Short-chain Acyl Research Articles

Integration of metabolomic and transcriptomic analyses reveals novel regulatory functions of the ChREBP transcription factor in energy metabolism.

Carbohydrate Response Element-Binding Protein (ChREBP) is a transcription factor that activates key genes involved in glucose, fructose, and lipid metabolism in response to carbohydrate feeding, but its other potential roles in metabolic homeostasis have not been as well studied. We used liver-selective GalNAc-siRNA technology to suppress expression of ChREBP in rats fed a high fat/high sucrose diet and characterized hepatic and systemic responses by integrating transcriptomic and metabolomic analyses. GalNAc-siChREBP-treated rats had lower levels of multiple short-chain acyl CoA metabolites compared to rats treated with GalNAc-siCtrl containing a non-targeting siRNA sequence. These changes were related to a sharp decrease in free CoA levels in GalNAc-siChREBP treated-rats, accompanied by lower expression of transcripts encoding enzymes and transporters involved in CoA biosynthesis. These activities of ChREBP likely contribute to its complex effects on hepatic lipid and energy metabolism. While core enzymes of fatty acid (FA) oxidation are induced by ChREBP knockdown, accumulation of liver acylcarnitines and circulating ketones indicate diversion of acetyl CoA to ketone production rather than complete oxidation in the TCA cycle. Despite strong suppression of pyruvate kinase and activation of pyruvate dehydrogenase, pyruvate levels were maintained, likely via increased expression of pyruvate transporters, and decreased expression of lactate dehydrogenase and alanine transaminase. GalNAc-siChREBP treatment increased hepatic citrate and isocitrate levels while decreasing levels of distal TCA cycle intermediates. The drop in free CoA levels, needed for the 2-ketoglutarate dehydrogenase reaction, as well as a decrease in transcripts encoding the anaplerotic enzymes pyruvate carboxylase, glutamate dehydrogenase, and aspartate transaminase likely contributed to these effects. GalNAc-siChREBP treatment caused striking increases in PRPP and ZMP/AICAR levels, and decreases in GMP, IMP, AMP, NaNM, NAD(P), and NAD(P)H levels, accompanied by reduced expression of enzymes that catalyze late steps in purine and NAD synthesis. ChREBP suppression also increased expression of a set of plasma membrane amino acid transporters, possibly as an attempt to replenish TCA cycle intermediates. In sum, combining transcriptomic and metabolomic analyses has revealed regulatory functions of ChREBP that go well beyond its canonical roles in control of carbohydrate and lipid metabolism to now include mitochondrial metabolism and cellular energy balance.

PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

Intracellular transport among organellar compartments occurs in two general ways, by membrane-bound carriers or membrane contacts. Specific circumstances that involve the coordination of these two modes of transport remain to be defined. Studying Coat Protein I (COPI) transport, we find that phosphatidylcholine with short acyl chains (sPC) is delivered through membrane contact from the endoplasmic reticulum (ER) to sites of COPI vesicle formation at the Golgi to support the fission stage. Phosphatidylinositol transfer protein beta (PITPβ) plays a key role in this process, with the elucidation of this role advancing a new understanding of how PITPβ acts, providing a mechanistic understanding of a specific circumstance when vesicular transport requires membrane contact, and contributing to a basic understanding of how transport carriers in a model intracellular pathway are formed.

Nanoprecipitation to produce hydrophobic cellulose nanospheres for water-in-oil Pickering emulsions

