Long-chain Acyl Research Articles

De Novo Lipogenesis Alters the Phospholipidome of Esophageal Adenocarcinoma.

The incidence of esophageal adenocarcinoma is rising, survival remains poor, and new tools to improve early diagnosis and precise treatment are needed. Cancer phospholipidomes quantified with mass spectrometry imaging (MSI) can support objective diagnosis in minutes using a routine frozen tissue section. However, whether MSI can objectively identify primary esophageal adenocarcinoma is currently unknown and represents a significant challenge, as this microenvironment is complex with phenotypically similar tissue-types. Here, we used desorption electrospray ionization-MSI (DESI-MSI) and bespoke chemometrics to assess the phospholipidomes of esophageal adenocarcinoma and relevant control tissues. Multivariate models derived from phospholipid profiles of 117 patients were highly discriminant for esophageal adenocarcinoma both in discovery (AUC = 0.97) and validation cohorts (AUC = 1). Among many other changes, esophageal adenocarcinoma samples were markedly enriched for polyunsaturated phosphatidylglycerols with longer acyl chains, with stepwise enrichment in premalignant tissues. Expression of fatty acid and glycerophospholipid synthesis genes was significantly upregulated, and characteristics of fatty acid acyls matched glycerophospholipid acyls. Mechanistically, silencing the carbon switch ACLY in esophageal adenocarcinoma cells shortened glycerophospholipid chains, linking de novo lipogenesis to the phospholipidome. Thus, DESI-MSI can objectively identify invasive esophageal adenocarcinoma from a number of premalignant tissues and unveils mechanisms of phospholipidomic reprogramming. SIGNIFICANCE: These results call for accelerated diagnosis studies using DESI-MSI in the upper gastrointestinal endoscopy suite, as well as functional studies to determine how polyunsaturated phosphatidylglycerols contribute to esophageal carcinogenesis.

Incidence of newborn screening disorders among 56632 infants in Central Saudi Arabia. A 6-year study.

Objectives:To determine the incidence of newborn screening (NBS) disorders and to study the key performance indicators of the program.Methods:This retrospective single-center study enrolled all infants who underwent NBS from January 2012 to December 2017 at Prince Sultan Military Medical City, Riyadh, Saudi Arabia. We screened 17 NBS disorders. Blood samples were collected 24 hours after birth. If the initial result was positive, a second sample was collected. True positive cases were immediately referred for medical management. Data were extracted from laboratory computerized and non-computerized records using case report forms.Results:During the study period, 56632 infants underwent NBS with a coverage rate of 100%. Thirty-eight cases were confirmed. The incidence of congenital hypothyroidism was 1:3775. The positive predictive value for the detection of congenital hypothyroidism was 11.8%. Propionic aciduria was the most common metabolic disorder, with an incidence of 1:14158. Very long-chain acyl CoA dehydrogenase deficiency and glutaric aciduria type 1 had an incidence of 1:18877 each. Phenylketonuria, biotinidase deficiency, maple syrup urine disease, and citrullinemia had an incidence of 1:28316 each. However, galactosemia and 3-methyl crotonyl carboxylase deficiency had the lowest incidence of 1:56632.Conclusion:The NBS coverage rate at our facility was 100%. Congenital hypothyroidism was the most frequently detected disorder with an incidence that matches worldwide figures. The incidence of other inherited disorders was consistent with regional figures.

1337-P: Metabolomic Profile Associated with Obstructive Sleep Apnea Severity in Obese Pregnant Women with Gestational Diabetes

