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1.  Oxidative Modifications of Mitochondria Complex II 
Summary
Increased superoxide (O2•−) and nitric oxide (NO) production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In the complex II, oxidative impairment, decreased protein S-glutathionylation, and increased protein tyrosine nitration at the 70 kDa subunit occurs in the post-ischemic myocardium (1–3). To gain the deeper insights into ROS–mediated oxidative modifications relevant in myocardial infarction, isolated complex II is subjected to in vitro oxidative modifications with GSSG (to induce cystine S-glutathionylation) or OONO− (to induce tyrosine nitration). Here, we describe the protocol to characterize the specific oxidative modifications at the 70 kDa subunit by nano-LC/MS/MS analysis. We further demonstrate the cellular oxidative modification with protein nitration/S-glutathionylation with immunofluorescence microscopy using the antibodies against 3-nitrotyrosine/glutathione and complex II 70 kDa polypeptide (AbGSC90) in myocytes under conditions of oxidative stress.
doi:10.1007/978-1-62703-386-2_12
PMCID: PMC3774787  PMID: 23606255
Mitochondria; Complex II; Nitration; S-glutathionylation; Protein Disulfide linkage; S-sulfonation
2.  Peroxynitrite-Mediated Oxidative Modifications of Complex II: Relevance in the Myocardial Infarction† 
Biochemistry  2010;49(11):2529-2539.
Increased O2•− and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In complex II, oxidative impairment and enhanced tyrosine nitration of the 70 kDa FAD-binding protein occurs in the post-ischemic myocardium, and is thought to be mediated by peroxynitrite (OONO−) in vivo (Chen et al. (2008) J. Biol. Chem. 283 27991–28003). To gain the deeper insights into the redox protein thiols involved in OONO− –mediated oxidative post-translational modifications relevant in myocardial infarction, isolated myocardial complex II was subjected to in vitro protein nitration with OONO−. This resulted in site-specific nitration at the 70 kDa polypeptide and impairment of complex II-derived electron transfer activity. Under reducing conditions, the gel band of the 70 kDa polypeptide was subjected to in-gel trypsin/chymotrypsin digestion and then LC/MS/MS analysis. Nitration of Y56 and Y142 was previously reported. Further analysis revealed that C267, C476, and C537 are involved in OONO− –mediated S-sulfonation. To identify the disulfide formation mediated by OONO−, the nitrated complex II was alkylated with iodoacetamide. In-gel proteolytic digestion and LC/MS/MS analysis were carried out under non-reducing conditions. The MS/MS data were examined with MassMatrix program, indicating that three cysteine pairs, C306-C312, C439-C444, and C288-C575 were involved in OONO− –mediated disulfide formation. Immuno-spin trapping with anti-DMPO antibody and subsequent MS was used to define oxidative modification with protein radical formation. An OONO− –dependent DMPO adduct was detected, and further LC/MS/MS analysis indicated C288 and C655 were involved in DMPO-binding. These results offered a complete profile of OONO− –mediated oxidative modifications that may be relevant in the disease model of myocardial infarction.
doi:10.1021/bi9018237
PMCID: PMC3754874  PMID: 20143804
3.  Protein Thiyl Radical Mediates S-glutathionylation of Complex I 
Free radical biology & medicine  2012;53(4):962-973.
Complex I is a critical site of O2•− production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, Complex I protein thiols at the 51 kDa and 75 kDa subunits are reversibly S-glutathionylated. The mechanism of Complex I S-glutathionylation is mainly obtained from insight into GSSG-mediated thiol-disulfide exchange, which would require a dramatic decline in the GSH/GSSG ratio. Intrinsic Complex I S-glutathionylation can be detected in the rat heart at a relatively high GSH/GSSG ratio (Chen, J., et al. J. Biol. Chem 285: 3168–3180, 2010). Thus, we hypothesized that reactive thiyl radical is more likely to mediate protein S-glutathionylation of Complex I. Here we employed immuno-spin trapping and tandem mass spectrometry (LC/MS/MS) to test the hypothesis in the 75 kDa subunit from S-glutathionylated Complex I. Under the conditions of O2•− production in the presence of GSH, we detected Complex I S-glutathionylation at the Cys-226, Cys-367, and Cys-727 of the 75 kDa subunit. Addition of a radical trap, 5, 5-dimethyl-1-Pyroline-N-Oxide (DMPO), significantly decreased Complex I S-glutathionylation, and subsequently increased the protein radical adduct of Complex I-DMPO as detected by immunoblotting using an anti-DMPO antibody. LC/MS/MS analysis indicated that Cys-226, Cys-554, and Cys-727 were involved in DMPO-binding, confirming that formation of the Complex I thiyl radical mediates S-glutathionylation. LC/MS/MS analysis also showed that Cys-554 and Cys-727 were S-sulfonated under conditions of O2•− generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O2•− generation, Complex I protein radical and S-glutathionylation were increased. Thus mediation of Complex I S-glutathionylation by the protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function.
