Michael Karas

Publications of Michael Karas

• Improving the precision of quantitative bottom-up proteomics based on stable isotope-labeled proteins.

  Authors: Anna Konopka, Martin E Boehm, Marion Rohmer, Dominic Baeumlisberger, Michael Karas, Wolf D Lehmann

  Analytical and bioanalytical chemistry. 04/2012;

  Stable isotope dilution-based quantitative proteomics with intact labeled proteins as internal standards in combination with a bottom-up approach, i.e., with quantification on the peptide level, isStable isotope dilution-based quantitative proteomics with intact labeled proteins as internal standards in combination with a bottom-up approach, i.e., with quantification on the peptide level, is an established method. To explore the technical precision of this approach, calmodulin-like protein 3 was prepared in non-labeled (light) and SILAC-type labeled (heavy) form by cell-free synthesis, mixed, digested with trypsin, and analyzed by UPLC-ESI-MS. In total, 16 light/heavy peptide pair ratios were determined. Pair-wise comparison of ratios of 12 peptides selected according to S/N ratios >50 revealed that the majority exhibited ratios, which were different at a high level of statistical significance (p < 0.001). HPLC-MALDI-MS ratio data confirmed this observation, thus excluding the ionization method as a source of the observed ratio differences. Variation of the digestion time from 0.25 to 4 h showed that the light/heavy ratios of most peptides decrease with time, indicating a kinetic isotope effect leading to preferred cleavage of light calmodulin-like protein 3. The subset of peptides with statistically identical ratios resulted in an average ratio with a RSD of 1.0 %. The light/heavy ratio calculated on the basis of these peptides probably provides the most accurate molar protein ratio.

• Identification of the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs.

  Authors: Jan Philip Wurm, Marco Griese, Ute Bahr, Martin Held, Alexander Heckel, Michael Karas, Jörg Soppa, Jens Wöhnert

  RNA (New York, N.Y.). 03/2012; 18(3):412-20.

  tRNAs from all three kingdoms of life contain a variety of modified nucleotides required for their stability, proper folding, and accurate decoding. One prominent example is the eponymoustRNAs from all three kingdoms of life contain a variety of modified nucleotides required for their stability, proper folding, and accurate decoding. One prominent example is the eponymous ribothymidine (rT) modification at position 54 in the T-arm of eukaryotic and bacterial tRNAs. In contrast, in most archaea this position is occupied by another hypermodified nucleotide: the isosteric N1-methylated pseudouridine. While the enzyme catalyzing pseudouridine formation at this position is known, the pseudouridine N1-specific methyltransferase responsible for this modification has not yet been experimentally identified. Here, we present biochemical and genetic evidence that the two homologous proteins, Mja_1640 (COG 1901, Pfam DUF358) and Hvo_1989 (Pfam DUF358) from Methanocaldococcus jannaschii and Haloferax volcanii, respectively, are representatives of the methyltransferase responsible for this modification. However, the in-frame deletion of the pseudouridine N1-methyltransferase gene in H. volcanii did not result in a discernable phenotype in line with similar observations for knockouts of other T-arm methylating enzymes.

• Heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors.

  Authors: Ye Gao, Björn Meyer, Lucie Sokolova, Klaus Zwicker, Michael Karas, Bernd Brutschy, Guohong Peng, Hartmut Michel

  Proceedings of the National Academy of Sciences of the United States of America. 02/2012; 109(9):3275-80.

  The cytochrome c oxidase Cox2 has been purified from native membranes of the hyperthermophilic eubacterium Aquifex aeolicus. It is a cytochrome ba(3) oxidase belonging to the family B of theThe cytochrome c oxidase Cox2 has been purified from native membranes of the hyperthermophilic eubacterium Aquifex aeolicus. It is a cytochrome ba(3) oxidase belonging to the family B of the heme-copper containing terminal oxidases. It consists of three subunits, subunit I (CoxA2, 63.9 kDa), subunit II (CoxB2, 16.8 kDa), and an additional subunit IIa of 5.2 kDa. Surprisingly it is able to oxidize both reduced cytochrome c and ubiquinol in a cyanide sensitive manner. Cox2 is part of a respiratory chain supercomplex. This supercomplex contains the fully assembled cytochrome bc(1) complex and Cox2. Although direct ubiquinol oxidation by Cox2 conserves less energy than ubiquinol oxidation by the cytochrome bc(1) complex followed by cytochrome c oxidation by a cytochrome c oxidase, ubiquinol oxidation by Cox2 is of advantage when all ubiquinone would be completely reduced to ubiquinol, e.g., by the sulfidequinone oxidoreductase, because the cytochrome bc(1) complex requires the presence of ubiquinone to function according to the Q-cycle mechanism. In the case that all ubiquinone has been reduced to ubiquinol its reoxidation by Cox2 will enable the cytochrome bc(1) complex to resume working.

