Structure, mechanism, and inhibition of Hedgehog acyltransferase.

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

Methyltransferase Setd2 prevents T cell-mediated autoimmune diseases via phospholipid remodeling.

Coordinated metabolic reprogramming and epigenetic remodeling are critical for modulating T cell function and differentiation. However, how the epigenetic modification controls Th17/Treg cell balance via metabolic reprogramming remains obscure. Here, we find that Setd2, a histone H3K36 trimethyltransferase, suppresses Th17 development but promotes iTreg cell polarization via phospholipid remodeling. Mechanistically, Setd2 up-regulates transcriptional expression of lysophosphatidylcholine acyltransferase 4 (Lpcat4) via directly catalyzing H3K36me3 of Lpcat4 gene promoter in T cells. Lpcat4-mediated phosphatidylcholine PC(16:0,18:2) generation in turn limits endoplasmic reticulum stress and oxidative stress. These changes decrease HIF-1α transcriptional activity and thus suppress Th17 but enhance Treg development. Consistent with this regulatory paradigm, T cell deficiency of Setd2 aggravates neuroinflammation and demyelination in experimental autoimmune encephalomyelitis due to imbalanced Th17/Treg cell differentiation. Overall, our data reveal that Setd2 acts as an epigenetic brake for T cell-mediated autoimmunity through phospholipid remodeling, suggesting potential targets for treating neuroinflammatory diseases.

Lysophospholipid remodeling mediated by the LplT and Aas protein complex in the bacterial envelope.

Lysophospholipid transporter LplT and acyltransferase Aas consist of a lysophospholipid-remodeling system ubiquitously found in gram-negative microorganisms. LplT flips lysophospholipid across the inner membrane which is subsequently acylated by Aas on the cytoplasmic membrane surface. Our previous study showed that the proper functioning of this system is important to protecting Escherichia coli from phospholipase-mediated host attack by maintaining the integrity of the bacterial cell envelope. However, the working mechanism of this system is still unclear. Herein, we report that LplT and Aas form a membrane protein complex in E. coli which allows these two enzymes to cooperate efficiently to move lysophospholipids across the bacterial membrane and catalyze their acylation. The direct interaction of LplT and Aas was demonstrated both in vivo and in vitro with a binding affinity of 2.3 µM. We found that a cytoplasmic loop of LplT adjacent to the exit of the substrate translocation pathway plays an important role in maintaining its interaction with Aas. Aas contains an acyl-acyl carrier protein synthase domain and an acyl-transferase domain. Its interaction with LplT is mediated exclusively by its transferase domain. Mutations within the three loops near the putative catalytic site of the transferase domain, respectively, disrupt its interaction with LplT and lysophospholipid acylation activity. These results support a hypothesis of the functional coupling mechanism, in which LplT directly interacts with the transferase domain of Aas for specific substrate membrane migration, providing synchronization of substrate translocation and biosynthetic events.

The glycerophosphocholine acyltransferase Gpc1 contributes to phosphatidylcholine biosynthesis, long-term viability, and embedded hyphal growth in Candida albicans.

Candida albicans is a commensal fungus, opportunistic pathogen, and the most common cause of fungal infection in humans. The biosynthesis of phosphatidylcholine (PC), a major eukaryotic glycerophospholipid, occurs through two primary pathways. In Saccharomyces cerevisiae and some plants, a third PC synthesis pathway, the PC deacylation/reacylation pathway (PC-DRP), has been characterized. PC-DRP begins with the acylation of the lipid turnover product, glycerophosphocholine (GPC), by the GPC acyltransferase, Gpc1, to form Lyso-PC. Lyso-PC is then acylated by lysolipid acyltransferase, Lpt1, to produce PC. Importantly, GPC, the substrate for Gpc1, is a ubiquitous metabolite available within the host. GPC is imported by C. albicans, and deletion of the major GPC transporter, Git3, leads to decreased virulence in a murine model. Here we report that GPC can be directly acylated in C. albicans by the protein product of orf19.988, a homolog of ScGpc1. Through lipidomic studies, we show loss of Gpc1 leads to a decrease in PC levels. This decrease occurs in the absence of exogenous GPC, indicating that the impact on PC levels may be greater in the human host where GPC is available. A gpc1Δ/Δ strain exhibits several sensitivities to antifungals that target lipid metabolism. Furthermore, loss of Gpc1 results in both a hyphal growth defect in embedded conditions and a decrease in long-term cell viability. These results demonstrate for the first time the importance of Gpc1 and this alternative PC biosynthesis route (PC-DRP) to the physiology of a pathogenic fungus.