doi:10.1016/j.freeradbiomed.2012.05.025
PMCID: PMC3418477  PMID: 22634394
Complex I; S-glutathionylation; Protein Thiyl Radical; Immuno-spin Trapping; Myocytes
4.  Proteomic Analysis Reveals New Cardiac-Specific Dystrophin-Associated Proteins 
PLoS ONE  2012;7(8):e43515.
Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.
doi:10.1371/journal.pone.0043515
PMCID: PMC3427372  PMID: 22937058
7.  The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine 
Nature  2011;471(7340):647-650.
Pyrrolysine, the 22nd amino acid to be found in the natural genetic code1–4, is necessary for all known pathways of methane formation from methylamines5,6. The residue is comprised of a methylated pyrroline carboxylate in amide linkage to the ε-amino group of L-lysine2,7,8. The three different methyltransferases that initiate methanogenesis from different methylamines9–11 have genes with an in-frame amber codon12,13 translated as pyrrolysine2,7,8. E. coli transformed with pylTSBCD from methanogenic Archaea can incorporate endogenously biosynthesized pyrrolysine into protein14. The decoding of UAG as pyrrolysine requires pylT1,6 which produces tRNAPyl (also called tRNACUA), and pylS1 encoding a pyrrolysyl-tRNA synthetase4,15,16. The pylBCD genes1 are each required for tRNA-independent pyrrolysine synthesis14. Pyrrolysine has been the last remaining genetically encoded amino acid with an unknown biosynthetic pathway. Here, we provide genetic and mass spectroscopic evidence for a pylBCD-dependent pathway in which pyrrolysine arises from two lysines. We show that a new UAG encoded residue, desmethylpyrrolysine, is made from lysine and exogenous D-ornithine in a pylC, then a pylD, dependent process, but is not further converted to pyrrolysine. These results indicate that the radical S-adenosyl-methionine (SAM) protein PylB mediates a lysine mutase reaction producing 3-methylornithine, which is then ligated to a second molecule of lysine by PylC before oxidation by PylD results in pyrrolysine. The discovery of lysine as sole precursor to pyrrolysine will further inform discussions of the evolution the genetic code and amino acid biosynthetic pathways, while intermediates of the pathway may provide new avenues by which the pyl system may be exploited for production of recombinant proteins with useful modified residues.
doi:10.1038/nature09918
PMCID: PMC3070376  PMID: 21455182
8.  Interspecific Proteomic Comparisons Reveal Ash Phloem Genes Potentially Involved in Constitutive Resistance to the Emerald Ash Borer 
PLoS ONE  2011;6(9):e24863.