In recent years, there has been growing interest in replacing petroleum-based water-in-oil (W/O) emulsifiers with sustainable and less toxic natural materials. Pickering emulsifiers are considered well-suited candidates due to their high interfacial activity and the ability to form emulsions with long-term stability. However, only sporadic examples of natural materials have been considered as inverse Pickering emulsifiers. This study describes the synthesis of a series of hydrophobic cellulose nanospheres by bulk modification with acyl groups of different chain lengths followed by nanoprecipitation, and their application as inverse emulsifiers. Modification with acyl groups of longer chain length (C16, C18) afforded lower degrees of substitution, but resulted in greater thermal stability than groups with shorter acyl chains (C12, C14). Formation of nanospheres with low aspect ratios and narrow size distributions required low initial cellulose concentrations (< 1% w/v), high volumetric ratios of antisolvent to solvent (> 10:1), and slow addition rates (< 20 mL/h). The modified cellulose nanospheres were able to reduce the interfacial tension between water and hexane from 45.8 mN/m to 31.1 mN/m, with an effect that increased with the number of carbons in the added acyl chains. The stearate-modified nanospheres exhibited superhydrophobic behavior, showing a contact angle of 156° ± 4° with water, and demonstrated emulsification performance comparable to the commonly used molecular surfactant sorbitan stearate. Our findings suggest that hydrophobically modified cellulose nanospheres have the potential to be a bio-derived alternative to traditional molecular W/O emulsifiers.Graphical

Cysteine S-acetylation is a post-translational modification involved in metabolic regulation.

Cysteine is a reactive amino acid central to the catalytic activities of many enzymes. It is also a common target of post-translational modifications (PTMs), such as palmitoylation. This longchain acyl PTM can modify cysteine residues and induce changes in protein subcellular localization. We hypothesized that cysteine could also be modified by short-chain acyl groups, such as cysteine S-acetylation. To test this, we developed sample preparation and non-targeted mass spectrometry protocols to analyze the mouse liver proteome for cysteine acetylation. Our findings revealed hundreds of sites of cysteine acetylation across multiple tissue types, revealing a previously uncharacterized cysteine acetylome. Cysteine acetylation shows a marked cytoplasmic subcellular localization signature, with tissue-specific acetylome patterns and specific changes upon metabolic stress. This study uncovers a novel aspect of cysteine biochemistry, highlighting short-chain modifications alongside known long-chain acyl PTMs. These findings enrich our understanding of the landscape of acyl modifications and suggest new research directions in enzyme activity regulation and cellular signaling in metabolism.

An In-Depth Study on the Inhibition of Quorum Sensing by Bacillus velezensis D-18: Its Significant Impact on Vibrio Biofilm Formation in Aquaculture.

Amid growing concerns about antibiotic resistance, innovative strategies are imperative in addressing bacterial infections in aquaculture. Quorum quenching (QQ), the enzymatic inhibition of quorum sensing (QS), has emerged as a promising solution. This study delves into the QQ capabilities of the probiotic strain Bacillus velezensis D-18 and its products, particularly in Vibrio anguillarum 507 communication and biofilm formation. Chromobacterium violaceum MK was used as a biomarker in this study, and the results confirmed that B. velezensis D-18 effectively inhibits QS. Further exploration into the QQ mechanism revealed the presence of lactonase activity by B. velezensis D-18 that degraded both long- and short-chain acyl homoserine lactones (AHLs). PCR analysis demonstrated the presence of a homologous lactonase-producing gene, ytnP, in the genome of B. velezensis D-18. The study evaluated the impact of B. velezensis D-18 on V. anguillarum 507 growth and biofilm formation. The probiotic not only controls the biofilm formation of V. anguillarum but also significantly restrains pathogen growth. Therefore, B. velezensis D-18 demonstrates substantial potential for preventing V. anguillarum diseases in aquaculture through its QQ capacity. The ability to disrupt bacterial communication and control biofilm formation positions B. velezensis D-18 as a promising eco-friendly alternative to conventional antibiotics in managing bacterial diseases in aquaculture.

Abridgement of Microbial Esterases and Their Eminent Industrial Endeavors.

