Stearate-derived very long-chain fatty acids are indispensable to tumor growth.

Reprogramming of lipid metabolism is emerging as a hallmark of cancer, yet involvement of specific fatty acids (FA) species and related enzymes in tumorigenesis remains unclear. While previous studies have focused on involvement of long-chain fatty acids (LCFAs) including palmitate in cancer, little attention has been paid to the role of very long-chain fatty acids (VLCFAs). Here, we show that depletion of acetyl-CoA carboxylase (ACC1), a critical enzyme involved in the biosynthesis of fatty acids, inhibits both de novo synthesis and elongation of VLCFAs in human cancer cells. ACC1 depletion markedly reduces cellular VLCFA but only marginally influences LCFA levels, including palmitate that can be nutritionally available. Therefore, tumor growth is specifically susceptible to regulation of VLCFAs. We further demonstrate that VLCFA deficiency results in a significant decrease in ceramides as well as downstream glucosylceramides and sphingomyelins, which impairs mitochondrial morphology and renders cancer cells sensitive to oxidative stress and cell death. Taken together, our study highlights that VLCFAs are selectively required for cancer cell survival and reveals a potential strategy to suppress tumor growth.

MSC-Derived Small Extracellular Vesicles Exert Cardioprotective Effect Through Reducing VLCFAs and Apoptosis in Human Cardiac Organoid IRI Model.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs' ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

Four-dimensional lipidomics profiling in X-linked adrenoleukodystrophy using trapped ion mobility mass spectrometry.

Lipids play pivotal roles in an extensive range of metabolic and physiological processes. In recent years, the convergence of trapped ion mobility spectrometry and MS has enabled 4D-lipidomics, a highly promising technology for comprehensive lipid analysis. 4D-lipidomics assesses lipid annotations across four distinct dimensions-retention time, collisional cross section, m/z (mass-to-charge ratio), and MS/MS spectra-providing a heightened level of confidence in lipid annotation. These advantages prove particularly valuable when investigating complex disorders involving lipid metabolism, such as adrenoleukodystrophy (ALD). ALD is characterized by the accumulation of very-long-chain fatty acids (VLCFAs) due to pathogenic variants in the ABCD1 gene. A comprehensive 4D-lipidomics strategy of ALD fibroblasts demonstrated significant elevations of various lipids from multiple classes. This indicates that the changes observed in ALD are not confined to a single lipid class and likely impacts a broad spectrum of lipid-mediated physiological processes. Our findings highlight the incorporation of mainly saturated and monounsaturated VLCFA variants into a range of lipid classes, encompassing phosphatidylcholines, triacylglycerols, and cholesterol esters. These include ultra-long-chain fatty acids with a length of up to thirty carbon atoms. Lipid species containing C26:0 and C26:1 were the most frequently detected VLCFA lipids in our study. Furthermore, we report a panel of 121 new candidate biomarkers in fibroblasts, exhibiting significant differentiation between controls and individuals with ALD. In summary, this study demonstrates the capabilities of a 4D-lipid profiling workflow in unraveling novel insights into the intricate lipid modifications associated with metabolic disorders like ALD.

A very long chain fatty acid responsive transcription factor, MYB93, regulates lateral root development in Arabidopsis.

Lateral roots (LRs) are critical to root system architecture development in plants. Although the molecular mechanisms by which auxin regulates LR development have been extensively studied, several additional regulatory systems are hypothesized to be involved. Recently, the regulatory role of very long chain fatty acids (VLCFAs) has been shown in LR development. Our analysis showed that LTPG1 and LTPG2, transporters of VLCFAs, are specifically expressed in the developing LR primordium (LRP), while the number of LRs is reduced in the ltpg1/ltpg2 double mutant. Moreover, late LRP development was hindered when the VLCFA levels were reduced by the VLCFA synthesis enzyme mutant, kcs1-5. However, the details of the regulatory mechanisms of LR development controlled by VLCFAs remain unknown. In this study, we propose a novel method to analyze the LRP development stages with high temporal resolution using a deep neural network and identify a VLCFA-responsive transcription factor, MYB93, via transcriptome analysis of kcs1-5. MYB93 showed a carbon chain length-specific expression response following treatment of VLCFAs. Furthermore, myb93 transcriptome analysis suggested that MYB93 regulated the expression of cell wall organization genes. In addition, we also found that LTPG1 and LTPG2 are involved in LR development through the formation of root cap cuticle, which is different from transcriptional regulation by VLCFAs. Our results suggest that VLCFA is a regulator of LRP development through transcription factor-mediated regulation of gene expression and the transportation of VLCFAs is also involved in LR development through root cap cuticle formation.