Background: Obstructive sleep apnea (OSA) is prevalent in obese women with gestational diabetes (GDM). In non-pregnant population, metabolomics profiles have been shown to differ between those with and without OSA. This study explored associations between OSA severity and metabolites in women with GDM. Methods: Eighty-two obese women with diet-controlled GDM had OSA assessment by a home monitor (mean gestational age, GA, 28 weeks). OSA severity was assessed by apnea hypopnea index (AHI). Metabolic profile was assayed from fasting serum samples via LC-MS using an untargeted approach. The metabolites were extracted and subjected to an Agilent 1290 UPLC coupled to an Agilent 6545 Q-TOF MS. Data were acquired using electrospray ionization (ESI) in positive and negative ion modes. The raw LC-MS data were processed using the OpenMS toolkit to detect and quantitate features and these LC-MS features were annotated using the Human Metabolite Database (HMDB) as a reference. The feature data were compared with OSA status, AHI, BMI and GA using limma in R. Correlation analyses of the continuous covariates (AHI, BMI, GA) were performed using Kendall’s Tau test. P-values were adjusted for multiple testing using Benjamini-Hochberg’s FDR correction. Results: Forty-three women (52.4%) had OSA, with a median AHI of 9.4. There were no significant differences in metabolomics profiles between those with and without OSA. However, differential analyses modeling in GA and pre-pregnancy BMI found 12 features that significantly associated with AHI. These features could be annotated to estradiols and phospholipids (e.g., Lysophospholipids, long-chain acyl carnitine, methyl-branched fatty acids) with higher levels related to higher AHI. Conclusion: Biomarkers including estradiols and phospholipids may be involved in pathogenesis of OSA in pregnant women with GDM. Targeted approach may help further identify these metabolites to better understand their role in OSA in this population. Disclosure S. Reutrakul: None. N. Chirakalwasan: None. S. Charoensri: None. S. Amnakkittikul: None. S. Saetung: None. H. Chen: None. G.E. Chlipala: None. Funding National Center for Advancing Translational Sciences (UL1TR002003); Mahidol University

Effects of Long-Chain Acyl Substituents on the Thermoplasticity and Mechanical Properties of Paramylon Mixed Esters

The moldability and robustness of thermoplastic paramylon mixed esters are highly related to primary structural parameters, such as the length of the long-chain acyl group and its degree of substitution. Short chain lengths and lower substitution degrees provide more robust molded thermoplastics, whereas longer chains and higher substitution degrees improve moldability. Herein, the choice of an appropriate long-chain acyl group (lauroyl or myristoyl) and the effect of the substitution degree were investigated by examining the thermal and mechanical properties of a series of paramylon acetate laurates and myristates and comparing them with those of their curdlan analogues. Melt volume flow rate measurements (180–220 °C) indicate that myristoyl substitution in paramylon results in a local thermoplasticity maximum at a substitution degree of 0.35 at higher temperatures (above ~ 200 °C). These measurements and dynamic viscoelastic analyses suggest that the myristoyl substitution serves an “internal plasticizer” in the higher strain range and higher temperature region (~ 200–230 °C), while it serves as a “viscosity increaser” in the lower strain range and lower temperature region (170–200 °C). Moreover, tensile and bending testing showed that this product exhibits superior mechanical strength. The successful realization of a good balance between moldability and robustness suggests that fine-tuning the chain length and substitution degree is an effective approach for producing thermoplastic polysaccharides with superior injection moldabilities and mechanical robustness.

Effect of Dendrobium nobile Lindl. alkaloids on myocardial lipid metabolism during cardiopulmonary bypass ischemia-reperfusion in dogs