Esterases are hydrolases that contribute to the hydrolysis of ester bonds into both water-soluble acyl esters and emulsified glycerol-esters containing short-chain acyl groups. They have garnered significant attention from biotechnologists and organic chemists due to their immense commercial value. Esterases, with their diverse and significant properties, have become highly sought after for various industrial applications. Synthesized ubiquitously by a wide range of living organisms, including animals, plants, and microorganisms, these enzymes have found microbial esterases to be the preferred choice in industrial settings. The cost-effective production of microbial esterases ensures higher yields, unaffected by seasonal variations. Their applications span diverse sectors, such as food manufacturing, leather tanneries, paper and pulp production, textiles, detergents, cosmetics, pharmaceuticals, biodiesel synthesis, bioremediation, and waste treatment. As the global trend shifts toward eco-friendly and sustainable practices, industrial processes are evolving with reduced waste generation, lower energy consumption, and the utilization of biocatalysts derived from renewable and unconventional raw materials. This review explores the background, structural characteristics, thermostability, and multifaceted roles of bacterial esterases in crucial industries, aiming to optimize and analyze their properties for continued successful utilization in diverse industrial processes. Additionally, recent advancements in esterase research are overviewed, showcasing novel techniques, innovations, and promising areas for further exploration.

Mimicking the inner mitochondrial membrane with curved supported lipid bilayers: A neutron reflectometry study

Cardiolipin (CL) is a phospholipid with an unusual molecular structure exhibiting four acyl chains bound to a polar headgroup. CL is found in curved regions within biological membranes, such as the poles of cylindrically shaped bacteria and the cristae in mitochondria. Like bacterial cells, mitochondria are characterised by two cell membranes. CL is exclusively found in the inner membrane of mitochondria (IMM), and it is vital for the proper functioning of this organelle. Together with CL, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main lipid components of the IMM. Interestingly, most of the CL in human tissues exhibits C18 acyl chains and alterations of its molecular structure are related to severe diseases. Here we investigated supported lipid bilayers (SLBs) composed of PC, PE and CL as model systems mimicking the IMM. In our experiments we used tetra-oleyl cardiolipin (TOCL), which exhibits four C18 acyl chains and is therefore close to the CL normally present in the IMM, and tetra-myristoyl cardiolipin (TMCL), which exhibits considerably shorter acyl chains, i.e. C14. All samples were investigated both in the presence and in the absence of Ca2+ ions. The structure of the produced samples was characterised by neutron reflectometry (NR). Our data indicate that all samples with TMCL were organised as a regular SLB with a small impact of TMCL on the bilayer structure. On the other hand, at a TOCL concentration above 10% mol and upon injection of Ca2+, we observed a large structural rearrangement of the initially formed SLB compatible with the formation of curved bilayer regions that protrude towards the bulk solvent. To the best of our knowledge, this is the first example where specular NR and off-specular scattering revealed buckled SLBs. This experimental evidence indicates the crucial role of CL acyl chain composition in favouring the proper folding of the IMM.

Antibacterial and Anti-Quorum Sensing Properties of Silver Nanoparticles Phytosynthesized Using Embelia ruminata.

The rise in antibiotic resistance (AR) poses an imminent threat to human health. Nanotechnology, together with mechanisms such as quorum sensing (QS), which relies on communication between bacterial cells, may decrease the selective pressure for AR. Thus, this study aimed to investigate the effectiveness of silver nanoparticles (AgNPs) synthesized at room temperature (Rt) and 80 °C using Embelia ruminata leaf, stem-bark, and fruit extracts as antibacterial and anti-QS agents. The phytosynthesized AgNPs solutions were subjected to various characterization assays and assessed for their antibacterial activities. Quantitative QS assays were performed using Chromobacterium subtsugae CV017 and Chromobacterium violaceum ATCC 12472. Synthesized AgNPs were spherical-to-near-spherical in shape, poly-dispersed, and crystalline, with a size range of 21.06-32.15 nm. Fruit AgNPs showed stronger antibacterial activity than AgNPs from other plant organs against selected bacterial strains. In the QS assays, fruit 80 °C AgNPs demonstrated the most significant violacein inhibition in an assay performed using the short-chain acyl homoserine lactone CV017 biosensor, while the leaf and fruit Rt AgNPs demonstrated the most violacein inhibition in an assay performed using the long-chain acyl homoserine lactone ATCC 12472 biosensor. The investigations carried out in this study lay the groundwork for future innovative research into antibacterial and anti-QS strategies using E. ruminata.

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.

HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 μM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 μM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

Solid–Liquid Phase Transitions of Triglycerides in Griebenschmalz, Smalec, and Fedt Studied Using 13C Solid-State NMR with Dynamics-Based Spectral Filtering

The consumer satisfaction of lard-based bread spreads depends on a delicate balance between a liquid fat phase, allowing the spread to flow, and solid fat crystals, providing the product with substance sometimes further enhanced by crispy pork cracklings. Here we apply 13C solid-state NMR with dynamics-based spectral filtering to characterize and follow the temperature dependence of the co-existing solid and liquid triglyceride phases in commercial German Griebenschmalz and Polish smalec, both containing cracklings, as well as home-made Danish fedt and, as a chemically more pure reference, German Schweineschmalz intended for baking. The NMR method allows detection of carbon atoms representative of saturated, unsaturated, and polyunsaturated acyl chains in both solid and liquid states. The results show that the solid comprises multiple crystal forms with different melting temperatures, while the liquid is at low temperature enriched in triglycerides with shorter acyl chains and higher degree of unsaturation, which become diluted with long-chain saturated triglycerides as the solids are melting. The obtained deeper understanding of the concomitant aspects of the phase transitions may pave the way for future efforts of rational optimization of fat blend composition to extend the temperature range over which the product contains sufficient amounts of both solids and liquids to give texture properties appealing to consumers.

Structural basis for plastic glycolipid recognition of the C-type lectin Mincle

Mincle (macrophage-inducible C-type lectin, CLEC4E) is a C-type lectin immune-stimulatory receptor for cord factor, trehalose dimycolate (TDM), which serves as a potent component of adjuvants. The recognition of glycolipids by Mincle, especially their lipid parts, is poorly understood. Here, we performed nuclear magnetic resonance analysis, revealing that titration of trehalose harboring a linear short acyl chain showed a chemical shift perturbation of hydrophobic residues next to the Ca-binding site. Notably, there were split signalsfor Tyr201 upon complex formation, indicating two binding modes for the acyl chain. In addition, most Mincle residues close to the Ca-binding site showed no observable signals, suggesting their mobility on an ∼ ms scale even after complex formation. Mutagenesis study supported two putative lipid-bindingmodes for branched acyl-chain TDM binding. These results provide novel insights into the plastic-binding modes of Mincle toward a wide range of glycol- and glycerol-lipids, important for rational adjuvant development.

Clinical and Gene Analysis of Fatty Acid Oxidation Disorders Found in Neonatal Tandem Mass Spectrometry Screening.

To investigate the clinical and gene mutation characteristics of fatty acid oxidative metabolic diseases found in neonatal screening. A retrospective analysis was performed on 29,948 neonatal blood tandem mass spectrometry screening samples from January 2018 to December 2021 in our neonatal screening centre. For screening positive, recall review is still suspected of fatty acid oxidation metabolic disorders in children as soon as possible to improve the genetic metabolic disease-related gene detection package to confirm the diagnosis. All diagnosed children were followed up to the deadline. Among 29,948 neonates screened by tandem mass spectrometry, 14 cases of primary carnitine deficiency, six cases of short-chain acyl coenzyme A dehydrogenase deficiency, two cases of carnitine palmitoyltransferase-I deficiency and one case of multiple acyl coenzyme A dehydrogenase deficiency were recalled. Except for two cases of multiple acyl coenzyme A dehydrogenase deficiency that exhibited [manifestations], the other 21 cases were diagnosed pre-symptomatically. Eight mutations of SLC22A5 gene were detected, including c.51C>G, c.403G>A, c.506G>A, c.1400C>G, c.1085C>T, c.706C>T, c.1540G>C and c.338G>A. Compound heterozygous mutation of CPT1A gene c.2201T>C, c.1318G>A, c.2246G>A, c.2125G>A and ETFA gene c.365G>A and c.699_701delGTT were detected, and new mutation sites were found. Neonatal tandem mass spectrometry screening is an effective method for identifying fatty acid oxidative metabolic diseases, but it should be combined with urine gas chromatography-mass spectrometry and gene sequencing technology. Our findings enrich the gene mutation profile of fatty acid oxidative metabolic disease and provide evidence for genetic counselling and prenatal diagnosis in families.

