in the FA elongation cycle complex are unknown. To reveal these, we generated and analyzed Ala-substituted mutants of 15 residues of Tsc13. An in vitro FA elongation assay showed that nine of these mutants were less active than WT protein, with E91A and Y256A being the least active. Growth complementation analysis, measurement of ceramide levels, and deuterium-sphingosine labeling revealed that the function of the E91A mutant was substantially impaired in vivo. In addition, we found that the activity of FA elongases, which catalyze the first step of the FA elongation cycle, were reduced in the absence of Tsc13. Similar results were observed in Tsc13 E91A-expressing cells, which is attributable to reduced interaction between the Tsc13 E91A mutant and the FA elongases Elo2/Elo3. Finally, we found that E94A and Y248A mutants of human TECR, which correspond to E91A and Y256A mutants of Tsc13, showed reduced and almost no activity, respectively. Based on these results and the predicted three-dimensional structure of Tsc13, we speculate that Tyr256/Tyr248 of Tsc13/TECR is the catalytic residue that supplies a proton to trans-2-enoyl-CoAs. Our findings provide a clue concerning the catalytic mechanism of Tsc13/TECR and the coordinated action in the FA elongation cycle complex.

In vivo adenine base editing rescues adrenoleukodystrophy in a humanized mouse model.

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent ß-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: ∼5.5%), spinal cord (∼5.1%), and adrenal gland (∼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.

Loss of nephric augmenter of liver regeneration facilitates acute kidney injury via ACSL4-mediated ferroptosis.

Ferroptosis, characterized by lipid accumulation in intracellular compartments, is related to acute kidney injury (AKI), but the mechanism remains obscure. In our previous study, the protective effect of augmenter of liver regeneration (ALR) on AKI was not fully clarified. In this study, we established an AKI mouse model by knocking out proximal tubule-specific ALR and an AKI cell model by inducing hypoxia, as well as enrolled AKI patients, to investigate the effects of ALR on ferroptosis and the progression of AKI. We found that ALR knockout aggravated ferroptosis and increased ROS accumulation and mitochondrial damage, whereas ALR overexpression attenuated ferroptosis through clearance of ROS and maintenance of mitochondrial morphology. Mechanistically, we demonstrated that ALR could directly bind to long-chain-fatty-acid-CoA ligase 4 (ACSL4) and further inhibit the expression of ACSL4 by interacting with certain regions. By resolution liquid chromatography coupled with triple quadruple mass spectrometry, we found that ALR could reduce the contents of polyunsaturated fatty acids, especially arachidonic acid. In addition, we showed that ALR binds to ACSL4 and attenuates oxylipin accumulation, exerting a protective effect against ferroptosis in AKI. Therefore, targeting renal ALR can attenuate ferroptosis and can offer a promising strategy for the treatment of AKI.

The 3-hydroxyacyl-CoA dehydratase 1/2 form complex with trans-2-enoyl-CoA reductase involved in substrates transfer in very long chain fatty acid elongation.

Very long-chain fatty acids (VLCFAs) are fatty acids with a carbon chain length greater than 18 carbons (>C18) and exhibit various functions, such as in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function, and anti-inflammation. VLCFAs are absorbed by dietary or elongated from endogenous hexadecanoyl acids (C16). Similar to long-chain fatty acid synthesis, VLCFAs elongation begins with acyl-CoA and malonyl-CoA as sources, and the length of the acyl chain is extended by two carbon units in each cycle. However, the VLCFAs elongation machinery is located in ER membrane and consists of four components, FA elongase (ELOVL), 3-ketoacyl-CoA reductase (KAR), 3-hydroxyacyl-CoA dehydratase (HACD), and trans-2-enoyl-CoA reductase (TECR), which is different with the long-chain fatty acid machinery fatty acid synthase (FAS) complex. Although the critical components in the elongation cycle are identified, the detailed catalytic and regulation mechanisms are still poorly understood. Here, we focused on the structural and biochemical analysis of TECR-associated VLCFA elongation reactions. Firstly, we identified a stable complex of human HACD2-TECR based on extensive in vitro characterizations. Combining computational modeling and biochemical analysis, we confirmed the critical interactions between TECR and HACD1/2. Then, we proposed the putative substrate binding sites and catalytic residues for TECR and HACD2. Besides, we revealed the structural similarities of HACD with ELOVLs and proposed the possible competition mechanism of TECR-associated complex formation.