The emerald ash borer (Agrilus planipennis) is an invasive wood-boring beetle that has killed millions of ash trees since its accidental introduction to North America. All North American ash species (Fraxinus spp.) that emerald ash borer has encountered so far are susceptible, while an Asian species, Manchurian ash (F. mandshurica), which shares an evolutionary history with emerald ash borer, is resistant. Phylogenetic evidence places North American black ash (F. nigra) and Manchurian ash in the same clade and section, yet black ash is highly susceptible to the emerald ash borer. This contrast provides an opportunity to compare the genetic traits of the two species and identify those with a potential role in defense/resistance. We used Difference Gel Electrophoresis (DIGE) to compare the phloem proteomes of resistant Manchurian to susceptible black, green, and white ash. Differentially expressed proteins associated with the resistant Manchurian ash when compared to the susceptible ash species were identified using nano-LC-MS/MS and putative identities assigned. Proteomic differences were strongly associated with the phylogenetic relationships among the four species. Proteins identified in Manchurian ash potentially associated with its resistance to emerald ash borer include a PR-10 protein, an aspartic protease, a phenylcoumaran benzylic ether reductase (PCBER), and a thylakoid-bound ascorbate peroxidase. Discovery of resistance-related proteins in Asian species will inform approaches in which resistance genes can be introgressed into North American ash species. The generation of resistant North American ash genotypes can be used in forest ecosystem restoration and urban plantings following the wake of the emerald ash borer invasion.
doi:10.1371/journal.pone.0024863
PMCID: PMC3174216  PMID: 21949771
9.  Shotgun Lipidomic Analysis of Human Meibomian Gland Secretions with Electrospray Ionization Tandem Mass Spectrometry 
The major molecular components of the lipids in normal human meibomian gland secretions were determined with shotgun electrospray ionization mass spectrometry and tandem mass spectrometry analysis.
Purpose.
The purpose of this investigation was to determine the major molecular components of the lipids in normal human meibomian gland secretions (meibum).
Methods.
The meibum samples were studied by direct infusion electrospray ionization (ESI), quadrupole time-of-flight mass spectrometry, and tandem mass spectrometry (MS/MS) analysis, in both positive and negative detection modes.
Results.
Hundreds of peaks were detected, among which the molecular compositions and subclasses of approximately 160 major peaks were confidently identified. The compositions and subclasses of these peaks were determined from collision-induced dissociation fragmentation patterns, high-resolution and high-mass-accuracy spectra, and references of literature reports. The major peaks detected in positive mode were those of nonpolar lipids, including wax esters, cholesteryl esters, triacylglycerols, and diesters, whereas in negative mode, the major peaks detected were those of polar lipids, including free fatty acids and (O-acyl)-ω-hydroxy fatty acids.
Conclusions.
The analysis of intact lipids in meibum with direct infusion ESI-MS/MS analysis has the advantages of minimal sample preparation (no chromatography or pre-separation needed), mild experimental conditions, high throughput, and high sensitivity.
doi:10.1167/iovs.10-5687
PMCID: PMC3055753  PMID: 20671273
11.  Proteomic Analysis of Protein Tyrosine Nitration after Ischemia Reperfusion Injury: Mitochondria as the Major Target 
Biochimica et biophysica acta  2008;1794(3):476-485.
Endothelial nitric oxide synthase-derived NO and its derivative, peroxynitrite (ONOO-), suppresses oxygen consumption by nitration of mitochondrial proteins after reperfusion. However, very few nitrated proteins are identified to date. In this paper, ischemia/reperfusion (I/R) injury was induced in mouse heart by ligation and release of the left anterior descending coronary artery. Western blotting showed that tyrosine nitration was higher in I/R hearts. Nitrated proteins were identified by capillary-liquid chromatography-nanospray tandem mass spectrometry. A total of 23 proteins were identified as being nitrated after I/R and 10 of them were from mitochondria. The nitrated mitochondrial proteins included 4 subunits from the oxidative phosphorylation system (the 24 and the 30 kDa subunits of complex I, the Rieske ISP of complex III, and the α subunit of ATP synthase), five enzymes in the matrix, and voltage-dependent anion channel. In purified complex I treated with ONOO-, 3-NT was identified locating at the residue of Y247 of the 30 kDa subunit and the residues of Y47, Y53 of the 49 kDa subunit. In conclusion, I/R induced protein nitration and mitochondrial proteins were the major targets. Selective nitration of proteins from the oxidative phosphorylation system at the beginning of reperfusion may contribute to the suppression of oxygen consumption.
