scholarly journals Mycobacterium smegmatis Erm(38) Is a Reluctant Dimethyltransferase

2005 ◽  
Vol 49 (9) ◽  
pp. 3803-3809 ◽  
Author(s):  
Christian Toft Madsen ◽  
Lene Jakobsen ◽  
Stephen Douthwaite
Keyword(s):  
Mycobacterium Smegmatis ◽  
Cell Walls ◽  
Pathogenic Bacteria ◽  
Specific Resistance ◽  
Methylation Pattern ◽  
Mass Spectrometry Analysis ◽  
23S Rrna ◽  
Resistance Pattern ◽  
Type I ◽  
Spectrometry Analysis

ABSTRACT The waxy cell walls of mycobacteria provide intrinsic tolerance to a broad range of antibiotics, and this effect is augmented by specific resistance determinants. The inducible determinant erm(38) in the nontuberculous species Mycobacterium smegmatis confers high resistance to lincosamides and some macrolides, without increasing resistance to streptogramin B antibiotics. This is an uncharacteristic resistance pattern falling between the type I and type II macrolide, lincosamide, and streptogramin B (MLSB) phenotypes that are conferred, respectively, by Erm monomethyltransferases and dimethyltransferases. Erm dimethyltransferases are typically found in pathogenic bacteria and confer resistance to all MLSB drugs by addition of two methyl groups to nucleotide A2058 in 23S rRNA. We show here by mass spectrometry analysis of the mycobacterial rRNA that Erm(38) is indeed an A2058-specific dimethyltransferase. The activity of Erm(38) is lethargic, however, and only a meager proportion of the rRNA molecules become dimethylated in M. smegmatis, while most of the rRNAs are either monomethylated or remain unmethylated. The methylation pattern produced by Erm(38) clarifies the phenotype of M. smegmatis, as it is adequate to confer resistance to lincosamides and 14-member ring macrolides such as erythromycin, but it is insufficient to raise the level of resistance to streptogramin B drugs above the already high intrinsic tolerance displayed by this species.

Pinpointing cysteine oxidation sites by high-resolution proteomics reveals a mechanism of redox-dependent inhibition of human STING

2021 ◽  
Vol 14 (680) ◽  
pp. eaaw4673
Author(s):  
Natalia Zamorano Cuervo ◽  
Audray Fortin ◽  
Elise Caron ◽  
Stéfany Chartier ◽  
Nathalie Grandvaux
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Disulfide Bonds ◽  
Redox Regulation ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Cysteine Oxidation ◽  
Spectrometry Analysis ◽  
Host Interactions ◽  
In Cells

Protein function is regulated by posttranslational modifications (PTMs), among which reversible oxidation of cysteine residues has emerged as a key regulatory mechanism of cellular responses. Given the redox regulation of virus-host interactions, the identification of oxidized cysteine sites in cells is essential to understand the underlying mechanisms involved. Here, we present a proteome-wide identification of reversibly oxidized cysteine sites in oxidant-treated cells using a maleimide-based bioswitch method coupled to mass spectrometry analysis. We identified 2720 unique oxidized cysteine sites within 1473 proteins with distinct abundances, locations, and functions. Oxidized cysteine sites were found in numerous signaling pathways, many relevant to virus-host interactions. We focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway, which is stimulated in response to the detection of cytosolic DNA by cGAS. We demonstrated the reversible oxidation of Cys148 and Cys206 of STING in cells. Molecular analyses led us to establish a model in which Cys148 oxidation is constitutive, whereas Cys206 oxidation is inducible by oxidative stress or by the natural ligand of STING, 2′3′-cGAMP. Our data suggest that the oxidation of Cys206 prevented hyperactivation of STING by causing a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This finding should aid the design of therapies targeting STING that are relevant to autoinflammatory disorders, immunotherapies, and vaccines.

scholarly journals Chemical characterization and mode of action of Ligustrum lucidum flower essential oil against food-borne pathogenic bacteria

2016 ◽  
Vol 11 (1) ◽  
pp. 269 ◽  
Author(s):  
Vivek K. Bajpai ◽  
Siddhartha Singh ◽  
Archana Mehta
Keyword(s):  
Essential Oil ◽  
Mode Of Action ◽  
Pathogenic Bacteria ◽  
Membrane Integrity ◽  
Chemical Characterization ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Ligustrum Lucidum ◽  
Food Borne