ELOVL6 promotes the progression of head and neck squamous cell carcinoma via activating WNT/ß-catenin pathway.

This study was to explore the role of ELOVL6 in the development of head and neck squamous cell carcinoma (HNSCC). Considering its previously identified oncogenic role in hepatocellular carcinoma. ELOVL6 gene expression, clinicopathological analysis, enrichment analysis, and immune infiltration analysis were based on the data from Gene Expression Omnibus and The Cancer Genome Atlas, with additional bioinformatics analyses performed. Human HNSCC tissue microarray and cell lines were used. The expression of ELOVL6 in HNSCC was detected by quantitative polymerase chain reaction, immunohistochemistry assay, and western blot analysis. The proliferation ability of HNSCC cells, invasion, and apoptosis were evaluated using cell counting kit-8 method, Transwell assay, and flow cytometry, respectively. Based on the data derived from the cancer databases and our HNSCC cell and tissue studies, we found that ELOVL6 was overexpressed in HNSCC. Moreover, ELOVL6 expression level had a positive correlation with clinicopathology of HNSCC. Gene set enrichment analysis showed that ELOVL6 affected the occurrence of HNSCC through WNT signaling pathway. Functional experiments demonstrated that ELOVL6 knockdown inhibited the proliferation and invasion of HNSCC cells while promoting apoptosis. Additionally, compound 3f, an agonist of WNT/ß-catenin signaling pathway, enhances the effect of ELOVL6 on the progression of HNSCC cells. ELOVL6 is upregulated in HNSCC and promotes the development of HNSCC cells by inducing WNT/ß-catenin signaling pathway. ELOVL6 stands a potential target for the treatment of HNSCC and a prognosis indicator of human HNSCC.

Major clinical events and healthcare resource use among patients with long-chain fatty acid oxidation disorders in the United States: Results from LC-FAOD Odyssey program.

Major clinical events (MCEs) related to long-chain fatty acid oxidation disorders (LC-FAOD) in triheptanoin clinical trials include inpatient or emergency room (ER) visits for three major clinical manifestations: rhabdomyolysis, hypoglycemia, and cardiomyopathy. However, outcomes data outside of LC-FAOD clinical trials are limited. The non-interventional cohort LC-FAOD Odyssey study examines data derived from US medical records and patient reported outcomes to quantify LC-FAOD burden according to management strategy including MCE frequency and healthcare resource utilization (HRU). Thirty-four patients were analyzed of which 21 and 29 patients had received triheptanoin and/or medium chain triglycerides (MCT), respectively. 36% experienced MCEs while receiving triheptanoin versus 54% on MCT. Total mean annualized MCE rates on triheptanoin and MCT were 0.1 and 0.7, respectively. Annualized disease-related inpatient and ER events were lower on triheptanoin (0.2, 0.3, respectively) than MCT (1.2, 1.0, respectively). Patients were managed more in an outpatient setting on triheptanoin (8.9 annualized outpatient visits) vs MCT (7.9). Overall, this shows that those with LC-FAOD in the Odyssey program experienced fewer MCEs and less HRU in inpatient and ER settings during triheptanoin-treated periods compared with the MCT-treated periods. The MCE rate was lower after initiation of triheptanoin, consistent with clinical trials.

Early diagnosis and treatment by newborn screening (NBS) or family history is associated with improved visual outcomes for long-chain 3-hydroxyacylCoA dehydrogenase deficiency (LCHADD) chorioretinopathy.

Long chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) is the only fatty acid oxidation disorder to develop a progressive chorioretinopathy resulting in vision loss; newborn screening (NBS) for this disorder began in the United States around 2004. We compared visual outcomes among 40 participants with LCHADD or trifunctional protein deficiency diagnosed symptomatically to those who were diagnosed via NBS or a family history. Participants completed ophthalmologic testing including measures of visual acuity, electroretinograms (ERG), fundal imaging, contrast sensitivity, and visual fields. Records were reviewed to document medical and treatment history. Twelve participants presented symptomatically with hypoglycemia, failure to thrive, liver dysfunction, cardiac arrest, or rhabdomyolysis. Twenty eight were diagnosed by NBS or due to a family history of LCHADD. Participants diagnosed symptomatically were older but had similar percent males and genotypes as those diagnosed by NBS. Treatment consisted of fasting avoidance, dietary long-chain fat restriction, MCT, C7, and/or carnitine supplementation. Visual acuity, rod- and cone-driven amplitudes on ERG, contrast sensitivity scores, and visual fields were all significantly worse among participants diagnosed symptomatically compared to NBS. In mixed-effects models, both age and presentation (symptomatic vs. NBS) were significant independent factors associated with visual outcomes. This suggests that visual outcomes were improved by NBS, but there was still lower visual function with advancing age in both groups. Early diagnosis and treatment by NBS is associated with improved visual outcomes and retinal function compared to participants who presented symptomatically. Despite the impact of early intervention, chorioretinopathy was greater with advancing age, highlighting the need for novel treatments.