Genome-wide identification and molecular evolution of elongation family of very long chain fatty acids proteins in Cyrtotrachelus buqueti.

To reveal the molecular function of elongation family of very long chain fatty acids(ELO) protein in Cyrtotrachelus buqueti, we have identified 15 ELO proteins from C.buqueti genome. 15 CbuELO proteins were located on four chromosomes. Their isoelectric points ranged from 9.22 to 9.68, and they were alkaline. These CbuELO proteins were stable and hydrophobic. CbuELO proteins had transmembrane movement, and had multiple phosphorylation sites. The secondary structure of CbuELO proteins was mainly α-helix. A total of 10 conserved motifs were identified in CbuELO protein family. Phylogenetic analysis showed that molecular evolutionary relationships of ELO protein family between C. buqueti and Tribolium castaneum was the closest. Developmental transcriptome analysis indicated that CbuELO10, CbuELO13 and CbuELO02 genes were key enzyme genes that determine the synthesis of very long chain fatty acids in pupae and eggs, CbuELO6 and CbuELO7 were that in the male, and CbuELO8 and CbuELO11 were that in the larva. Transcriptome analysis under different temperature conditions indicated that CbuELO1, CbuELO5, CbuELO12 and CbuELO14 participated in regulating temperature stress responses. Transcriptome analysis at different feeding times showed CbuELO12 gene expression level in all feeding time periods was significant downregulation. The qRT-PCR experiment verified expression level changes of CbuELO gene family under different temperature and feeding time conditions. Protein-protein interaction analysis showed that 9 CbuELO proteins were related to each other, CbuELO1, CbuELO4 and CbuELO12 had more than one interaction relationship. These results lay a theoretical foundation for further studying its molecular function during growth and development of C. buqueti.

3-ketoacyl-CoA synthase 19 contributes to the biosynthesis of seed lipids and cuticular wax in Arabidopsis and abiotic stress tolerance.

Very-long-chain fatty acids (VLCFAs) are essential precursors for plant membrane lipids, cuticular waxes, suberin, and storage oils. Integral to the fatty acid elongase (FAE) complex, 3-ketoacyl-CoA synthases (KCSs) function as crucial enzymes in the VLCFA pathway, determining the chain length of VLCFA. This study explores the in-planta role of the KCS19 gene. KCS19 is predominantly expressed in leaves and stem epidermis, sepals, styles, early silique walls, beaks, pedicels, and mature embryos. Localized in the endoplasmic reticulum, KCS19 interacts with other FAE proteins. kcs19 knockout mutants displayed reduced total wax and wax crystals, particularly alkanes, while KCS19 overexpression increased these components and wax crystals. Moreover, the cuticle permeability was higher for the kcs19 mutants compared to the wild type, rendering them more susceptible to drought and salt stress, whereas KCS19 overexpression enhanced drought and salt tolerance. Disrupting KCS19 increased C18 species and decreased C20 and longer species in seed fatty acids, indicating its role in elongating C18 to C20 VLCFAs, potentially up to C24 for seed storage lipids. Collectively, KCS19-mediated VLCFA synthesis is required for cuticular wax biosynthesis and seed storage lipids, impacting plant responses to abiotic stress.

Tetracosanoic acids produced by 3-ketoacyl-CoA synthase 17 are required for synthesizing seed coat suberin in Arabidopsis.