Objective: To explore the effects and mechanisms of Dendrobium nobile Lindl. alkaloids (DNLA) on myocardial lipid metabolism during ischemia-reperfusion in dogs undergoing cardiopulmonary bypass (CPB). Methods: Twenty-four healthy hybrid dogs, half male and half female, were randomly divided into sham group, model group, solvent control group and treatment group (DNLA, 6 mg/kg) (n=6), all of which were established with CPB. Except for the sham group, the aorta of the other groups was occluded for 60 min and then reopened. The uptake rate of free fatty acids, the concentration of long-chain acyl coenzyme A (LCACoA), mRNA and protein expression of fatty acid translocase enzyme/CD36 (FAT/CD36) in myocardial tissue and the cardiac function indexes were measured at 4 time points: before cardiopulmonary bypass (T1), 15 min (T2), 60 min (T3), and 90 min (T4) after reperfusion in each group. Results: Before CPB, there were no statistically significant differences in the uptake rate of free fatty acids, the concentration of LCACoA and mRNA expression of FAT/CD36 in myocardial tissue in each group (P>0.05). After the opening of the aorta, the above indexes in model group [(35.8±4.7)%, (8.55±1.51) nmol/g, 3.23±0.68] and treatment group [(27.4±2.7)%, (6.10±1.38) nmol/g, 2.20±0.56] were higher than those in sham group [(19.6±3.9)%, (4.16±0.81)nmol/g, 1.19±0.52], which were the highest at T2, and then gradually decreased (all P<0.05). Compared with the model group, the increase of above indicators in the treatment group was significantly lower at T2 (all P<0.05). Before CPB, there was no statistically significant differences in cardiac function indexes [left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and±dp/dtmax] among the groups (P>0.05). After the aorta was opened, the above indexes in model group [(76.5±9.1) mmHg, (31.1±2.9) mmHg, (1.2±0.4) mmHg/ms, (-0.9±0.1) mmHg/ms] and treatment group [(92.9±8.7) mmHg, (25.3±3.6) mmHg, (1.8±0.4) mmHg/ms, (-1.3±0.1) mmHg/ms] were lower than those in sham group [(165.5±12.9) mmHg, (6.5±0.5) mmHg, (3.3±0.6) mmHg/ms, (-2.9±0.3) mmHg/ms] (all P<0.05), but the impairment degree of cardiac function indicators in treatment group was significantly lower than that those in model group (all P<0.05). Conclusion: During CPB in dogs, DNLA can inhibit the abnormal expression of FAT/CD36, decrease the uptake of free fatty acids, and reduce the abnormal accumulation of LCACoA in myocardium,thereby alleviating the myocardial injury after ischemia-reperfusion.

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeoleiVT8.

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.

Exogenous Fatty Acids Remodel Staphylococcus aureus Lipid Composition through Fatty Acid Kinase.

Staphylococcus aureus can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of S. aureus in the absence of fakA We found the fakA mutant to have increased abundance of lipids containing longer acyl chains. Since S. aureus does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and fakA mutant bacteria grown in mouse skin homogenate. We show that wild-type S. aureus can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology.IMPORTANCE Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is unclear. Our results reveal lipid composition changes in response to fatty acid addition and when S. aureus is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to S. aureus Furthermore, using mouse skin homogenates as a surrogate for in vivo conditions, we showed that S. aureus can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function.

Palmitoylome profiling indicates that androgens promote the palmitoylation of metabolism-related proteins in prostate cancer-derived LNCaP cells

To explore potential therapeutic targets other than androgen-deprivation treatment for prostate cancer by screening the proteins induced by androgen at palmitoylation modification level in LNCaP cells. The LNCaP cells were treated with androgen (Methyltrienolone, R1881, 5 nmol/L) or dimethyl sulfoxide (DMSO) for 24 h, and then labeled with alkynyl palmitic acid Alk-C16 (100 μmol/L). After that, the cells were collected, lysed, the total protein was extracted, agarose beads labeled with azide (1 mmol/L) were added, and the click-chemistry reaction was carried out at room temperature for 1 h. The covalent bond formed by click-chemistry reaction of azide and alkynyl group was used to enrich the palmitoylated proteins on agarose beads. Label-free quantitation (LFQ) was used to compare the protein palmitoylation level of R1881 treated and untreated cells to screen the proteins induced by androgen at palmitoylation modification level. In this experiment, 907 potential palmitoylated proteins (mascot score>2, P<0.05) were identified, among which 430 proteins had LFQ values not zero at least twice. Among the 430 proteins, the palmitoylation levels of 92 candidates were increased by androgen treatment, and their LFQ values were significantly upregulated (>1.5-fold, P<0.05) in ≥2 samples of androgen-treated vs. untreated LNCaP cells. We also used the software of cytoscape to classify the 92 proteins, and found that the known functional proteins of them could be divided into three categories: metabolism related, protein folding related and translation initiation related. Among them, metabolism related proteins included lipid metabolism (6), glucose metabolism (7) and respiratory electron transport chain (8), and a small amount of amino acid metabolism (2) and other metabolism related proteins (2). Notably, the ratio of LFQ of cytochrome b-c1 complex subunit 2 (UQCRC2) was significantly (>3-fold, P<0.05) higher in androgen-treated cells compared with untreated cells, indicating that the palmitoylation level of UQCRC2 was enhanced by androgen most significantly than that of others. The second was long-chain acyl CoA dehydrogenase (ACADVL) related to lipid metabolism and glucose 6-phosphate dehydrogenase (PGD) related to glucose metabolism, but the LFQ ratio of them was less than 3-fold. The research on palmitoylation mechanism of metabolism, especially the proteins related to respiratory electron transport chain, will provide a new guidance for the diagnosis and treatment of prostate cancer and the development of targeted drugs.