Increased neurotoxicity of high-density lipoprotein secreted from murine reactive astrocytes deficient in a peroxisomal very-long-chain fatty acid transporter Abcd1.

X-linked adrenoleukodystrophy (X-ALD) is a genetic neurodegenerative disorder caused by pathogenic variants in ABCD1, resulting in the accumulation of very-long-chain fatty acids (VLCFAs) in tissues. The etiology of X-ALD is unclear. Activated astrocytes play a pathological role in X-ALD. Recently, reactive astrocytes have been shown to induce neuronal cell death via saturated lipids in high-density lipoprotein (HDL), although how HDL from reactive astrocytes exhibits neurotoxic effects has yet to be determined. In this study, we obtained astrocytes from wild-type and Abcd1-deficient mice. HDL was purified from the culture supernatant of astrocytes, and the effect of HDL on neurons was evaluated in vitro. To our knowledge, this study shows for the first time that HDL obtained from Abcd1-deficient reactive astrocytes induces a significantly higher level of lactate dehydrogenase (LDH) release, a marker of cell damage, from mouse primary cortical neurons as compared to HDL from wild-type reactive astrocytes. Notably, HDL from Abcd1-deficient astrocytes contained significantly high amounts of VLCFA-containing phosphatidylcholine (PC) and LysoPC. Activation of Abcd1-deficient astrocytes led to the production of HDL containing decreased amounts of PC with arachidonic acid in sn-2 acyl moieties and increased amounts of LysoPC, presumably through cytosolic phospholipase A2 α upregulation. These results suggest that compositional changes in PC and LysoPC in HDL, due to Abcd1 deficiency and astrocyte activation, may contribute to neuronal damage. Our findings provide novel insights into central nervous system pathology in X-ALD.

Biological characteristics of a new long-chain fatty acid transport protein 1 from Trichinella spiralis and its participation in lipid metabolism, larval moulting, and development.

Long-chain fatty acid transport protein 1 (FATP1) is a member of the fatty acid transporter family. It facilitates transmembrane transport of fatty acids and participates in lipid metabolism. Lipids are essential components of the cell and organelle membranes of Trichinella spiralis. The nematode has lost the capacity to synthesise the necessary lipids de novo and has instead evolved to obtain fatty acids and their derivatives from its host. This study aims to ascertain the primary biological characteristics and roles of T. spiralis FATP1 (TsFATP1) in lipid metabolism, larval moulting, and the development of this nematode. The results show that TsFATP1 is highly expressed at enteral T. spiralis stages, mainly localised at the cuticle, the stichosome and the intrauterine embryos of the parasite. The silencing of the TsFATP1 gene by TsFATP1-specific dsRNA significantly decreases the expression levels of TsFATP1 in the worm. It reduces the contents of ATP, triglycerides, total cholesterol, and phospholipids both in vitro and in vivo. RNAi inhibits lipid metabolism, moulting, and the growth of this nematode. The results demonstrate that TsFATP1 plays an essential role in lipid metabolism, moulting, and the development of T. spiralis. It could also be a target candidate for the anti-Trichinella vaccine and drugs.

Syntheses of the ω-pyridinium-containing very-long-chain ceramides PyrCer(24:1(15Z)) and PyrCer(24:0) and their anticancer activity.