Neuroinvasive virus facilitates viral replication by employing lipid droplets to reduce arachidonic acid-induced ferroptosis.

Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.

A serine carboxypeptidase-like acyltransferase catalyzes consecutive four-step reactions of hydrolyzable tannin biosynthesis in Camellia oleifera.

Hydrolyzable tannins (HTs), a class of polyphenolic compounds found in dicotyledonous plants, are widely used in food and pharmaceutical industries because of their beneficial effects on human health. Although the biosynthesis of simple HTs has been verified at the enzymatic level, relevant genes have not yet been identified. Here, based on the parent ion-fragment ion pairs in the feature fragment data obtained using UPLC-Q-TOF-/MS/MS, galloyl phenolic compounds in the leaves of Camellia sinensis and C. oleifera were analyzed qualitatively and quantitatively. Correlation analysis between the transcript abundance of serine carboxypeptidase-like acyltransferases (SCPL-ATs) and the peak area of galloyl products in Camellia species showed that SCPL3 expression was highly correlated with HT biosynthesis. Enzymatic verification of the recombinant protein showed that CoSCPL3 from C. oleifera catalyzed the four consecutive steps involved in the conversion of digalloylglucose to pentagalloylglucose. We also identified the residues affecting the enzymatic activity of CoSCPL3 and determined that SCPL-AT catalyzes the synthesis of galloyl glycosides. The findings of this study provide a target gene for germplasm innovation of important cash crops that are rich in HTs, such as C. oleifera, strawberry, and walnut.

Identification of a 1-acyl-glycerol-3-phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis.

Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant in vivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA in vitro and were not able to rescue the growth defect of E. coli PlsC mutant in vivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

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

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

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.

Acat1/Soat1 knockout extends the mutant Npc1 mouse lifespan and ameliorates functional deficiencies in multiple organelles of mutant cells.

Multiple membrane organelles require cholesterol for proper function within cells. The Niemann-Pick type C (NPC) proteins export cholesterol from endosomes to other membrane compartments, including the endoplasmic reticulum (ER), plasma membrane (PM), trans-Golgi network (TGN), and mitochondria, to meet their cholesterol requirements. Defects in NPC cause malfunctions in multiple membrane organelles and lead to an incurable neurological disorder. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), a resident enzyme in the ER, converts cholesterol to cholesteryl esters for storage. In mutant NPC cells, cholesterol storage still occurs in an NPC-independent manner. Here we report the interesting finding that in a mutant Npc1 mouse (Npc1nmf), Acat1 gene (Soat1) knockout delayed the onset of weight loss, motor impairment, and Purkinje neuron death. It also improved hepatosplenic pathology and prolonged lifespan by 34%. In mutant NPC1 fibroblasts, ACAT1 blockade (A1B) increased cholesterol content associated with TGN-rich membranes and mitochondria, while decreased cholesterol content associated with late endosomes. A1B also restored proper localization of syntaxin 6 and golgin 97 (key proteins in membrane trafficking at TGN) and improved the levels of cathepsin D (a key protease in lysosome and requires Golgi/endosome transport for maturation) and ABCA1 (a key protein controlling cholesterol release at PM). This work supports the hypothesis that diverting cholesterol from storage can benefit multiple diseases that involve cholesterol deficiencies in cell membranes.

Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase.

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

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

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

Deciphering the biosynthesis of a novel lipid in Mycobacterium tuberculosis expands the known roles of the nitroreductase superfamily.

Mycobacterium tuberculosis's (Mtb) success as a pathogen is due in part to its sophisticated lipid metabolic programs, both catabolic and biosynthetic. Several of Mtb lipids have specific roles in pathogenesis, but the identity and roles of many are unknown. Here, we demonstrated that the tyz gene cluster in Mtb, previously implicated in resistance to oxidative stress and survival in macrophages, encodes the biosynthesis of acyl-oxazolones. Heterologous expression of tyzA (Rv2336), tyzB (Rv2338c) and tyzC (Rv2337c) resulted in the biosynthesis of C12:0-tyrazolone as the predominant compound, and the C12:0-tyrazolone was identified in Mtb lipid extracts. TyzA catalyzed the N-acylation of l-amino acids, with highest specificity for l-Tyr and l-Phe and lauroyl-CoA (kcat/KM = 5.9 ± 0.8 × 103 M-1s-1). In cell extracts, TyzC, a flavin-dependent oxidase (FDO) of the nitroreductase (NTR) superfamily, catalyzed the O2-dependent desaturation of the N-acyl-L-Tyr produced by TyzA, while TyzB, a ThiF homolog, catalyzed its ATP-dependent cyclization. The substrate preference of TyzB and TyzC appear to determine the identity of the acyl-oxazolone. Phylogenetic analyses revealed that the NTR superfamily includes a large number of broadly distributed FDOs, including five in Mtb that likely catalyze the desaturation of lipid species. Finally, TCA1, a molecule with activity against drug-resistant and persistent tuberculosis, failed to inhibit the cyclization activity of TyzB, the proposed secondary target of TCA1. Overall, this study identifies a novel class of Mtb lipids, clarifies the role of a potential drug target, and expands our understanding of the NTR superfamily.

Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1.

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

S-acylation of Sprouty and SPRED proteins by the S-acyltransferase zDHHC17 involves a novel mode of enzyme-substrate interaction.

S-acylation is an essential post-translational modification, which is mediated by a family of 23 zDHHC enzymes in humans. Several thousand proteins are modified by S-acylation; however, we lack a detailed understanding of how enzyme-substrate recognition and specificity is achieved. Previous work showed that the ankyrin repeat domain of zDHHC17 (ANK17) recognizes a short linear motif, known as the zDHHC ANK binding motif (zDABM) in substrate protein SNAP25, as a mechanism of substrate recruitment prior to S-acylation. Here, we investigated the S-acylation of the Sprouty and SPRED family of proteins by zDHHC17. Interestingly, although Sprouty-2 (Spry2) contains a zDABM that interacts with ANK17, this mode of binding is dispensable for S-acylation, and indeed removal of the zDABM does not completely ablate binding to zDHHC17. Furthermore, the related SPRED3 protein interacts with and is efficiently S-acylated by zDHHC17, despite lacking a zDABM. We undertook mutational analysis of SPRED3 to better understand the basis of its zDABM-independent interaction with zDHHC17. This analysis found that the cysteine-rich SPR domain of SPRED3, which is the defining feature of all Sprouty and SPRED proteins, interacts with zDHHC17. Surprisingly, the interaction with SPRED3 was independent of ANK17. Our mutational analysis of Spry2 was consistent with the SPR domain of this protein containing a zDHHC17-binding site, and Spry2 also showed detectable binding to a zDHHC17 mutant lacking the ANK domain. Thus, zDHHC17 can recognize its substrates through zDABM-dependent and/or zDABM-independent mechanisms, and some substrates display more than one mode of binding to this enzyme.

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

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

The acyltransferase Gpc1 is both a target and an effector of the unfolded protein response in Saccharomyces cerevisiae.

The unfolded protein response (UPR) is sensitive to proteotoxic and membrane bilayer stress, both of which are sensed by the ER protein Ire1. When activated, Ire1 splices HAC1 mRNA, producing a transcription factor that targets genes involved in proteostasis and lipid metabolism, among others. The major membrane lipid phosphatidylcholine (PC) is subject to phospholipase-mediated deacylation, producing glycerophosphocholine (GPC), followed by reacylation of GPC through the PC deacylation/reacylation pathway (PC-DRP). The reacylation events occur via a two-step process catalyzed first by the GPC acyltransferase Gpc1, followed by acylation of the lyso-PC molecule by Ale1. However, whether Gpc1 is critical for ER bilayer homeostasis is unclear. Using an improved method for C14-choline-GPC radiolabeling, we first show that loss of Gpc1 results in abrogation of PC synthesis through PC-DRP and that Gpc1 colocalizes with the ER. We then probe the role of Gpc1 as both a target and an effector of the UPR. Exposure to the UPR-inducing compounds tunicamycin, DTT, and canavanine results in a Hac1-dependent increase in GPC1 message. Further, cells lacking Gpc1 exhibit increased sensitivity to those proteotoxic stressors. Inositol limitation, known to induce the UPR via bilayer stress, also induces GPC1 expression. Finally, we show that loss of GPC1 induces the UPR. A gpc1Δ mutant displays upregulation of the UPR in strains expressing a mutant form of Ire1 that is unresponsive to unfolded proteins, indicating that bilayer stress is responsible for the observed upregulation. Collectively, our data indicate an important role for Gpc1 in yeast ER bilayer homeostasis.

New insights into the function of plant tannase with promiscuous acyltransferase activity.