• Perfusion reversed-phase high-performance liquid chromatography for protein separation from detergent-containing solutions: An alternative to gel-based approaches.

  Authors: Jan Gorka, Marion Rohmer, Sandra Bornemann, Dimitrios G Papasotiriou, Dominic Baeumlisberger, Tabiwang N Arrey, Ute Bahr, Michael Karas

  Analytical biochemistry. 02/2012; 424(2):97-107.

  Detergents are frequently used for the solubilization of membrane proteins during and after purification steps. Unfortunately some of these detergents impair chromatographic separations and massDetergents are frequently used for the solubilization of membrane proteins during and after purification steps. Unfortunately some of these detergents impair chromatographic separations and mass spectrometry (MS) analysis. Perfusion reversed-phase high-performance liquid chromatography (RP-HPLC) using POROS materials is suited for separating intact proteins solubilized by detergents due to the particles' highly diffusive pores and chemical stability. In this article, the use of perfusive reversed-phase material packed into small inner diameter capillary columns is presented as a cheap, rapid, and efficient method for the removal of different types of detergents from protein solutions. The ability to purify and separate the subunits of membrane protein complexes with self-packed capillary columns is exemplified for bovine cytochrome bc(1) complex. Even highly hydrophobic subunits can be detected in collected fractions by intact mass measurements and identified after proteolytic digestion and matrix-assisted laser desorption/ionization tandem MS (MALDI MS/MS). The comparison with a gel-based approach shows that this method is a valuable alternative for purification and separation of intact proteins with subsequent MS analysis and that hydrophobic proteins are even better represented in the LC-based approach.

• Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416.

  Authors: Michael Hörnig, Stavroula Markoutsa, Ann-Kathrin Häfner, Sven George, Joanna M Wisniewska, Carmen B Rödl, Bettina Hofmann, Thorsten Maier, Michael Karas, Oliver Werz, Dieter Steinhilber

  Biochimica et biophysica acta. 11/2011; 1821(2):279-86.

  U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30nM. 5-LO catalyzes the conversionU73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.

• Fast quantitative determination of melamine and its derivatives by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

  Authors: Anne Arnold, Tabiwang N Arrey, Michael Karas, Markus Persike

  Rapid communications in mass spectrometry : RCM. 10/2011; 25(19):2844-50.

  A simple, sensitive and fast method for the determination of melamine and its derivatives in milk powder using matrix-assisted laser desorption/ionization time-of-flight mass spectrometryA simple, sensitive and fast method for the determination of melamine and its derivatives in milk powder using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed. Neither time-consuming sample preparation, nor special target plates, or other extra equipment are necessary. The common matrix sinapinic acid (SA) was used with a dried-droplet preparation. Detection limits (signal-to-noise (S/N) ratio = 3) for standard solutions of melamine, ammeline and cyanuric acid were 10, 25 and 10 µg/L, respectively. The limit of quantification (LOQ) for melamine was 25 µg/L and excellent linearity (R(2): 0.9990) was maintained over the range of 10-2000 µg/L. Ammeline and cyanuric acid were analyzed with an LOQ of 50 µg/L and also excellent linearity (R(2): 0.9997 and R(2): 0.9998). Good accuracy and precision were obtained for all concentrations within the range of the standard curve. The developed method was successfully used for the determination of melamine, ammeline and cyanuric acid in milk powder samples with a simple sample preparation. The LOQ of melamine was 0.5 µg/g. Ammeline and cyanuric acid were detectable at 0.5 and 5 µg/g. This method showed excellent accuracy, precision and linearity and significantly reduces the needed analysis time, as only approximately 10 s/sample measuring time is required. To the authors' knowledge, this is the first published method to quantify melamine and derivatives by MALDI-TOF-MS.

• Nano-liquid chromatography-direct electron ionization mass spectrometry: improving performance by a new ion source adapter.

  Authors: Cornelia Flender, Christian Wolf, Peter Leonhard, Michael Karas

  Journal of mass spectrometry : JMS. 10/2011; 46(10):1004-10.

  A novel interface adapter has been designed to provide a new way of directly coupling a nano-liquid chromatograph to an electron ionization mass spectrometer. It connects the transfer capillaryA novel interface adapter has been designed to provide a new way of directly coupling a nano-liquid chromatograph to an electron ionization mass spectrometer. It connects the transfer capillary coming from the liquid chromatograph to the ionization chamber and can be easily screwed into the ion source. Liquid coming from the column passes through the heated adapter flow path and is vaporized. A continuous flow of new liquid pushes the vapor into the ionization chamber where it is ionized and continues on to the mass analyzer. The advantages of the new adapter are reduced ice formation inside the ion source and less clogging of the transfer capillary. Improvements achieved are demonstrated on the basis of caffeine and steroid analysis. The limits of detection of selected steroids are compared with and without the adapter. The adapter improves the detection limit of the system by a factor of 2 and precision from ≤15% to ≤9% relative standard deviation. No derivatization procedure is necessary before the analysis of small polar compounds. The resulting spectra are reproducible, easily interpretable, and database searchable. The new method is robust, delivers reproducible results, and provides a highly efficient alternative to existing methods in the field of pharmaceutical analysis.