Ceramides, crucial sphingolipids in cellular biology, play various roles ranging from structural membrane integrity to signaling pathway regulation. Structurally, a ceramide consists of a fatty acid connected to a sphingoid base. The characteristics of the fatty acid chain, including length and saturation, determine the physiological properties of the ceramide. Ceramides typically fall into the following categories based on chain length: medium, long, very-long, and ultra-long. Among them, two very-long-chain ceramides, Cer(24:1(15Z)) and Cer(24:0), have been extensively studied, and they are known for their regulatory functions. However, the hydrophobic natures of ceramides, arising from their long hydrocarbon chain impedes their solubilities and levels of cellular delivery. Although ω-pyridinium ceramide analogs (ω-PyrCers) have been developed to address this issue, ω-PyrCers with very-long fatty acid chains or unsaturation have not been developed, presumably due to limited access to the corresponding ω-bromo fatty acids required in their syntheses. In this study, we prepared the ω-PyrCers of Cer(24:1(15Z)) and Cer(24:0), PyrCer(24:1(15Z)) and PyrCer(24:0), respectively. The key in the synthesis is the Wittig reaction to prepare the ω-bromo fatty acid with an appropriate chain length and (Z)-double bond position. Preliminary evaluation of the PyrCer(24:1(15Z)) and PyrCer(24:0) revealed their potential in hepatocellular carcinoma treatment.

Low expression of ELOVL6 may be involved in fat loss in white adipose tissue of cancer-associated cachexia.

BACKGROUND: Cancer-associated cachexia (CAC) arises from malignant tumors and leads to a debilitating wasting syndrome. In the pathophysiology of CAC, the depletion of fat plays an important role. The mechanisms of CAC-induced fat loss include the enhancement of lipolysis, inhibition of lipogenesis, and browning of white adipose tissue (WAT). However, few lipid-metabolic enzymes have been reported to be involved in CAC. This study hypothesized that ELOVL6, a critical enzyme for the elongation of fatty acids, may be involved in fat loss in CAC. METHODS: Transcriptome sequencing technology was used to identify CAC-related genes in the WAT of a CAC rodent model. Then, the expression level of ELOVL6 and the fatty acid composition were analyzed in a large clinical sample. Elovl6 was knocked down by siRNA in 3T3-L1 mouse preadipocytes to compare with wild-type 3T3-L1 cells treated with tumor cell conditioned medium. RESULTS: In the WAT of patients with CAC, a significant decrease in the expression of ELOVL6 was found, which was linearly correlated with the extent of body mass reduction. Gas chromatographic analysis revealed an increase in palmitic acid (C16:0) and a decrease in linoleic acid (C18:2n-6) in these tissue samples. After treatment with tumor cell-conditioned medium, 3T3-L1 mouse preadipocytes showed a decrease in Elovl6 expression, and Elovl6-knockdown cells exhibited a reduction in preadipocyte differentiation and lipogenesis. Similarly, the knockdown of Elovl6 in 3T3-L1 cells resulted in a significant increase in palmitic acid (C16:0) and a marked decrease in oleic acid (C18:1n-9) content. CONCLUSION: Overall, the expression of ELOVL6 was decreased in the WAT of CAC patients. Decreased expression of ELOVL6 might induce fat loss in CAC patients by potentially altering the fatty acid composition of adipocytes. These findings suggest that ELOVL6 may be used as a valuable biomarker for the early diagnosis of CAC and may hold promise as a target for future therapies.

Fatty Acid Metabolism in Peroxisomes and Related Disorders.

One of the functions of peroxisomes is the oxidation of fatty acids (FAs). The importance of this function in our lives is evidenced by the presence of peroxisomal disorders caused by the genetic deletion of proteins involved in these processes. Unlike mitochondrial oxidation, peroxisomal oxidation is not directly linked to ATP production. What is the role of FA oxidation in peroxisomes? Recent studies have revealed that peroxisomes supply the building blocks for lipid synthesis in the endoplasmic reticulum and facilitate intracellular carbon recycling for membrane quality control. Accumulation of very long-chain fatty acids (VLCFAs), which are peroxisomal substrates, is a diagnostic marker in many types of peroxisomal disorders. However, the relationship between VLCFA accumulation and various symptoms of these disorders remains unclear. Recently, we developed a method for solubilizing VLCFAs in aqueous media and found that VLCFA toxicity could be mitigated by oleic acid replenishment. In this chapter, we present the physiological role of peroxisomal FA oxidation and the knowledge obtained from VLCFA-accumulating peroxisome-deficient cells.

The Checkpoints of Intestinal Fat Absorption in Obesity.