Very long-chain fatty acids (VLCFAs) are precursors for the synthesis of membrane lipids, cuticular waxes, suberins, and storage oils in plants. 3-Ketoacyl CoA synthase (KCS) catalyzes the condensation of C2 units from malonyl-CoA to acyl-CoA, the first rate-limiting step in VLCFA synthesis. In this study, we revealed that Arabidopsis KCS17 catalyzes the elongation of C22-C24 VLCFAs required for synthesizing seed coat suberin. Histochemical analysis of Arabidopsis plants expressing GUS (ß-glucuronidase) under the control of the KCS17 promoter revealed predominant GUS expression in seed coats, petals, stigma, and developing pollen. The expression of KCS17:eYFP (enhanced yellow fluorescent protein) driven by the KCS17 promoter was observed in the outer integument1 of Arabidopsis seed coats. The KCS17:eYFP signal was detected in the endoplasmic reticulum of tobacco epidermal cells. The levels of C22 VLCFAs and their derivatives, primary alcohols, α,ω-alkane diols, ω-hydroxy fatty acids, and α,ω-dicarboxylic acids increased by ~2-fold, but those of C24 VLCFAs, ω-hydroxy fatty acids, and α,ω-dicarboxylic acids were reduced by half in kcs17-1 and kcs17-2 seed coats relative to the wild type (WT). The seed coat of kcs17 displayed decreased autofluorescence under UV and increased permeability to tetrazolium salt compared with the WT. Seed germination and seedling establishment of kcs17 were more delayed by salt and osmotic stress treatments than the WT. KCS17 formed homo- and hetero-interactions with KCR1, PAS2, and ECR, but not with PAS1. Therefore, KCS17-mediated VLCFA synthesis is required for suberin layer formation in Arabidopsis seed coats.

PASTICCINO2 interacting with Golgi Anti-Apoptotic Proteins resists endoplasmic reticulum stress dependent on very long-chain fatty acids.

The endoplasmic reticulum (ER) is crucial for maintaining cell homeostasis because it is the primary site for synthesizing secreted and transmembrane proteins and lipids. The unfolded protein response (UPR) is activated to restore ER homeostasis under ER stress. However, the relationship between lipids and the ER stress response in plants is not well understood. Arabidopsis Golgi anti-apoptotic proteins (GAAPs) are involved in resisting ER stress. To elucidate the function of GAAPs, PASTICCINO2 (PAS2), involved in very long-chain fatty acid (VLCFA) synthesis, was found to interact with GAAPs and IRE1. Single pas2 and gaap1/gaap2pas2 double mutants exhibited increased seedling damage and impaired UPR response under chronic ER stress. Site mutation combined with genetic analysis revealed that the role of PAS2 in resisting ER stress depended on its VLCFA synthesis domain. VLCFA contents were upregulated under ER stress, which required GAAPs. Exogenous VLCFAs partially restored the defect in UPR upregulation caused by PAS2 or GAAP mutations under chronic ER stress. These findings demonstrate that the association of PAS2 with GAAPs confers plant resistance to ER stress by regulating VLCFA synthesis and the UPR. This provides a basis for further studies on the connection between lipids and cell fate decisions under stress.

A leucine-rich repeat receptor kinase as a regulator in the cuticular wax deposition in sorghum.

Cuticular wax (CW) is the first defensive barrier of plants that forms a waterproof barrier, protects the plant from desiccation, and defends against insects, pathogens, and UV radiation. Sorghum, an important grass crop with high heat and drought tolerance, exhibits a much higher wax load than other grasses and the model plant Arabidopsis. In this study, we explored the regulation of sorghum CW biosynthesis using a bloomless mutant. The CW on leaf sheaths of bloomless 41 (bm41) mutant showed significantly reduced very long-chain fatty acids (VLCFAs), triterpenoids, alcohols, and other wax components, with an overall 86% decrease in total wax content compared to the wild-type. Notably, the 28-carbon and 30-carbon VLCFAs were decreased in the mutants. Using bulk segregant analysis, we identified the causal gene of the bloomless phenotype as a leucine-rich repeat transmembrane protein kinase. Transcriptome analysis of the wild-type and bm41 mutant leaf sheaths revealed BM41 as a positive regulator of lipid biosynthesis and steroid metabolism. BM41 may regulate CW biosynthesis by regulating the expression of the gene encoding 3-ketoacyl-CoA synthase 6. Identification of BM41 as a new regulator of CW biosynthesis provides fundamental knowledge for improving grass crops' heat and drought tolerance by increasing CW.