Enhancement of Sphingolipid Synthesis Improves Osmotic Tolerance of Saccharomyces cerevisiae.

To enhance the growth performance of Saccharomyces cerevisiae under osmotic stress, mutant XCG001, which tolerates up to 1.5 M NaCl, was isolated through adaptive laboratory evolution (ALE). Comparisons of the transcriptome data of mutant XCG001 and the wild-type strain identified ELO2 as being associated with osmotic tolerance. In the ELO2 overexpression strain (XCG010), the contents of inositol phosphorylceramide (IPC; t18:0/26:0), mannosylinositol phosphorylceramide [MIPC; t18:0/22:0(2OH)], MIPC (d18:0/22:0), MIPC (d20:0/24:0), mannosyldiinositol phosphorylceramide [M(IP)2C; d20:0/26:0], M(IP)2C [t18:0/26:0(2OH)], and M(IP)2C [d20:0/26:0(2OH)] increased by 88.3 times, 167 times, 63.3 times, 23.9 times, 27.9 times, 114 times, and 208 times at 1.0 M NaCl, respectively, compared with the corresponding values of the control strain XCG002. As a result, the membrane integrity, cell growth, and cell survival rate of strain XCG010 increased by 24.4% ± 1.0%, 21.9% ± 1.5%, and 22.1% ± 1.1% at 1.0 M NaCl, respectively, compared with the corresponding values of the control strain XCG002 (wild-type strain with a control plasmid). These findings provided a novel strategy for engineering complex sphingolipids to enhance osmotic tolerance.IMPORTANCE This study demonstrated a novel strategy for the manipulation of membrane complex sphingolipids to enhance S. cerevisiae tolerance to osmotic stress. Elo2, a sphingolipid acyl chain elongase, was related to osmotic tolerance through transcriptome analysis of the wild-type strain and an osmosis-tolerant strain generated from ALE. Overexpression of ELO2 increased the content of complex sphingolipid with longer acyl chain; thus, membrane integrity and osmotic tolerance improved.

Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

Cardiac Palmitome Sheds New Light on the Structural and Functional Roles of S-Palmitoylation in Cardiac Myocytes

S-palmitoylation, linking saturated long-chain fatty acyls (commonly palmitoyl) to cysteine thiol through thioester bonds, is a reversible form of post-translational modification that affords dynamic control of protein-protein and protein-lipid interactions. It is estimated that 10-12% of human proteome is S-palmitoylatable, involved in diverse cellular functions including: cell adhesion, protein localization, cytoskeletal and membrane organization, intracellular signaling cascade, regulation of protein and nucleotide metabolism, and generation of precursor metabolites and energy. Changes in the expression of palmitoylating enzymes have been observed in heart failure patients. Yet the scope of protein S-palmitoylation in the heart is unknown. Global search for S-palmitoylatable proteins in the heart (cardiac palmitome) and functional classification. We used hydroxylamine (HA)-cleavage of thioester bonds, followed by thiol-reactive-resin-assisted-capture to isolate S-palmitoylated proteins from human (non-failing), dog and rat hearts ([+] samples). No HA-cleavage samples served as [-] controls. Samples were subject to LC/MS-MS for protein identification. Proteins detected in [+] (sum of peptide-spectra matches ‘PSM’ > 2) but not in [-], or with [+]/[-] ratio of PSM > 2, were retained as putative components of cardiac palmitome. Cardiac palmitome shares the general functional classification with previously reported non-cardiac palmitomes. However, comparing the protein list in our human cardiac palmitome (n=696) to the combined protein list from 17 human non-cardiac palmitomes (SwissPalm release 3, n=4587) showed that 169 proteins are unique to the former. These proteins are tailored to the cardiac function, e.g. RyR2, myomesin-3, Cx43, and obscurin. Current efforts are focused on validating the cardiac-selective palmitome. S-palmitoylation is involved in a broad scope of cardiac protein function. Future work should explore how S-palmitoylation impacts on cardiac function in normal and disease.