In this chapter, intestinal lipid transport, which plays a central role in fat homeostasis and the development of obesity in addition to the mechanisms of fatty acids and monoacylglycerol absorption in the intestinal lumen and reassembly of these within the enterocyte was described. A part of the resynthesized triglycerides (triacylglycerols; TAG) is repackaged in the intestine to form the hydrophobic core of chylomicrons (CMs). These are delivered as metabolic fuels, essential fatty acids, and other lipid-soluble nutrients, from enterocytes to the peripheral tissues following detachment from the endoplasmic reticulum membrane. Moreover, the attitudes of multiple receptor functions in dietary lipid uptake, synthesis, and transport are highlighted. Additionally, intestinal fatty acid binding proteins (FABPs), which increase the cytosolic flux of fatty acids via intermembrane transfer in enterocytes, and the functions of checkpoints for receptor-mediated fatty acid signaling are debated. The importance of the balance between storage and secretion of dietary fat by enterocytes in determining the physiological fate of dietary fat, including regulation of blood lipid concentrations and energy balance, is mentioned. Consequently, promising checkpoints regarding how intestinal fat processing affects lipid homeostatic mechanisms and lipid stores in the body and the prevention of obesity-lipotoxicity due to excessive intestinal lipid absorption are evaluated. In this context, dietary TAG digestion, pharmacological inhibition of TAG hydrolysis, the regulation of long-chain fatty acid uptake traffic into adipocytes, intracellular TAG resynthesis, the enlargement of cytoplasmic lipid droplets in enterocytes and constitutional alteration of their proteome, CD36-mediated conversion of diet-derived fatty acid into cellular lipid messengers and their functions are discussed.

Mechanism of Obesity-Related Lipotoxicity and Clinical Perspective.

The link between cellular exposure to fatty acid species and toxicity phenotypes remains poorly understood. However, structural characterization and functional profiling of human plasma free fatty acids (FFAs) analysis has revealed that FFAs are located either in the toxic cluster or in the cluster that is transcriptionally responsive to lipotoxic stress and creates genetic risk factors. Genome-wide short hairpin RNA screen has identified more than 350 genes modulating lipotoxicity. Hypertrophic adipocytes in obese adipose are both unable to expand further to store excess lipids in the diet and are resistant to the antilipolytic action of insulin. In addition to lipolysis, the inability of packaging the excess lipids into lipid droplets causes circulating fatty acids to reach toxic levels in non-adipose tissues. Deleterious effects of accumulated lipid in non-adipose tissues are known as lipotoxicity. Although triglycerides serve a storage function for long-chain non-esterified fatty acid and their products such as ceramide and diacylglycerols (DAGs), overloading of palmitic acid fraction of saturated fatty acids (SFAs) raises ceramide levels. The excess DAG and ceramide load create harmful effects on multiple organs and systems, inducing chronic inflammation in obesity. Thus, lipotoxic inflammation results in ß cells death and pancreatic islets dysfunction. Endoplasmic reticulum stress stimuli induce lipolysis by activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk) 1/2 signaling in adipocytes. However, palmitic acid-induced endoplasmic reticulum stress-c-Jun N-terminal kinase (JNK)-autophagy axis in hypertrophic adipocytes is a pro-survival mechanism against endoplasmic reticulum stress and cell death induced by SFAs. Endoplasmic reticulum-localized acyl-coenzyme A (CoA): glycerol-3-phosphate acyltransferase (GPAT) enzymes are mediators of lipotoxicity, and inhibiting these enzymes has therapeutic potential for lipotoxicity. Lipotoxicity increases the number of autophagosomes, which engulf palmitic acid, and thus suppress the autophagic turnover. Fatty acid desaturation promotes palmitate detoxification and storages into triglycerides. As therapeutic targets of glucolipotoxicity, in addition to caloric restriction and exercise, there are four different pharmacological approaches, which consist of metformin, glucagon-like peptide 1 (GLP-1) receptor agonists, peroxisome proliferator-activated receptor-gamma (PPARγ) ligands thiazolidinediones, and chaperones are still used in clinical practice. Furthermore, induction of the brown fat-like phenotype with the mixture of eicosapentanoic acid and docosahexaenoic acid appears as a potential therapeutic application for treatment of lipotoxicity.

Thermophilic and mesophilic digestion of high-solid oily food waste: How to ensure long-term and stable continuous operation?