Potential Clinical Benefit of Very Long Chain Fatty Acid Supplementation in Spinocerebellar Ataxia Type 34.

Spinocerebellar ataxia type 34 (SCA34) is a dominantly inherited disease that causes late-onset ataxia, in association with skin lesions in the form of erythrokeratodermia variabilis. It is caused by mutations in the ELOVL4 gene, which encodes for the ELOVL4 protein and has the function of lengthening very long chain (VLC) fatty acids (FA), which are important components of central myelin. The aim of this work was to review the medical literature on the biochemical abnormalities of SCA34, and based on the obtained information, to propose supplementation of deficient FAs. A review of the ad hoc medical literature was performed. Plasma levels of the ELOVL4 products C32, C34 and C36 FA have not been reported in SCA34 yet. However, pathogenic variants of ELOVL4 revealed deficient biosynthesis of C28, C30, C32, C34 and C36 FA compared to WT in cell cultures, and the levels of ceramides and phosphatidylcholines containing ≥ 34 C FA were decreased compared to WT in HeLa cells expressing mutant SCA34 proteins. Besides, a pathological study of SCA34 revealed myelin destruction and loss of oligodendrocytes in cerebral and cerebellar white matter. Levels of VLC-FA should be determined, to identify specifically deficient FAs in SCA34. Cerebellar ataxia could possibly be improved by administration of the deficient FAs, as found in SCA38 with supplementation of docosahexaenoic acid. The authors suggest investigators with access to SCA34, to take into consideration this therapeutic hypothesis, and try to verify the potential efficacy of administration of VLCFA in this disease.

Evolution and molecular basis of substrate specificity in a 3-ketoacyl-CoA synthase gene cluster from Populus trichocarpa.

Very long chain fatty acids (VLCFAs) are precursors to sphingolipids, glycerophospholipids, and plant cuticular waxes. In plants, members of a large 3-ketoacyl-CoA synthase (KCS) gene family catalyze the substrate-specific elongation of VLCFAs. Although it is well understood that KCSs have evolved to use diverse substrates, the underlying molecular determinants of their specificity are still unclear. In this study, we exploited the sequence similarity of a KCS gene cluster from Populus trichocarpa to examine the evolution and molecular determinants of KCS substrate specificity. Functional characterization of five members (PtKCS1, 2, 4, 8, 9) in yeast showed divergent product profiles based on VLCFA length, saturation, and position of the double bond. In addition, homology models, rationally designed chimeras, and site-directed mutants were used to identify two key regions (helix-4 and position 277) as being major determinants of substrate specificity. These results were corroborated with chimeras involving a more distantly related KCS, PtCER6 (the poplar ortholog of the Arabidopsis CER6), and used to show that helix-4 is necessary for the modulatory effect of PtCER2-like5 on KCS substrate specificity. The role of position 277 in limiting product length was further tested by substitution with smaller amino acids, which shifted specificity toward longer products. Finally, treatment with KCS inhibitors (K3 herbicides) showed varying inhibitor sensitivities between the duplicated paralogs despite their sequence similarity. Together, this work sheds light on the molecular mechanisms driving substrate diversification in the KCS family and lays the groundwork for tailoring the production of specific VLCFAs.

A very-long-chain fatty acid synthesis gene, SD38, influences plant height by activating ethylene biosynthesis in rice.

