Human intestinal acyl-CoA synthetase 5 is sensitive to the inhibitor triacsin C

AIM:To investigate whether human acyl-CoA synthetase 5(ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS:The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes.Ni 2+-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C.In addition,ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored.ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence.The ACSL assay mix included TrisHCl(pH 7.4),ATP,CoA,EDTA,DTT,MgCl 2,[9,103 H] palmitic acid,and triton X-100.The 200 μL reaction was initiated with the addition of solubilized,purified recombinant proteins or cellular lysates.Reactions were terminated after 10,30 or 60 min of incubation with Doles medium.RESULTS:Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl betaD-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni 2+-affinity chromatography.Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5,as well as recombinant rat ACSL1(sensitive control),but not recombinant rat ACSL5(insensitive control).The IC50 for human ACSL5 was about 10 μmol/L.The inhibitory triacsin C effect was similar for different incubation times(10,30 and 60 min) and was not modified by the N-or C-terminal location of the 6xHis-tag.In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment,stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed.In both models,ACSL5 peak activity was found at pH 7.5 and pH 9.5,corresponding to the properties of recombinant human ACSL5 protein.In the presence of triacsin C(25 μmol/L),total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.CONCLUSION:The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.

Front line defenders of the ecological niche!Screening the structural diversity of peptaibiotics from saprotrophic and fungicolous Trichoderma/Hypocrea species

Approximately 950 individual sequences of nonribosomally biosynthesised peptides are produced by the genus Trichoderma/Hypocreathat belong to a perpetually growing class of mostly linear antibiotic oligopeptides,which are rich in the non-proteinogenic α-aminoisobutyric acid(Aib).Thus,they are comprehensively named peptaibiotics.Notably,peptaibiotics represent ca.80% of the total inventory of secondarymetabolites currently known from Trichoderma/Hypocrea.Their unique membrane-modifying bioactivity results from amphipathicity and helicity,thus making them ideal candidates in assisting both colonisation and defence of the natural habitats by their fungal producers.Despite this,reports on the in vivo-detection of peptaibiotics have scarcely been published in the past.In order to evaluate the significance of peptaibiotic production for a broader range of potential producers,we screened nine specimens belonging to seven hitherto uninvestigated fungicolous or saprotrophic Trichoderma/Hypocrea species by liquid chromatography coupled to electrospray high resolution mass spectrometry.Sequences of peptaibiotics found were independently confirmed by analysing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens.Of the nine species examined,five were screened positive for peptaibiotics.A total of 78 peptaibiotics were sequenced,56(=72%)of which are new.Notably,dihydroxyphenylalaninol and O-prenylated tyrosinol,two C-terminal residues,which have not been reported for peptaibiotics before,were found as well as new and recurrent sequences carrying the recently described tyrosinol residue at their C-terminus.The majority of peptaibiotics sequenced are 18-or 19-residue peptaibols.Structural homologies with‘classical representatives’of subfamily 1(SF1)-peptaibiotics argue for the formation of transmembrane ion channels,which are prone to facilitate the producer capture and defence of its substratum.

Antidiabetic profiling of veramycins,polyketides accessible by biosynthesis,chemical synthesis and precursor-directed modification

Seven new polyketides,termed veramycins,were isolated from a Streptomyces sp.from the Sanofi microbial strain collection along with their known congeners NFAT-133 and TM-123.Veramycin A,anα-pyrone congener of TM-123 and NFAT-133 showed an increased baseline deoxy-glucose uptake in the absence of insulin in a modified L6 rat skeletal muscle cell line(L6 GLUT4 AS160-like cells).In addition,both compounds slightly increased the sensitivity to insulin in this cell line.Total syntheses of NFAT-133,TM-123 and veramycin A were accomplished starting from a central building block,which bears the three contiguous stereogenic centers of this polyketide family.Our approach enables an efficient,selective and flexible access to all possible isomers of the stereotriad for further exploration of this series as a potential anti-diabetic lead structure as exemplified by the synthesis of an NFAT-133 epimer.Finally,the corresponding biosynthetic gene cluster(BGC)was identified by genome sequencing and gene inactivation.Based on feeding experiments,a biosynthetic pathway was proposed,which enabled access to new veramycin A analogs by precursor-directed biosynthesis.