<p class="Abstract">Current research analyzes the chemical composition of <em>Ligustrum lucidum</em> flower essential oil obtained by the hydrodistillation, and examines its anti-microbial mode of action against food-borne pathogenic bacteria. Gas chromatography-mass spectrometry analysis of the oil resulted in the determination of 44 different compounds, representing 85.2% of the total oil. The oil (1 mg/disc) showed significant antibacterial effect as diameters of inhibition zones (14.6 ± 0.2 – 19.7 ± 0.3 mm), as well as minimum inhibitory and minimum bactericidal concentrations values (250–1000 and 250–2000 µg/mL), respectively. Based on the susceptibility, <em>L. lucidum</em> flower oil revealed its mode of action on membrane integrity as confirmed by increased release of extracellular ATP (2.5 and 2.2 pg/mL), leakage of potassium ions (950 and 900 mM/L), loss of 260-nm absorbing materials (4.2 and 3.9 optical density), and increase in relative electrical conductivity (10.6 and 9.8%) against <em>Staphylococcus aureus</em> KCTC-1621 (Gram-positive) and <em>Salmonella enterica</em> ATCC-4731 (Gram-negative), respectively.</p><p> </p>

scholarly journals Identification of Extracellular N-Acylhomoserine Lactone Acylase from a Streptomyces sp. and Its Application to Quorum Quenching

2005 ◽  
Vol 71 (5) ◽  
pp. 2632-2641 ◽  
Author(s):  
Sun-Yang Park ◽  
Hye-Ok Kang ◽  
Hak-Sun Jang ◽  
Jung-Kee Lee ◽  
Bon-Tag Koo ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Pathogenic Bacteria ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Amide Bond ◽  
Mass Spectrometry Analysis ◽  
Penicillin G ◽  
Spectrometry Analysis ◽  
Acyl Chains

ABSTRACT N-Acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.

rBAT-b0,+AT heterodimer is the main apical reabsorption system for cystine in the kidney

2002 ◽  
Vol 283 (3) ◽  
pp. F540-F548 ◽  
Author(s):  
Esperanza Fernández ◽  
Montserrat Carrascal ◽  
Ferran Rousaud ◽  
Joaquín Abián ◽  
Antonio Zorzano ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Expression Patterns ◽  
Human Kidney ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Spectrometry Analysis ◽  
Sds Page ◽  
Heterologous Cell ◽  
Dibasic Amino Acids

Mutations in the rBAT and b0,+AT genes cause type I and non-type I cystinuria, respectively. The disulfide-linked rBAT-b0,+AT heterodimer mediates high-affinity transport of cystine and dibasic amino acids (b0,+-like activity) in heterologous cell systems. However, the significance of this heterodimer for cystine reabsorption is unknown, as direct evidence for such a complex in vivo is lacking and the expression patterns of rBAT and b0,+AT along the proximal tubule are opposite. We addressed this issue by biochemical means. Western blot analysis of mouse and human kidney brush-border membranes showed that rBAT and b0,+AT were solely expressed as heterodimers of identical size and that both proteins coprecipitated. Moreover, quantitative immunopurification of b0,+AT followed by SDS-PAGE and mass spectrometry analysis established that b0,+AT heterodimerizes exclusively with rBAT. Together with cystine reabsorption data, our results demonstrate that a decreasing expression gradient of heterodimeric rBAT-b0,+AT along the proximal tubule is responsible for virtually all apical cystine reabsorption. As a corollary of the above, there should be an excess of rBAT expression over that of b0,+AT protein in the kidney. Indeed, complete immunodepletion of b0,+AT did not coprecipitate >20–30% of rBAT. Therefore, another rBAT-associated subunit may be present in latter parts of the proximal tubule.

scholarly journals Holomycin, an antibiotic secondary metabolite, is required for biofilm formation of the native producer Photobacterium galatheae S2753

2021 ◽  
Author(s):  
Sheng-Da Zhang ◽  
Thomas Isbrandt ◽  
Laura Louise Lindqvist ◽  
Thomas Ostenfeld Larsen ◽  
Lone Gram
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Genome Mining ◽  
Core Gene ◽  
Mass Spectrometry Analysis ◽  
Distinct Advantage ◽  
Spectrometry Analysis ◽  
Cell Extracts ◽  
Antibiotic Compounds ◽  
Ecological Importance