As an important trait in crop breeding, plant height is associated with lodging resistance and yield. With the identification and cloning of several semi-dwarfing genes, increasing numbers of semi-dwarf cultivars have emerged, which has led to a 'green revolution' in rice (Oryza sativa) production. In this study, we identified a rice semi-dwarf mutant, semi-dwarf 38 (sd38), which showed significantly reduced cell length. SD38 encodes a fatty acid elongase, ß-ketoacyl-CoA synthase, which is involved in the synthesis of very-long-chain fatty acids (VLCFAs). Expression analysis showed that SD38 was localized on the membrane of the endoplasmic reticulum, and was expressed in all analyzed tissues with differential abundance. The mutation of SD38 affected lipid metabolism in the sd38 mutant. A functional complementarity test in Saccharomyces cerevisiae indicated that SD38 was capable of complementing the deficiency of ELO3p activity in BY4741-elo3 knockout yeast cells by participating in the synthesis of C24:0 VLCFA. Significant changes were observed in the expression of genes involved in ethylene synthesis, which resulted in reduced content of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the sd38 mutant. Exogenously supplied VLCFA (C24:0) increased the expression levels of OsACS3, OsACS4, and OsACO7 and the plant height of sd38 mutant seedlings, similar to the effect of exogenous application of ACC and ethephon. These results reveal a relationship among VLCFAs, ethylene biosynthesis, and plant height and improve our understanding of plant height development in crops.

SCA34 caused by ELOVL4 L168F mutation is a lysosomal lipid storage disease sharing pathology features with neuronal ceroid lipofuscinosis and peroxisomal disorders.

Spinocerebellar ataxia 34 (SCA34) is a late-onset progressive ataxia caused by a mutation in ELOVL4, a gene involved in the biosynthesis of very long-chain fatty acids (VLCFAs). We performed post-mortem neuropathological examinations on four SCA34 patients with the ELOVL4 L168F mutation and compared the findings to age-matched controls. Specific gross findings of SCA34 were limited to pontocerebellar atrophy. On light microscopy, pontine base showed neuronal loss and storage of an autofluorescent lipopigment positive on oil red O, PAS and Hale's colloidal iron and negative on Alcian blue and Luxol fast blue (LFB). Among the swollen neurons were abundant CD68+ /CD163+ /IBA1- macrophages laden with a material with similar histochemical profile as in neurons except for the lack of autofluorescence and oil red O positivity and the presence of needle-like birefringent inclusions. Normal resting IBA1 + microglia were generally absent from pontine base nuclei but present in normal numbers elsewhere in the pons. In dentate nucleus neurons, atrophy was milder than in the pontine base and the coarser storage material was LFB-positive, closely resembling lipofuscin. On electron microscopy, dentate nucleus neurons showed neuronal storage of tridimensionally organized trilaminar spicules within otherwise normal lipofuscin, while in the more affected pontine base neurons, lipofuscin was almost completely replaced by the storage material. Storage macrophages were tightly packed with stacks of unorganized trilaminar spicules, reminiscent of the storage material seen in peroxisomal disorders and thought to represent VLCFAs incorporated in complex polar lipids. In summary, we provide histochemical and ultrastructural evidence that SCA34 is a lipid storage disease, the first among the currently known SCAs, and that the storage lipid is accumulating within neuronal lipofuscin. Our findings suggest that the storage lipid is similar to the one accumulating in non-neuronal cells in peroxisomal disorders and provide the first ultrastructural description of this type of material within neurons.

Increased expression of ELOVL7 contributes to production of inflammatory cytokines in THP-1 cell-derived M1-like macrophages.

The elevation of intracellular very long-chain fatty acids (VLCFAs) augments pro-inflammatory activity of macrophages. VLCFAs are considered to function as regulators in macrophage inflammatory responses; however, the precise mechanism of regulating the production of VLCFAs is unclear. In this study, we focused on elongation of the very­long­chain fatty acid protein (ELOVL) family, rate-determining enzymes for VLCFA synthesis, in macrophages. ELOVL7 mRNA was upregulated in human monocytic THP-1 cell-derived M1-like macrophages. Metascape analysis using the RNA-seq data set showed the involvement of NF-κB and STAT1 in transcriptional regulation of ELOVL7 highly correlated genes. Gene ontology (GO) enrichment analysis suggested that ELOVL7 highly correlated genes were closely associated with multiple pro-inflammatory responses, including response to virus and positive regulation of NF-κB signaling. Consistent with RNA-seq analysis, the NF-κB inhibitor BAY11-7082, but not the STAT1 inhibitor fludarabine, canceled ELOVL7 upregulation in M1-like macrophages. ELOVL7 knockdown decreased interleukin (IL)-6 and IL-12/IL-23 p40 production. Moreover, RNA-seq analysis of plasmacytoid dendritic cells (pDCs) revealed that ELOVL7 was upregulated in pDCs treated with TLR7 and TLR9 agonists. In conclusion, we propose that ELOVL7 is a novel pro-inflammatory gene that is upregulated by inflammatory stimuli, and regulates M1-like macrophage and pDC functions.