Harnessing Clickable Acylated Glycerol Probes as Chemical Tools for Tracking Glycerolipid Metabolism.

We report the use of clickable monoacylglycerol (MAG) analogs as probes for the labeling of glycerolipids during lipid metabolism. Incorporation of azide tags onto the glycerol region was pursued to develop probes that would label glycerolipids, in which the click tag would not be removed through processes including acyl chain and headgroup remodeling. Analysis of clickable MAG probes containing acyl chains of different length resulted in widely variable cell imaging and cytotoxicity profiles. Based on these results, we focused on a probe bearing a short acyl chain (C4 -MAG-N3 ) that was found to infiltrate natural lipid biosynthetic pathways to produce click-tagged versions of both neutral and phospholipid products. Alternatively, strategic blocking of the glycerol sn-3 position in probe C4 -MEG-N3 served to deactivate phospholipid tagging and focus labeling on neutral lipids. This work shows that lipid metabolic labeling profiles can be tuned based on probe structures and provides valuable tools for evaluating alterations to lipid metabolism in cells.

Biochemical characterization of an acetylesterase from Bacillus subtilis and its application for 7-aminocephalosporanic acid deacetylation.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA), which could be converted from 7-aminocephalosporanic acid (7-ACA), is a crucial starting material that is used for synthesizing industrial semisynthetic β-lactam antibiotics. Enzymes involved in the conversion from 7-ACA to D-7-ACA present critical resources in the pharmaceutical industry. In the present study, a putative acetylesterase, EstSJ, identified from Bacillus subtilis KATMIRA1933, was first heterologously expressed in Escherichia coli BL21(DE3) cells and biochemically characterized. EstSJ belongs to carbohydrate esterase family 12 and is active on short-chain acyl esters from p-NPC2 to p-NPC6. Multiple sequence alignments showed that EstSJ was also an SGNH family esterase with a typical GDS(X) motif at its N-terminal end and a catalytic triad composed of Ser186-Asp354-His357. The purified EstSJ displayed the highest specific activity of 1,783.52 U mg-1 at 30°C and pH 8.0, and was stable within the pH range of 5.0-11.0. EstSJ can deacetylate the C3' acetyl group of 7-ACA to generate D-7-ACA, and the deacetylation activity was 4.50 U mg-1. Based on the structural and molecular docking with 7-ACA, the catalytic active sites (Ser186-Asp354-His357) together with four substrate-binding residues (Asn259, Arg295, Thr355, and Leu356) of EstSJ are revealed. This study provided a promising 7-ACA deacetylase candidate that could be applied to produce D-7-ACA from 7-ACA in the pharmaceutical industry.

Milk metabolomics analyses of lactating dairy cows with divergent residual feed intake reveals physiological underpinnings and novel biomarkers.