Finding the gas pedal on a slow sirtuin.

The class III histone deacetylase sirtuin 6 (SIRT6) modulates numerous functions in the cell by deacetylating histone lysine residues. Interestingly, SIRT6's efficiency in in vitro experiments is far greater against substrates carrying long-chain fatty acyl modifications such as myristoylated lysine compared with acetylated counterparts, but the deacetylase activity can be stimulated by fatty acids and small-molecule allosteric modulators. A new study helps to explain this puzzling activation using a novel activator, thorough kinetic investigation, and mutagenesis studies. These data help elucidate the molecular requirements for activation of SIRT6 and provide a foundation for development of activators for therapeutic purposes.

SIRT6 cooperates with SIRT5 to regulate bovine preadipocyte differentiation and lipid metabolism via the AMPKα signaling pathway

Preadipocyte differentiation and lipid synthesis are critical steps for intramuscular fat (IMF) deposition and lipid metabolism homeostasis. IMF content of beef not only determines the ratio of muscle to adipose, but also determines the beef quality, flavor, and sensory characteristics. Maintaining lipid metabolism homeostasis is the key means of preventing and treating diabetes, obesity, and other metabolic diseases. SIRT6, which is an ADP-ribosyltransferase and NAD+-dependent deacetylase of acetyl and long-chain fatty acyl groups, playing central roles in lipid and glucose metabolism, is closely related to the occurrence of diabetes and obesity caused by overnutrition and aging. This study was based on bovine preadipocyte differentiation and an obese mice model, and comprehensively used transcriptome sequencing (RNA-seq) and morphological identification methods to explore the effects of inhibition of SIRT6 on differentiation and lipid synthesis, and related molecular mechanisms. Additionally, the feedback synergistic regulation of SIRT5 and SIRT6 on differentiation and lipid deposition was analyzed. The results showed that in the differentiation process of bovine preadipocytes, inhibition of SIRT5 significantly promoted SIRT6 expression. In addition, SIRT6 inhibited bovine preadipocyte differentiation and lipid synthesis, cooperating with SIRT5 to decrease lipid deposition, and repressed cell cycle arrest of preadipocytes. Moreover, in vivo verification experiments also obtained consistent results. Furthermore, SIRT6 inhibited preadipocyte differentiation and lipid deposition by activating the adenosine monophosphate activated protein kinase alpha (AMPKα) pathway. The above results provided a novel approach for understanding the functions of SIRT6 in regulating bovine adipocyte differentiation and lipid metabolism, as well as a new target for the treatment of diabetes and obesity in a clinical setting.

Combinatorial Enzymatic Synthesis of Unnatural Long-Chain β-Branch Pyrones by a Highly Promiscuous Enzyme.

In this study, we described in detail a combinatorial enzymatic synthesis approach to produce a series of unnatural long-chain β-branch pyrones. We attempted to investigate the catalytic potential of a highly promiscuous enzyme type III PKS to catalyze the non-decarboxylative condensation reaction by two molecules of fatty acyl diketide-N-acetylcysteines (diketide-NACs) units. Two non-natural long-chain (C16, C18) fatty acyl diketide-NACs were prepared successfully for testing the ability of non-decarboxylative condensation. In vitro, 12 novel naturally unavailable long-chain β-branch pyrones were generated by one-pot formation and characterized by ultraviolet–visible spectroscopy and high-resolution liquid chromatography–mass spectrometry. Interestingly, enzymatic kinetics result displays that this enzyme exhibits the remarkable compatibility to various non-natural long-chain substrates. These results would be useful to deeply understand the catalytic mechanism of this enzyme and further extend the application of enzymatic synthesis of non-natural products.

Bacteriocin enterocin CRL35 is a modular peptide that induces non-bilayer states in bacterial model membranes

The mechanism of action of the anti-Listeria peptide enterocin CRL35 was studied with biophysical tools by using lipid mixtures that mimicked Gram-positive plasma membranes. Langmuir monolayers and infrared spectroscopy indicated that the peptide readily interacted with phospholipid assembled in monolayers and bilayers to produce a dual effect, depending on the acyl chains. Indeed, short chain mixtures were disordered by enterocin CRL35, but the gel-phases of membranes composed by longer acyl chains were clearly stabilized by the bacteriocin. Structural and functional studies indicated that non-bilayer states were formed when liposomes were co-incubated with enterocin CRL35, whereas significant permeabilization could be detected when bilayer and non-bilayer states co-existed. Results can be explained by a two-step model in which the N-terminal of the peptide firstly docks enterocin CRL35 on the lipid surface by means of electrostatic interactions; then, C-terminal triggers membrane perturbation by insertion of hydrophobic α-helix.