doi:10.1016/j.bbapap.2008.12.008
PMCID: PMC2637933  PMID: 19150419
Peroxynitrite; Nitric oxide; Oxidative phosphorylation; Energy Metabolism; Oxygen consumption
12.  Mass Spectrometry Profiles Superoxide-Induced Intra-molecular Disulfide in the FMN-binding Subunit of Mitochondrial Complex I 
Protein thiols with regulatory functions play a critical role in maintaining the homeostasis of the redox state in mitochondria. One major host of regulatory cysteines in mitochondria is complex I, with the thiols primarily located on its 51 kDa FMN-binding subunit. In response to oxidative stress, these thiols are expected to form intra-molecular disulfide bridges as one of their oxidative post-translational modifications. Here, to test this hypothesis and gain insights into the molecular pattern of disulfide in complex I, the isolated bovine complex I was prepared. Superoxide (O2•−) is generated by complex I under the conditions of enzyme turnover. O2•−-induced intra-molecular disulfide formation at the 51 kDa subunit was determined by tandem mass spectrometry and database searching, with the latter accomplished by adaptation of the in-house developed database search engine, MassMatrix [Xu H., et. al J. Proteome Res. (2008) 7, 138–44]. LC/MS/MS analysis of tryptic/chymotryptic digests of the 51 kDa subunit from alkylated complex I revealed that four specific cysteines (C125, C142, C187, and C206) of the 51 kDa subunit were involved in the formation of mixed intra-molecular disulfide linkages. In all, three cysteine pairs were observed: C125/C142, C187/C206, and C142/C206. The formation of disulfide bond was subsequently inhibited by superoxide dismutase, indicating the involvement of O2•−. These results elucidated by mass spectrometry indicates that the residues of C125, C142, C187, and C206 are the specific regulatory cysteines of complex I, and they participate in the oxidative modification with disulfide formation under the physiological or pathophysiological conditions of oxidative stress.
doi:10.1016/j.jasms.2008.08.004
PMCID: PMC2614441  PMID: 18789718
13.  Biomarker Discovery for Lupus Nephritis Through Longitudinal Urine Proteomics 
Kidney international  2008;74(6):799-807.
Lupus nephritis is a frequent and serious complication of systemic lupus erythematosus (SLE). Treatment often requires the use of immunosuppression, and may be associated with severe side effects. The ability to predict relapse, relapse severity, and recovery could be used to more effectively implement therapy and reduce toxicity. We postulated that a proteomic analysis of the low-molecular weight urine proteome using serial urine samples obtained before, during, and after SLE nephritis flares would demonstrate potential biomarkers of SLE renal flare. This study was undertaken to test our hypothesis.
Urine from 25 flare cycles of 19 WHO Class III, IV, and V SLE nephritis patients was used. Urine samples included a baseline, and pre-flare, flare, and post-flare specimens. The urines were fractionated to remove proteins larger than 30 kDa, and spotted onto weak cation exchanger (CM10) protein chips for analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS).
SELDI-TOF MS screening showed 176 protein ions between 2-20 kDa of which 27 were found to be differentially-expressed between specific flare intervals. On-chip peptide sequencing by integrated tandem mass spectrometry was used to positively identify selected differentially-expressed protein ions. The identified proteins included the 20 and 25 amino acid isoforms of hepcidin, a fragment of α1-antitrypsin, and an albumin fragment. Hepcidin 20 increased 4 months pre-flare and returned to baseline at renal flare, whereas hepcidin 25 decreased at renal flare and returned to baseline 4 months post-flare.
Using SELDI-TOF urine protein profiling in lupus nephritis, several candidate biomarkers of renal flare were found. To verify these candidates as true biomarkers, further identification and validation are needed in an independent SLE cohort.
doi:10.1038/ki.2008.316
PMCID: PMC2614389  PMID: 18596723
lupus nephritis; biomarker; SELDI
14.  Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase 
Biochemistry  2007;46(19):5754-5765.
The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2·-) and O2·--derived oxidants change the redox status of the mitochondrial GSH pool. An electron transport protein, Mitochondrial Complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of Complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated Complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O2·- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact Complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of Complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibiton of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when Complex I is exposed to redox change in the GSH pool.
doi:10.1021/bi602580c
PMCID: PMC2527596  PMID: 17444656
15.  Histone H4 Acetylation Dynamics Determined by Stable Isotope Labeling with Amino Acids in Cell Culture and Mass Spectrometry 
Analytical biochemistry  2006;363(1):22-34.