Physiological effects of inactivation and the roles of Elovl3/ELOVL3 in maintaining ocular homeostasis.

Recently, elongase of very long chain fatty acids-3 (ELOVL3) was demonstrated to play a pivotal role in physiology and biochemistry of the ocular surface by maintaining a proper balance in the lipid composition of meibum. The goal of this study was to further investigate the effects of ELOVL3 ablation in homozygous Elovl3-knockout mice (E3hom) in comparison with age and sex matched wild-type controls (E3wt). Slit lamp examination of the ocular surface of mice, and histological examination of their ocular tissues, highlighted a severe negative impact of Elovl3 inactivating mutation on the Meibomian glands (MG) and conjunctiva of mice. MG transcriptomes of the E3hom and E3wt mice were assessed and revealed a range of up- and downregulated genes related to lipid biosynthesis, inflammation, and stress response, compared with E3wt mice. Heat stage polarized light microscopy was used to assess melting characteristics of normal and abnormal meibum. The loss of Elovl3 led to a 8°C drop in the melting temperature of meibum in E3hom mice, and increased its fluidity. Also noted were the excessive accumulation of lipid material and tears around the eye and severe ocular inflammation, among other abnormalities.

D-bifunctional protein deficiency caused by splicing variants in a neonate with severe peroxisomal dysfunction and persistent hypoglycemia.

D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient's father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.

Overexpression of ß-Ketoacyl CoA Synthase 2B.1 from Chenopodium quinoa Promotes Suberin Monomers' Production and Salt Tolerance in Arabidopsis thaliana.

Very-long-chain fatty acids (VLCFAs) are precursors for the synthesis of various lipids, such as triacylglycerols, sphingolipids, cuticular waxes, and suberin monomers, which play important roles in plant growth and stress responses. However, the underlying molecular mechanism regulating VLCFAs' biosynthesis in quinoa (Chenopodium quinoa Willd.) remains unclear. In this study, we identified and functionally characterized putative 3-ketoacyl-CoA synthases (KCSs) from quinoa. Among these KCS genes, CqKCS2B.1 showed high transcript levels in the root tissues and these were rapidly induced by salt stress. CqKCS2B.1 was localized to the endoplasmic reticulum. Overexpression of CqKCS2B.1 in Arabidopsis resulted in significantly longer primary roots and more lateral roots. Ectopic expression of CqKCS2B.1 in Arabidopsis promoted the accumulation of suberin monomers. The occurrence of VLCFAs with C22-C24 chain lengths in the overexpression lines suggested that CqKCS2B.1 plays an important role in the elongation of VLCFAs from C20 to C24. The transgenic lines of overexpressed CqKCS2B.1 showed increased salt tolerance, as indicated by an increased germination rate and improved plant growth and survival under salt stress. These findings highlight the significant role of CqKCS2B.1 in VLCFAs' production, thereby regulating suberin biosynthesis and responses to salt stress. CqKCS2B.1 could be utilized as a candidate gene locus to breed superior, stress-tolerant quinoa cultivars.

Synthesis of C20-38 Fatty Acids in Plant Tissues.