Plant tannases (TAs) or tannin acyl hydrolases, a class of recently reported carboxylesterases in tannin-rich plants, are involved in the degalloylation of two important groups of secondary metabolites: flavan-3-ol gallates and hydrolyzable tannins. In this paper, we have made new progress in studying the function of tea (Camellia sinensis) (Cs) TA-it is a hydrolase with promiscuous acyltransferase activity in vitro and in vivo and promotes the synthesis of simple galloyl glucoses and flavan-3-ol gallates in plants. We studied the functions of CsTA through enzyme analysis, protein mass spectrometry, and metabolic analysis of genetically modified plants. Firstly, CsTA was found to be not only a hydrolase but also an acyltransferase. In the two-step catalytic reaction where CsTA hydrolyzes the galloylated compounds epigallocatechin-3-gallate or 1,2,3,4,6-penta-O-galloyl-ß-d-glucose into their degalloylated forms, a long-lived covalently bound Ser159-linked galloyl-enzyme intermediate is also formed. Under nucleophilic attack, the galloyl group on the intermediate is transferred to the nucleophilic acyl acceptor (such as water, methanol, flavan-3-ols, and simple galloyl glucoses). Then, metabolic analysis suggested that transient overexpression of TAs in young strawberry (Fragaria × ananassa) fruits, young leaves of tea plants, and young leaves of Chinese bayberry (Myrica rubra) actually increased the total contents of simple galloyl glucoses and flavan-3-ol gallates. Overall, these findings provide new insights into the promiscuous acyltransferase activity of plant TA.

Identification of key genes for triacylglycerol biosynthesis and storage in herbaceous peony (Paeonia lactifolra Pall.) seeds based on full-length transcriptome.

BACKGROUND: The herbaceous peony (Paeonia lactiflora Pall.) is extensively cultivated in China due to its root being used as a traditional Chinese medicine known as 'Radix Paeoniae Alba'. In recent years, it has been discovered that its seeds incorporate abundant unsaturated fatty acids, thereby presenting a potential new oilseed plant. Surprisingly, little is known about the full-length transcriptome sequencing of Paeonia lactiflora, limiting research into its gene function and molecular mechanisms. RESULTS: A total of 484,931 Reads of Inserts (ROI) sequences and 1,455,771 full-Length non-chimeric reads (FLNC) sequences were obtained for CDS prediction, TF analysis, SSR analysis and lncRNA identification. In addition, gene function annotation and gene structure analysis were performed. A total of 4905 transcripts were related to lipid metabolism biosynthesis pathway, belonging to 28 enzymes. We use these data to identify 10 oleosin (OLE) and 5 diacylglycerol acyltransferase (DGAT) gene members after de-redundancy. The analysis of physicochemical properties and secondary structure showed them similarity in gene family respectively. The phylogenetic analysis showed that the distribution of OLE and DGAT family members was roughly the same as that of Arabidopsis. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed expression changes in different seed development stages, and showed a trend of increasing and then decreasing. CONCLUSION: In summary, these results provide new insights into the molecular mechanism of triacylglycerol (TAG) biosynthesis and storage during the seedling stage in Paeonia lactiflora. It provides theoretical references for selecting and breeding oil varieties and understanding the functions of oil storage as well as lipid synthesis related genes in Paeonia lactiflora.

Comparative Analysis of Shikonin and Alkannin Acyltransferases Reveals Their Functional Conservation in Boraginaceae.

Shikonin and its enantiomer, alkannin, are bioactive naphthoquinones produced in several plants of the family Boraginaceae. The structures of these acylated derivatives, which have various short-chain acyl moieties, differ among plant species. The acylation of shikonin and alkannin in Lithospermum erythrorhizon was previously reported to be catalyzed by two enantioselective BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1). However, the mechanisms by which various shikonin and alkannin derivatives are produced in Boraginaceae plants remain to be determined. In the present study, evaluation of six Boraginaceae plants identified 23 homologs of LeSAT1 and LeAAT1, with 15 of these enzymes found to catalyze the acylation of shikonin or alkannin, utilizing acetyl-CoA, isobutyryl-CoA or isovaleryl-CoA as an acyl donor. Analyses of substrate specificities of these enzymes for both acyl donors and acyl acceptors and determination of their subcellular localization using Nicotiana benthamiana revealed a distinct functional differentiation of BAHD acyltransferases in Boraginaceae plants. Gene expression of these acyltransferases correlated with the enantiomeric ratio of produced shikonin/alkannin derivatives in L. erythrorhizon and Echium plantagineum. These enzymes showed conserved substrate specificities for acyl donors among plant species, indicating that the diversity in acyl moieties of shikonin/alkannin derivatives involved factors other than the differentiation of acyltransferases. These findings provide insight into the chemical diversification and evolutionary processes of shikonin/alkannin derivatives.