Whilst the effects of antibiotics on microorganisms are widely studied, it remains less well-understood how antibiotics affect the physiology of the native producing organisms. Here, using a marine bacterium Photobacterium galatheae S2753 that produces the antibiotic holomycin, we generated a holomycin deficient strain by in-frame deletion of hlmE, the core gene responsible for holomycin production. Mass spectrometry analysis of cell extracts confirmed that ΔhlmE did not produce holomycin and that the mutant was devoid of antibacterial activity. Biofilm formation of ΔhlmE was significantly reduced compared to that of the wild-type S2753 and was restored in an hlmE complementary mutant. Consistently, exogenous holomycin, but not its dimethylated and less antibacterial derivative, S,S’-dimethyl holomycin, restored the biofilm formation of ΔhlmE. Furthermore, zinc starvation was found essential for both holomycin production and biofilm formation of S2753, although the molecular mechanism remains elusive. Collectively, these data suggest that holomycin promotes biofilm formation of S2753 via its ene-disulfide group. Lastly, the addition of holomycin in sub-inhibitory concentrations also enhanced the biofilm of four other Vibrionaceae strains. P. galatheae likely gains an ecological advantage from producing holomycin as both an antibiotic and a biofilm stimulator, which facilitates the nutrition acquisition and protects P. galatheae from environmental stresses. Studying the function of antibiotic compounds in the native producer will shed light on their role in nature and could potentially point to novel bioprospecting strategies. Importance Despite the societal impact of antibiotics, their ecological functions remain elusive and have mostly been studied by exposing non-producing bacteria to sub-inhibitory concentrations. Here, we studied the effects of the antibiotic holomycin on its native producer, Photobacterium galatheae S2753, a Vibrionaceae bacterium. Holomycin provides a distinct advantage to S2753 both as an antibiotic and by enhancing biofilm formation in the producer. Vibrionaceae species successfully thrive in global marine ecosystems, where they play critical ecological roles as free-living, symbiotic, or pathogenic bacteria. Genome mining has demonstrated that many have the potential to produce several bioactive compounds, including P. galaltheae. To unravel the contribution of the microbial metabolites to the development of marine microbial ecosystems, better insight into the function of these compounds in the producing organisms is needed. Our finding provides a model to pursue this and highlights the ecological importance of antibiotics to the fitness of the producing organisms.

scholarly journals Inhibition of PRMT1 Mediated FLT3 Arginine Methylation As a Potent Therapeutic Strategy for MLL-r ALL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 892-892
Author(s):  
Yinghui Zhu ◽  
Xin He ◽  
Haojie Dong ◽  
Jie Sun ◽  
Hanying Wang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Combination Treatment ◽  
Kinase Inhibitors ◽  
Arginine Methylation ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Mrna Levels ◽  
Spectrometry Analysis ◽  
Dismal Prognosis

Abstract Mixed-lineage leukemia-rearranged (MLL-r) ALL, seen in 70% of infant ALL, has a dismal prognosis compared to those with wild type MLL1 gene. Transcriptional profiling has identified Fms-like receptor tyrosine kinase 3 (FLT3) as one of the most significantly upregulated genes in MLL-r ALL. The highly expressed FLT3 protein is activated by the autocrine ligand, making the kinase a therapeutic target. FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412, although effective in kinase inhibition, partially impair survival of MLL-r ALL cells and clinical trial results are not promising, promoting us to ask whether FLT3 regulates the ALL cells survival also through a kinase-independent mechanism. Herein, we report the finding of dimethylated arginines on FLT3, detected through mass spectrometry analysis of a MLL-r ALL specimen and a MLL-r ALL line SEM. The most conserved and enriched of dimethylated arginines are residues R972/R973. Using home-made arginine methylation (R-Me) antibody, we found that PRMT1, which is responsible for most type I arginine methyltransferases activity, catalyzes FLT3 methylation. Immunoblot (IB) analysis validated the expression of FLT3 R-Me in MLL-r ALL samples (6 out of 6) and MLL-r ALL lines (4 out of 4). Analysis of the GEO dataset (GSE13204) revealed that PRMT1 mRNA levels are increased in MLL-r ALL relative to normal cells (MLL-r, n=70 vs. normal, n=73, p<0.0001). We studied FLT3 R-Me biological function using two approaches that specifically blocked FLT3 methylation levels: cells expressing FLT3 methylation deficient construct (R972/973K, arginine [R] to lysine [K]) exhibited reduced survival (BaF3: FLT3-WT 98.5±0.11% vs. R972/973K 71.5±0.53%, p=0.0004); knockdown of PRMT1 in SEM cells also had an inhibitory effect (siCtrl 95.1±0.1% vs. siPRMT1 74.7±0.5%, p=0.0007). Moreover, the type I arginine methyltransferase inhibitor MS023 (5 µM) treatment markedly induced apoptosis of primary ALL cells but spared normal counterparts from healthy donors (ALL: vehicle 10.4±0.4% vs. MS023 23.7±0.8%, n=4; p<0.0001; normal CD19+: 8.3±0.3% vs. 8.2±0.1%, n=3, p=0.86). Interestingly, inhibition of FLT3 methylation decreased FLT3 phosphorylation at tyrosine 969 (Y969) but not Y589/591 or Y842. Expression of R972/973K decreased FLT3 downstream signaling like phospho-STAT5 and -AKT to a greater extent than that of Y969F mutant (Y to phenylalanine [F] substitution, mimics loss of Y phosphorylation). Next, FLT3 WT, R972/973K or Y969F transduced primary MLL-r ALL cells were transplanted into NSGS mice for analysis of leukemia development (n=6/group). Mice transplanted with FLT3 Y969F MLL-r ALL had longer survival relative to FLT-WT injected animals (p=0.0031), and the median survival was further extended in mice injected with R972/973K mutant compared with FLT3 Y969F MLL-r ALL (p=0.0007). Additionally, PKC412 treatment alone did not alter FLT3 R-Me, and high FLT3 methylation level in SEM cells was not affected by FLT3 ligand stimulation, confirming that the function of R-Me is independent of FLT3 phosphorylation. Importantly, we observed that the combination of MS023 with PKC412 significantly induced a higher rate of apoptosis in primary MLL-r ALL cells compared with each drug alone (control, 10±0.43%, MS023, 21.1±1.2%, PKC412, 21.5±0.11%, combination, 39.8±2.9%, PKC412 vs combination, p<0.01, n=4). We further tested the effects of in vivo administration of MS023 plus PKC412 on primary MLL-r ALL cells xenografted in NSGS mice. Following engraftment >1% in peripheral blood, mice were subdivided into four groups and treated with vehicle, PKC412 (100 mg/kg, i.g.), MS023 (80 mg/kg, i.p, bid), or the combination (n=7/group) for 4 weeks. The BM tumor burden of CD45+ CD19+ cells was reduced in single drug-treated mice cohorts, with further reduction after combination treatment (vehicle, 94.4±0.5%, PKC412, 50.2±6.3%, MS023, 55.6±4.5%, combination, 30.7±4.9%, PKC412 vs. combination, p<0.001). Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced BM engraftment at 16 weeks compared to PKC412 treatment alone (PKC412, 62.2±4.9%, combination, 8.4±5.1%, n=5, p<0.0001), indicating reduced leukemia initiating capacity. Our results support further exploring the molecular function of FLT3 R-Me. We will determine whether PRMT1 and FLT3 methylation are potential druggable targets in MLL-r ALL. Disclosures Konopleva: Stemline Therapeutics: Research Funding.