Commonly high lipid in food waste confronts anaerobic digestion with improved energy production and also inhibition risk from the intermediate long chain fatty acids (LCFAs). Combined with operation challenges from anaerobic digestion of food waste itself, coping strategies are necessitated to ensure stable operation for oily food waste (OFW). A parallel thermophilic (TD) and mesophilic digestion (MD) of high-solid OFW was conducted and operated continuously for a long term. It was clarified that challenges were mainly from acidification, trace metal deficiency and LCFA inhibition. Acidification resulted in an abrupt pH decline to even below 6.00, and over 75% drop of biogas production rate. In addition to the requirements of saturated strong alkali to maintain an appropriate range, supplementation of trace metals were proven effective in counteracting the sharp decrease of biogas production rate. The TD was observed more competent in coping with the acidification than the MD, while the TD needed more supplementation of trace metals at approximately 0.10 mg Fe/g chemical oxygen demand (COD)added, 0.01 mg Co/g CODadded and 0.01 mg Ni/g CODadded. The TD was more adaptable in LCFA conversion due to the stronger ability of overcoming the palmitic acid (C16:0) accumulation. The MD experienced a prolonged recovery period owing to LCFA inhibition shortly after acidification. Similar operation performance was ultimately achieved for the TD and MD by the counteractions, with a methane yield and volatile solids (VS) removal efficiency at about 0.60 L/g VSadded and 75.0%, respectively. In summary, combined pH control and trace metal supplementation, and prevention and recovery of LCFA inhibition were necessary for the stability insurance of a long-term continuous digestion of oily food waste.

ELOVL4 Mutations That Cause Spinocerebellar Ataxia-34 Differentially Alter Very Long Chain Fatty Acid Biosynthesis.

The FA Elongase-4 (ELOVL4) enzyme mediates biosynthesis of both very long chain (VLC)-PUFAs and VLC-saturated FA (VLC-SFAs). VLC-PUFAs play critical roles in retina and sperm function, whereas VLC-SFAs are predominantly associated with brain function and maintenance of the skin permeability barrier. While some ELOVL4 mutations cause Autosomal Dominant Stargardt-like Macular Dystrophy (STGD3), other ELOVL4 point mutations, such as L168F and W246G, affect the brain and/or skin, leading to Spinocerebellar Ataxia-34 (SCA34) and Erythrokeratodermia variabilis. The mechanisms by which these ELOVL4 mutations alter VLC-PUFA and VLC-SFA biosynthesis to cause the different tissue-specific pathologies are not well understood. To understand how these mutations alter VLC-PUFA and VLC-SFA biosynthesis, we expressed WT-ELOVL4, L168F, and W246G ELOVL4 variants in cell culture and supplemented the cultures with VLC-PUFA or VLC-SFA precursors. Total lipids were extracted, converted to FA methyl esters, and quantified by gas chromatography. We showed that L168F and W246G mutants were capable of VLC-PUFA biosynthesis. W246G synthesized and accumulated 32:6n3, while L168F exhibited gain of function in VLC-PUFA biosynthesis as it made 38:5n3, which we did not detect in WT-ELOVL4 or W246G-expressing cells. However, compared with WT-ELOVL4, both L168F and W246G mutants were deficient in VLC-SFA biosynthesis, especially the W246G protein, which showed negligible VLC-SFA biosynthesis. These results suggest VLC-PUFA biosynthetic capabilities of L168F and W246G in the retina, which may explain the lack of retinal phenotype in SCA34. Defects in VLC-SFA biosynthesis by these variants may be a contributing factor to the pathogenic mechanism of SCA34 and Erythrokeratodermia variabilis.

Class A lysophosphatidic acid acyltransferase 2 from Camelina sativa promotes very long-chain fatty acids accumulation in phospholipid and triacylglycerol.