The opportunity to select for feed efficient cows has been limited by inability to cost-effectively record individual feed efficiency on an appropriate scale. This study investigated the differences in milk metabolite profiles between high- and low residual feed intake (RFI) categories and identified biomarkers of residual feed intake and models that can be used to predict residual feed intake in lactating Holsteins. Milk metabolomics analyses were undertaken at early, mid and late lactation stages and residual feed intake was calculated in 72 lactating dairy cows. Cows were ranked and grouped into high residual feed intake (RFI >0.5 SD above the mean, n = 20) and low residual feed intake (RFI <0.5 SD below the mean, n = 20). Milk metabolite profiles were compared between high residual feed intake (least efficient) and low residual feed intake (most efficient) groups. Results indicated that early lactation was predominantly characterized by significantly elevated levels of medium chain acyl carnitines and glycerophospholipids in high residual feed intake cows. Citrate cycle and glycerophospholipid metabolism were the associated pathways enriched with the significantly different metabolites in early lactation. At mid lactation short and medium chain acyl carnitines, glycerophospholipids and amino acids were the main metabolite groups differing according to residual feed intake category. Late lactation was mainly characterized by increased levels of amino acids in high residual feed intake cows. Amino acid metabolism and biosynthesis pathways were enriched for metabolites that differed between residual feed intake groups at the mid and late lactation stages. Receiver operating characteristic curve analysis identified candidate biomarkers: decanoylcarnitine (area under the curve: AUC = 0.81), dodecenoylcarnitine (AUC = 0.81) and phenylalanine (AUC = 0.85) at early, mid and late stages of lactation, respectively. Furthermore, panels of metabolites predicted residual feed intake with validation coefficient of determination (R 2) of 0.65, 0.37 and 0.60 at early, mid and late lactation stages, respectively. The study sheds light on lactation stage specific metabolic differences between high-residual feed intake and low-residual feed intake lactating dairy cows. Candidate biomarkers that distinguished divergent residual feed intake groups and panels of metabolites that predict individual residual feed intake phenotypes were identified. This result supports the potential of milk metabolites to select for more efficient cows given that traditional residual feed intake phenotyping is costly and difficult to conduct in commercial farms.

A novel thermostable and salt-tolerant carboxylesterase involved in the initial aerobic degradation pathway for pyrethroids in Glycomyces salinus

The long-term and excessive use of pyrethroid pesticides poses substantial health risks and ecosystem concerns. Several bacteria and fungi have been reported that could degrade pyrethroids. The ester-bond hydrolysis using hydrolases is the initial regulatory metabolic reaction of pyrethroids. However, the thoroughly biochemical characterization of hydrolases involved in this process is limited. Here, a novel carboxylesterase, designated as EstGS1 that could hydrolyze pyrethroid pesticides was characterized. EstGS1 showed low sequence identity (<27.03%) compared to other reported pyrethroid hydrolases and belonged to the hydroxynitrile lyase family that preferred short short-chain acyl esters (C2 to C8). EstGS1 displayed the maximal activity of 213.38 U/mg at 60 °C and pH 8.5 using pNPC2 as substrate, with Km and Vmax were 2.21 ± 0.72 mM and 212.90 ± 41.78 µM/min, respectively. EstGS1 is a halotolerant esterase and remains stable in 5.1 M NaCl. Based on molecular docking and mutational analysis, the catalytic triad of S74-D181-H212 and three other substrate-binding residues I108, S159, and G75 are critical for the enzymatic activity of EstGS1. Additionally, 61 and 40 mg/L of deltamethrin and λ-cyhalothrin were hydrolyzed by 20 U of EstGS1 in 4 h. This work presents the first report on a pyrethroid pesticide hydrolase characterized from a halophilic actinobacteria.

Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae.

Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.

The Anti-Tubercular Aminolipopeptide Trichoderin A Displays Selective Toxicity against Human Pancreatic Ductal Adenocarcinoma Cells Cultured under Glucose Starvation.

Pancreatic ductal adenocarcinoma remains a highly debilitating condition with no effective disease-modifying interventions. In our search for natural products with promising anticancer activity, we identified the aminolipopeptide trichoderin A as a potential candidate. While it was initially isolated as an antitubercular peptide, we provide evidence that it is also selectively toxic against BxPC-3 and PANC-1 human pancreatic ductal adenocarcinoma cells cultured under glucose deprivation. This has critical implications for the pancreatic ductal adenocarcinoma, which is characterized by nutrient deprivation due to its hypovascularized network. We have also successfully simplified the trichoderin A peptide backbone, allowing greater accessibility to the peptide for further biological testing. In addition, we also conducted a preliminary investigation into the role of peptide lipidation at the N-terminus. This showed that analogues with longer fatty acyl chains exhibited superior cytotoxicity than those with shorter acyl chains. Further structural optimization of trichoderin A is anticipated to improve its biological activity, whilst ongoing mechanistic studies to elucidate its intracellular mechanism of action are conducted in parallel.

SiRNA delivery to lymphatic endothelial cells via ApoE-mediated uptake by lipid nanoparticles.

Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.