Development Of Novel Liposome-Encapsulated Combretastatin A4 Acylated Derivatives: Prodrug Approach For Improving Antitumor Efficacy.

PurposeThe objective of the present study was to develop a liposomal drug delivery system based on combretastatin A4 (CA4) prodrugs modified with varying alkyl chains and investigate the in vitro drug conversion from prodrug and in vivo antitumor effect.MethodsThe prodrug of CA4 was synthesized with stearyl chloride (18-carbon chain), palmitoyl chloride (16-carbon chain), myristoyl chloride (14-carbon chain), decanoyl chloride (10-carbon chain), and hexanoyl chloride (6-carbon chain) at the 3′-position of the CA4. Subsequently, it was encapsulated with liposomes through the thin-film evaporation method. Furthermore, the characteristics of prodrug-liposome were evaluated using in vitro drug release, conversion, and cytotoxicity assays, as well as in vivo pharmacokinetic, antitumor, and biodistribution studies.ResultsThe liposome system with loaded CA4 derivatives was successfully developed with nano-size and electronegative particles. The rate of in vitro drug release and conversion was reduced as the fatty acid carbon chain lengthened. On the contrary, in vivo antitumor effects were improved with the enlargement of the fatty acid carbon chain. The results of the in vivo pharmacokinetic and tissue distribution studies indicated that the reduced rate of CA4 release with a long carbon chain could prolong the circulation time and increase the drug concentration in the tumor tissue.ConclusionThese results suggested that the release or hydrolysis of the parent drug from the prodrug was closely related with the in vitro and in vivo properties. The slow drug release of CA4 modified with longer acyl chain could prolong the circulation time and increase the concentration of the drug in the tumor tissue. These effects play a critical role in increasing the antitumor efficacy.

Identification of adaptive inhibitors of Cryptosporidium parvum fatty acyl-coenzyme A synthetase isoforms by virtual screening.

Cryptosporidiosis is a significant cause of gastroenteritis in both humans and livestock in developing countries. The only FDA-approved drug available against the same is nitazoxanide, with questionable efficacy in malnourished children and immunocompromised patients. Recent in vitro studies have indicated the viability of Triacsin C as a potential drug candidate, which targets the parasite's long-chain fatty acyl coenzyme A synthetase enzyme (LC-FACS), a critical component of the fatty acid metabolism pathway. We have used this molecule as a baseline to propose more potent versions thereof. We have applied a combined approach of substructure replacement, literature search, and database screening to come up with 514 analogs of Triacsin C. A virtual screening protocol was carried out which lead us to identify a potential hit compound. This was further subjected to a 100-ns molecular dynamics simulation in complex to determine its stability and binding characteristics. After which, the ADME/tox properties were predicted to assess its viability as a drug. The molecule R134 was identified as the best hit due to its highest average binding affinity, stability in complex when subjected to MD simulations, and reasonable predicted ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties comparable to those of the Triacsin C parent molecule. We have proposed R134 as a putative drug candidate against the Cryptosporidium parvum LC-FACS enzyme isoforms, following an in silico protocol. We hope the results will be helpful when planning future in vitro experiments for identifying drugs against Cryptosporidium.