This paper describes an integrated approach that couples Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to Acetic acid-Urea Polyacrylamide Gel Electrophoresis (AU-PAGE) and Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) for the quantitation and dynamics of histone H4 acetylation. The 697 acute lymphoblastic cell lines were grown in regular media (Lys-D0) and media in which lysine was substituted with deuterium-labeled lysine (Lys-D4). HDAC activity was inhibited by addition of the HDAC inhibitor depsipeptide to the culture media for different exposure times. Histones were extracted from cells pooled from unlabeled, untreated cells and labeled, treated cells, followed by AU-PAGE separation. Gel bands corresponding to different acetylation states of H4 were excised, in-gel digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Detailed information was obtained for both the change of histone H4 acetylation specific to N-terminus and global transformation of H4 from one acetylation state to another following treatment with the HDAC inhibitor depsipeptide. The kinetics of H4 acetylation was also assessed. The current study provides quantitative basis for developing potential therapies by using epigenetic regulation and the developed methodology can be applied to quantitation of change for other histone modifications induced by external stimuli.
doi:10.1016/j.ab.2006.12.031
PMCID: PMC1993805  PMID: 17286952
16.  Mass spectrometry-based proteomic analyses of contact lens deposition 
Molecular Vision  2008;14:291-297.
Purpose
The purpose of this report is to describe the contact lens deposition proteome associated with two silicone hydrogel contact lenses and care solutions using a mass spectrometric-based approach.
Methods
This was a randomized, controlled, examiner-masked crossover clinical trial that included 48 participants. Lenses and no-rub care solutions evaluated included galyfilcon A (Acuvue Advance, Vistakon Inc., Jacksonville, FL), lotrafilcon B (O2 Optix, CIBA Vision Inc., Duluth, GA), AQuify (CIBA Vision Inc.), and ReNu MoistureLoc (Bausch and Lomb Inc., Rochester, NY). After two weeks of daily wear in each lens-solution combination, the left lens was removed by the examiner (using gloves and forceps) and placed in a protein precipitation buffer (acetone). The precipitate was quantitated for total protein concentration (per lens), and proteins were then identified using liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) and peptide sequencing.
Results
Between 7.32 and 9.76 µg/lens of protein was observed on average from each lens-solution combination. There were 19 total unique proteins identified across the two lens materials, and six proteins were identified in all four lens-solution combinations including lipocalin, lysozyme, lacritin, lactoferrin, proline rich 4, and Ig Alpha. Lotrafilcon B was associated with 15 individual proteins (across both care solutions), and 53% of these proteins were observed in at least 50% of the analyses. Galyfilcon A was associated with 13 individual proteins, and 38.5% of these proteins were observed in at least 50% of the analyses. There were three unique proteins identified from galyfilcon A and four unique proteins identified from lotrafilcon B.
Conclusions
The total amount of proteins identified from silicone hydrogel materials is much less than the amount from traditional soft lens materials. For the most part, the deposition proteome across these lenses is similar, although the different polymer characteristics might be associated with some variability in observance of the less frequently identified proteins.
PMCID: PMC2254969  PMID: 18334948
17.  Investigation of the human tear film proteome using multiple proteomic approaches 
Molecular Vision  2008;14:456-470.
Purpose
The purpose of this work was to examine the tear film proteome using a combination of one-dimensional (1D) and two dimensional (2D) gel electrophoresis and mass spectrometry-based techniques and to explore the effect of the tear collection methods on the tear proteome.
Methods
Tear samples from eight normal non-contact lens wearing human subjects collected by Drummond glass microcapillary and Schirmer strips were subjected to 1D-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), 2D-SDS–PAGE, and 2D LC-MS/MS (Multidimensional protein identification technology - MudPIT). Bands or cores from the 1D- and 2D-SDS–PAGE were cut, digested with trypsin, and analyzed by tandem mass spectrometry for identification by the generation of sequence tags.