scholarly journals Preservation of collagen in the soft tissues of frozen mammoths

2021 ◽  
Author(s):  
Shunji Hattori ◽  
Tomomi Kiriyama-Tanaka ◽  
Masashi Kusubata ◽  
Yuki Taga ◽  
Testuya Ebihara ◽  
...  
Keyword(s):  
Soft Tissues ◽  
Mass Spectrometry Analysis ◽  
Fibrillar Structure ◽  
Collagen Molecule ◽  
Type I ◽  
Spectrometry Analysis ◽  
Type Iv ◽  
Cross Striation ◽  
Main Protein ◽  
Biochemical Analyses

We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths ( Mammuthus primigenius ) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography–mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years.

Abstract 323: Arginine Methylation Regulates Epicardial Cell Plasticity

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Jian Xu ◽  
Juan Oses-Prieto ◽  
Alma Burlingame ◽  
Rik Derynck
Keyword(s):  
Complex Formation ◽  
Arginine Methylation ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Receptor Complex ◽  
Kinase Activation ◽  
Spectrometry Analysis ◽  
Transcriptional Reprogramming ◽  
Epicardial Cells ◽  
Substrate Phosphorylation

Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. TGFbeta family ligands induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases following type I and type II receptor complex formation. Here we show that arginine methylation, which regulates gene expression, yet also modifies some signaling mediators, initiates TGFbeta-induced Smad signaling. TGFbeta-induced receptor complex formation promotes presentation of the methyltransferase PRMT1 to the inhibitory Smad7, resulting in Smad7 methylation at a specific arginine as revealed by mass spectrometry analysis and confirmed with a mutagenesis approach. Antibodies specific for asymmetric di-methylated arginine further confirmed Smad7 methylation and Smad7 recruitment to the cell membrane upon TGFbeta stimulation. TGFbeta-induced and PRMT1-mediated Smad7 methylation leads to Smad7 dissociation from TGFbeta type I receptors, allowing activation of effector Smads through phosphorylation. This PRMT1-mediated Smad7 methylation is required in TGFbeta-regulated epicardial plasticity, as apparent by the role of PRMT1 in TGFbeta-induced epithelial-to-mesenchymal trans-differentiation and transcriptional reprogramming in mouse epicardial cells.

scholarly journals Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 792
Author(s):  
Olivier Tremblay ◽  
Zachary Thow ◽  
A. Rod Merrill
Keyword(s):  
Host Cell ◽  
In Silico ◽  
Pathogenic Bacteria ◽  
Erwinia Amylovora ◽  
Genomic Sequence ◽  
Whooping Cough ◽  
Sequence Data ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Adp Ribosyltransferase

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora, called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

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