Structural analysis of a Vibrio phospholipase reveals an unusual Ser–His–chloride catalytic triad

Phospholipases can disrupt host membranes and are important virulence factors in many pathogens. VvPlpA is a phospholipase A2 secreted by Vibrio vulnificus and essential for virulence. Its homologs, termed thermolabile hemolysins (TLHs), are widely distributed in Vibrio bacteria, but no structural information for this virulence factor class is available. Herein, we report the crystal structure of VvPlpA to 1.4-Å resolution, revealing that VvPlpA contains an N-terminal domain of unknown function and a C-terminal phospholipase domain and that these two domains are packed closely together. The phospholipase domain adopts a typical SGNH hydrolase fold, containing the four conserved catalytic residues Ser, Gly, Asn, and His. Interestingly, the structure also disclosed that the phospholipase domain accommodates a chloride ion near the catalytic His residue. The chloride is five-coordinated in a distorted bipyramid geometry, accepting hydrogen bonds from a water molecule and the amino groups of surrounding residues. This chloride substitutes for the most common Asp/Glu residue and forms an unusual Ser-His-chloride catalytic triad in VvPlpA. The chloride may orient the catalytic His and stabilize the charge on its imidazole ring during catalysis. Indeed, VvPlpA activity depended on chloride concentration, confirming the important role of chloride in catalysis. The VvPlpA structure also revealed a large hydrophobic substrate-binding pocket that is capable of accommodating a long-chain acyl group. Our results provide the first structure of the TLH family and uncover an unusual Ser-His-chloride catalytic triad, expanding our knowledge on the biological role of chloride.

A novel LuxR-type solo of Sinorhizobium meliloti, NurR, is regulated by the chromosome replication coordinator, DnaA and activates quorum sensing.

The genome of Sinorhizobium meliloti, a model for studying plant-bacteria symbiosis, contains eight genes coding for LuxR-like proteins. Two of these, SinR and ExpR, are essential for quorum sensing (QS). Roles and regulation surrounding the others are mostly unknown. Here, we reveal the DNA recognition sequence and regulon of the LuxR-like protein SMc00877. Unlike ExpR, which uses the long-chain acyl homoserine lactones (AHLs) as inducers, SMc00877 functioned independently of AHLs and was even functional in Escherichia coli. A target of SMc00877 is SinR, the major regulator of AHL production in S. meliloti. Disruption of SMc00877 decreased AHL production. A weaker production of AHLs resulted in smaller microcolonies, starting from single cells, and delayed AHL-dependent regulation. SMc00877 was expressed only in growing cells in the presence of replete nutrients. Therefore, we renamed it NurR (nutrient sensitive LuxR-like regulator). We traced this nutrient-sensitive expression to transcription control by the DNA replication initiation factor, DnaA, which is essential for growth. These results indicate that NurR has a role in modulating the threshold of QS activation according to growth. We propose growth behavior as an additional prerequisite to population density for the activation of QS in S. meliloti.

High Membranous Expression of Fatty Acid Transport Protein 4 Is Associated with Tumorigenesis and Tumor Progression in Clear Cell Renal Cell Carcinoma.

The increased requirement of fatty acids forces cancer cells to enhance uptake of fatty acids from the extracellular milieu, in addition to de novo lipogenesis. Coexpression of cluster of differentiation 36 (CD36) with fatty acid transport protein 4 (FATP4) or long-chain acyl CoA synthetase 1 (ACSL1) synergistically activated fatty acid uptake in experimental models. In this study, we investigated the immunohistochemical expression of CD36, FATP4, and ACSL1 in 180 cases of clear cell renal cell carcinoma (RCC) in comparison with 80 specimens of the normal kidney. We also examined the clinical implication of these three fatty acid transporters in RCC, which was validated by an open-access The Cancer Genome Atlas data analysis. Both CD36 and FATP4 revealed higher membranous expressions in RCC tumor cells than in normal cells. In contrast, ACSL1 expression was remarkably reduced in RCC tumor cells compared to normal cells. CD36, FATP4, and ACSL1 showed high expressions in 74 (41.1%), 85 (47.2%), and 72 (40.0%) out of 180 RCC cases, respectively. Clinically, high FATP4 in tumor cells was associated with female gender (p = 0.05), high TNM stage (p = 0.039), tumor necrosis (p = 0.009), and tumor recurrence (p = 0.037), while high ACSL1 was only related to female gender (p = 0.023). CD36 expression revealed no correlation with the clinicopathologic parameters of RCC. Increased FATP4 expression displayed an association with short recurrence-free survival (p = 0.003). In conclusion, the high FATP4 expression was clinically associated with poor prognostic factors of RCC. Overexpression of membranous FATP4 and CD36 combined with reduced cytoplasmic expression of ACSL1 might be a tumor-specific feature of RCC, contributing to the tumorigenesis and tumor progression.