Results
In total (across sampling and proteomic methods), 97 unique proteins were observed, and a significant number of the spots/bands in the PAGE were from posttranslational modifications. Fifty-four unique proteins were identified from proteins extracted from the Schirmer strips in comparison to 13 unique proteins identified from capillary tubes, and 30 unique proteins were identified by both collection methods. Secreted (serum) proteins were predominantly observed from tears collected by capillary whereas a combination of cellular and serum proteins were identified from tear film collected by Schirmer strips.
Conclusions
Overall, these results suggest that the tear film collection and the proteomic method impacts the proteins present in the tear film and that care should be exercised in choosing a tear collection method to best correlate to the experiment being conducted or the hypothesis that is being tested.
PMCID: PMC2268847  PMID: 18334958
18.  Isolation and Identification of a Paenibacillus polymyxa Strain That Coproduces a Novel Lantibiotic and Polymyxin▿ †  
A new bacterial strain, displaying potent antimicrobial properties against gram-negative and gram-positive pathogenic bacteria, was isolated from food. Based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as Paenibacillus polymyxa and it was designated as strain OSY-DF. The antimicrobials produced by this strain were isolated from the fermentation broth and subsequently analyzed by liquid chromatography-mass spectrometry. Two antimicrobials were found: a known antibiotic, polymyxin E1, which is active against gram-negative bacteria, and an unknown 2,983-Da compound showing activity against gram-positive bacteria. The latter was purified to homogeneity, and its antimicrobial potency and proteinaceous nature were confirmed. The antimicrobial peptide, designated paenibacillin, is active against a broad range of food-borne pathogenic and spoilage bacteria, including Bacillus spp., Clostridium sporogenes, Lactobacillus spp., Lactococcus lactis, Leuconostoc mesenteroides, Listeria spp., Pediococcus cerevisiae, Staphylococcus aureus, and Streptococcus agalactiae. Furthermore, it possesses the physico-chemical properties of an ideal antimicrobial agent in terms of water solubility, thermal resistance, and stability against acid/alkali (pH 2.0 to 9.0) treatment. Edman degradation, mass spectroscopy, and nuclear magnetic resonance were used to sequence native and chemically modified paenibacillin. While details of the tentative sequence need to be elucidated in future work, the peptide was unequivocally characterized as a novel lantibiotic, with a high degree of posttranslational modifications. The coproduction of polymyxin E1 and a lantibiotic is a finding that has not been reported earlier. The new strain and associated peptide are potentially useful in food and medical applications.
doi:10.1128/AEM.02023-06
PMCID: PMC1797129  PMID: 17071789
19.  Identification of a Novel Anaplasma marginale Appendage-Associated Protein That Localizes with Actin Filaments during Intraerythrocytic Infection  
Infection and Immunity  2004;72(12):7257-7264.
The rickettsial pathogen Anaplasma marginale assembles an actin filament bundle during intracellular infection. Unlike other bacterial pathogens that generate actin filament tails, A. marginale infects mature erythrocytes, and the F-actin appendages are assembled on the cytoplasmic surface of a vacuole containing several organisms. To identify A. marginale molecules associated with these filaments, two complementary approaches were used: matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and tandem mass spectrometry of A. marginale proteins identified with an appendage-specific monoclonal antibody and expression screening of an A. marginale phage library. Amino acid and nucleotide sequences were mapped to a full-length gene in the genome of the St. Maries strain of A. marginale; the correct identification was confirmed by expression of full-length recombinant protein and its reactivity with appendage-specific antibodies. Interestingly, there is marked variation in the abilities of diverse A. marginale strains to assemble the F-actin appendages. Comparison of four strains, the Florida, Illinois, St. Maries, and Virginia strains, revealed substantial polymorphism in the gene encoding the appendage-associated protein, with amino acid sequence identity of as low as 34% among strains. However, this variation does not underlie the differences in expression, as there is no specific polymorphism associated with loss of ability to assemble actin appendages. In contrast, the ability to assemble an actin filament bundle reflected dramatic strain-specific differences in the expression level of the appendage-associated protein. Understanding how this protein influences the cycle of invasion, replication, and egress in the host cell may provide new insights into pathogen-host interactions.
doi:10.1128/IAI.72.12.7257-7264.2004
PMCID: PMC529098  PMID: 15557651

Results 1-19 (19)