Very-long-chain fatty acids (VLCFA) are involved in a number of important plant physiological functions. Disorders in the expression of genes involved in the synthesis of VLCFA lead to a number of phenotypic consequences, ranging from growth retardation to the death of embryos. The elongation of VLCFA in the endoplasmic reticulum (ER) is carried out by multiple elongase complexes with different substrate specificities and adapted to the synthesis of a number of products required for a number of metabolic pathways. The information about the enzymes involved in the synthesis of VLCFA with more than 26 atoms of Carbon is rather poor. Recently, genes encoding enzymes involved in the synthesis of both regular-length fatty acids and VLCFA have been discovered and investigated. Polyunsaturated VLCFA in plants are formed mainly by 20:1 elongation into new monounsaturated acids, which are then imported into chloroplasts, where they are further desaturated. The formation of saturated VLCFA and their further transformation into a number of aliphatic compounds included in cuticular waxes and suberin require the coordinated activity of a large number of different enzymes.

AKR2A interacts with KCS1 to improve VLCFAs contents and chilling tolerance of Arabidopsis thaliana.

Arabidopsis thaliana AKR2A plays an important role in plant responses to cold stress. However, its exact function in plant resistance to cold stress remains unclear. In the present study, we found that the contents of very long-chain fatty acids (VLCFAs) in akr2a mutants were decreased, and the expression level of KCS1 was also reduced. Overexpression of KCS1 in the akr2a mutants could enhance VLCFAs contents and chilling tolerance. Yeast-2-hybrid and bimolecular fluorescence complementation (BIFC) results showed that the transmembrane motif of KCS1 interacts with the PEST motif of AKR2A both in vitro and in vivo. Overexpression of KCS1 in akr2a mutants rescued akr2a mutant phenotypes, including chilling sensitivity and a decrease of VLCFAs contents. Moreover, the transgenic plants co-overexpressing AKR2A and KCS1 exhibited a greater chilling tolerance than the plants overexpressing AKR2A or KCS1 alone, as well as the wild-type. AKR2A knockdown and kcs1 knockout mutants showed the worst performance under chilling conditions. These results indicate that AKR2A is involved in chilling tolerance via an interaction with KCS1 to affect VLCFA biosynthesis in Arabidopsis.

Drug discovery for X-linked adrenoleukodystrophy: An unbiased screen for compounds that lower very long-chain fatty acids.

X-linked adrenoleukodystrophy (XALD) is a genetic neurologic disorder with multiple phenotypic presentations and limited therapeutic options. The childhood cerebral phenotype (CCALD), a fatal demyelinating disorder affecting about 35% of patients, and the adult-onset adrenomyeloneuropathy (AMN), a peripheral neuropathy affecting 40%-45% of patients, are both caused by mutations in the ABCD1 gene. Both phenotypes are characterized biochemically by elevated tissue and plasma levels of saturated very long-chain fatty acids (VLCFA), and an increase in plasma cerotic acid (C26:0), along with the clinical presentation, is diagnostic. Administration of oils containing monounsaturated fatty acids, for example, Lorenzo's oil, lowers patient VLCFA levels and reduced the frequency of development of CCALD in presymptomatic boys. However, this therapy is not currently available. Hematopoietic stem cell transplant and gene therapy remain viable therapies for boys with early progressive cerebral disease. We asked whether any existing approved drugs can lower VLCFA and thus open new therapeutic possibilities for XALD. Using SV40-transformed and telomerase-immortalized skin fibroblasts from an XALD patient, we conducted an unbiased screen of a library of approved drugs and natural products for their ability to decrease VLCFA, using measurement of C26:0 in lysophosphatidyl choline (C26-LPC) by tandem mass spectrometry as the readout. While several candidate drugs were initially identified, further testing in primary fibroblast cell lines from multiple CCALD and AMN patients narrowed the list to one drug, the anti-hypertensive drug irbesartan. In addition to lowering C26-LPC, levels of C26:0 and C28:0 in total fibroblast lipids were reduced. The effect of irbesartan was dose dependent between 2 and 10 µM. When male XALD mice received orally administered irbesartan at a dose of 10 mg/kg/day, there was no reduction in plasma C26-LPC. However, irbesartan failed to lower mouse fibroblast C26-LPC consistently. The results of these studies indicate a potential therapeutic benefit of irbesartan in XALD that should be validated by further study.