ABSTRACT
Thioesters of coenzyme A (CoA) carrying different acyl chains (acyl-CoAs) are central intermediates of many metabolic pathways and donor molecules for protein lysine acylation. Acyl-CoA species largely differ in terms of cellular concentrations and physico-chemical properties, rendering their analysis challenging. Here, we compare several approaches to quantify cellular acyl-CoA concentrations in normal and ischemic rat liver, using HPLC and LC-MS/MS for multi-acyl-CoA analysis, as well as NMR, fluorimetric and spectrophotometric techniques for the quantification of acetyl-CoAs. In particular, we describe a simple LC-MS/MS protocol that is suitable for the relative quantification of short and medium-chain acyl-CoA species. We show that ischemia induces specific changes in the short-chain acyl-CoA relative concentrations, while mild ischemia (1-2 min), although reducing succinyl-CoA, has little effects on acetyl-CoA, and even increases some acyl-CoA species upstream of the tricarboxylic acid cycle. In contrast, advanced ischemia (5-6 min) also reduces acetyl-CoA levels. Our approach provides the keys to accessing the acyl-CoA metabolome for a more in-depth analysis of metabolism, protein acylation and epigenetics.
Subject(s)
Acyl Coenzyme A , Tandem Mass Spectrometry , Rats , Animals , Acetyl Coenzyme A/analysis , Chromatography, Liquid/methods , Acyl Coenzyme A/metabolism , Coenzyme A/analysis , Ischemia , Liver/metabolismABSTRACT
Discovery after discovery, host-associated microbiota reveal a growing list of positive effects on host homeostasis by contributing to host nutrition, improving hosts' immune systems and protecting hosts against pathogens. In that context, a collection of oyster associated bacteria producing antibacterial compounds have been established to evaluate their role in non-host-derived immunity. Here, we described alterins; potent anti-Gram negative compounds produced by Pseudoalteromonas hCg-6 and hCg-42 isolated from different healthy oyster hemolymph. The strains hCg-6 and hCg-42 produce a set of at least seven antibacterial compounds, ranging from 926 to 982 Da structurally characterized as cyclolipopeptides (CLPs). Alterins share the same cationic heptapeptidic cycle connected via an amido bond to different hydrophobic hydrocarbon tails. Their MICs disclosed a potent antibacterial activity directed against Gram-negative bacteria including oyster and human pathogens that may confer a beneficial defense mechanism to the host but also represents an untapped source of new antibiotics. The alterins' mechanisms of action have been deciphered: after binding to lipopolysaccharides (LPS), alterins provoke a membrane depolarization and permeabilization leading to bacterial lysis. As hCg-6 and hCg-42 produced a set of natural derivatives, the structure/activity relationship linked to the carbon tail is clarified. We showed that the hydrocarbon tail determines the LPS-binding properties of alterins and consequently their antibacterial activities. Its length and saturation seem to play a major role in this interaction.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Gram-Negative Bacteria/drug effects , Lipopeptides/pharmacology , Lipopolysaccharides/metabolism , Ostreidae/microbiology , Peptides, Cyclic/pharmacology , Pseudoalteromonas/metabolism , Animals , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/metabolism , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/metabolism , Gram-Negative Bacteria/growth & development , Hemolymph/microbiology , Host-Pathogen Interactions , Lipopeptides/isolation & purification , Lipopeptides/metabolism , Microbial Sensitivity Tests , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/metabolism , Structure-Activity RelationshipABSTRACT
Selective glycosylation of the C-6 fluorinated galactofuranosyl acceptor 2 was studied with four galactofuranosyl donors. It was highlighted that this electron-withdrawing atom strongly impacted the behavior of the acceptor, thus leading to unprecedented glycosylation pathways. Competition between expected glycosylation of 2, ring expansion of this acceptor and furanosylation, and intermolecular aglycon transfer was observed. Further investigation of the fluorinated synthetic compounds showed that the presence of fluorine atom contributed to increase the inhibition of the growth of Leishmania tarentolae, a non-pathogenic strain of Leishmania.