Very long-chain fatty acids (VLCFAs) are important industrial raw materials and can be produced by genetically modified oil plants. For a long time, class A lysophosphatidic acid acyltransferase (LPAT) was considered unable to promote the accumulation of VLCFA in oil crops. The bottlenecks that the transgenic high VLCFA lines have an oil content penalty and the low amount of VLCFA in phosphatidylcholine remains intractable. In the present study, a class A LPAT2 from Camelina sativa (CsaLPAT2) promoting VLCFAs accumulation in phospholipid was found. Overexpression of CsaLPAT2 alone in Arabidopsis seeds significantly increased the VLCFA content in triacylglycerol, including C20:0, C20:2, C20:3, C22:0, and C22:1. The proportion of phosphatidic acid molecules containing VLCFAs in transgenic seeds reached up to 45%, which was 2.8-fold greater than that in wild type. The proportion of phosphatidylcholine and diacylglycerol molecules containing VLCFAs also increased significantly. Seed size in CsaLPAT2 transgenic lines showed a slight increase without an oil content penalty. The total phospholipid content in the seed of the CsaLPAT2 transgenic line was significantly increased. Furthermore, the function of class A LPAT in promoting the accumulation of VLCFAs is conserved in the representative oil crops of Brassicaceae, such as C. sativa, Arabidopsis thaliana, Brassica napus, Brassica rapa, and Brassica oleracea. The findings of this study provide a promising gene resource for the production of VLCFAs.

Beyond THC and Endocannabinoids.

Research in the cannabinoid field, namely on phytocannabinoids, the endogenous cannabinoids anandamide and 2-arachidonoyl glycerol and their metabolizing and synthetic enzymes, the cannabinoid receptors, and anandamide-like cannabinoid compounds, has expanded tremendously over the last few years. Numerous endocannabinoid-like compounds have been discovered. The Cannabis plant constituent cannabidiol (CBD) was found to exert beneficial effects in many preclinical disease models ranging from epilepsy, cardiovascular disease, inflammation, and autoimmunity to neurodegenerative and kidney diseases and cancer. CBD was recently approved in the United States for the treatment of rare forms of childhood epilepsy. This has triggered the development of many CBD-based products for human use, often with overstated claims regarding their therapeutic effects. In this article, the recently published research on the chemistry and biological effects of plant cannabinoids (specifically CBD), endocannabinoids, certain long-chain fatty acid amides, and the variety of relevant receptors is critically reviewed.

Metabolic engineering of oleaginous yeast in the lipogenic phase enhances production of nervonic acid.

Insufficient biosynthesis efficiency during the lipogenic phase can be a major obstacle to engineering oleaginous yeasts to overproduce very long-chain fatty acids (VLCFAs). Taking nervonic acid (NA, C24:1) as an example, we overcame the bottleneck to overproduce NA in an engineered Rhodosporidium toruloides by improving the biosynthesis of VLCFAs during the lipogenic phase. First, evaluating the catalytic preferences of three plant-derived ketoacyl-CoA synthases (KCSs) rationally guided reconstructing an efficient NA biosynthetic pathway in R. toruloides. More importantly, a genome-wide transcriptional analysis endowed clues to strengthen the fatty acid elongation (FAE) module and identify/use lipogenic phase-activated promoter, collectively addressing the stagnation of NA accumulation during the lipogenic phase. The best-designed strain exhibited a high NA content (as the major component in total fatty acid [TFA], 46.3%) and produced a titer of 44.2 g/L in a 5 L bioreactor. The strategy developed here provides an engineering framework to establish the microbial process of producing valuable VLCFAs in oleaginous yeasts.

Nervonic acid and its sphingolipids: Biological functions and potential food applications.

Nervonic acid, a 24-carbon fatty acid with only one double bond at the 9th carbon (C24:1n-9), is abundant in the human brain, liver, and kidney. It not only functions in free form but also serves as a critical component of sphingolipids which participate in many biological processes such as cell membrane formation, apoptosis, and neurotransmission. Recent studies show that nervonic acid supplementation is not only beneficial to human health but also can improve the many medical conditions such as neurological diseases, cancers, diabetes, obesity, and their complications. Nervonic acid and its sphingomyelins serve as a special material for myelination in infants and remyelination patients with multiple sclerosis. Besides, the administration of nervonic acid is reported to reduce motor disorder in mice with Parkinson's disease and limit weight gain. Perturbations of nervonic acid and its sphingolipids might lead to the pathogenesis of many diseases and understanding these mechanisms is critical for investigating potential therapeutic approaches for such diseases. However, available studies about this aspect are limited. In this review, relevant findings about functional mechanisms of nervonic acid have been comprehensively and systematically described, focusing on four interconnected functions: cellular structure, signaling, anti-inflammation, lipid mobilization, and their related diseases.