• High-reactivity matrices increase the sensitivity of matrix enhanced secondary ion mass spectrometry.

  Authors: Fabian N Svara, Andras Kiss, Thorsten W Jaskolla, Michael Karas, Ron M A Heeren

  Analytical chemistry. 09/2011; 83(21):8308-13.

  Secondary ion mass spectrometry (SIMS) is a desorption/ionization method in which ions are generated by the impact of a primary ion beam on a sample. Classic matrix assisted laser desorption andSecondary ion mass spectrometry (SIMS) is a desorption/ionization method in which ions are generated by the impact of a primary ion beam on a sample. Classic matrix assisted laser desorption and ionization (MALDI) matrices can be used to increase secondary ion yields and decrease fragmentation in a SIMS experiment, which is referred to as matrix enhanced SIMS (ME-SIMS). Contrary to MALDI, the choice of matrices for ME-SIMS is not constrained by their photon absorption characteristics. This implies that matrix compounds that exhibit an insufficient photon absorption coefficient have the potential of working well with ME-SIMS. Here, we evaluate a set of novel derivatives of the classical MALDI matrices α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) for usability in ME-SIMS. This evaluation was carried out using peptide mixtures of different complexity and demonstrates significant improvements in signal intensity for several compounds with insufficient UV absorption at the standard MALDI laser wavelengths. Our study confirms that the gas-phase proton affinity of a matrix compound is a key physicochemical characteristic that determines its performance in a ME-SIMS experiment. As a result, these novel matrices improve the performance of matrix enhanced secondary ion mass spectrometry experiments on complex peptide mixtures.

• Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: lipidome meets proteome.

  Authors: Karlheinz Grillitsch, Melanie Connerth, Harald Köfeler, Tabiwang N Arrey, Benjamin Rietschel, Brigitte Wagner, Michael Karas, Günther Daum

  Biochimica et biophysica acta. 07/2011; 1811(12):1165-76.

  In the yeast Saccharomyces cerevisiae as in other eukaryotes non-polar lipids are a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerolsIn the yeast Saccharomyces cerevisiae as in other eukaryotes non-polar lipids are a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerols (TG) and steryl esters (SE) are stored in so-called lipid particles/droplets (LP) as biologically inert form of fatty acids and sterols. To understand LP structure and function in more detail we investigated the molecular equipment of this compartment making use of mass spectrometric analysis of lipids (TG, SE, phospholipids) and proteins. We addressed the question whether or not lipid and protein composition of LP influence each other and performed analyses of LP from cells grown on two different carbon sources, glucose and oleate. Growth of cells on oleate caused dramatic cellular changes including accumulation of TG at the expense of SE, enhanced the amount of glycerophospholipids and strongly increased the degree of unsaturation in all lipid classes. Most interestingly, oleate as a carbon source led to adaptation of the LP proteome resulting in the appearance of several novel LP proteins. Localization of these new LP proteins was confirmed by cell fractionation. Proteomes of LP variants from cells grown on glucose or oleate, respectively, were compared and are discussed with emphasis on the different groups of proteins detected through this analysis. In summary, we demonstrate flexibility of the yeast LP lipidome and proteome and the ability of LP to adapt to environmental changes.

• 100% protein sequence coverage: a modern form of surrealism in proteomics.

  Authors: Bjoern Meyer, Dimitrios G Papasotiriou, Michael Karas

  Amino acids. 07/2011; 41(2):291-310.

  This review intends not only to discuss the current possibilities to gain 100% sequence coverage for proteins, but also to point out the critical limits to such an attempt. The aim of 100% sequenceThis review intends not only to discuss the current possibilities to gain 100% sequence coverage for proteins, but also to point out the critical limits to such an attempt. The aim of 100% sequence coverage, as the review title already implies, seems to be rather surreal if the complexity and dynamic range of a proteome is taken into consideration. Nevertheless, established bottom-up shotgun approaches are able to roughly identify a complete proteome as exemplary shown by yeast. However, this proceeding ignores more or less the fact that a protein is present as various protein species. The unambiguous identification of protein species requires 100% sequence coverage. Furthermore, the separation of the proteome must be performed on the protein species and not on the peptide level. Therefore, top-down is a good strategy for protein species analysis. Classical 2D-electrophoresis followed by an enzymatic or chemical cleavage, which is a combination of top-down and bottom-up, is another interesting approach. Moreover, the review summarizes further top-down and bottom-up combinations and to which extent middle-down improves the identification of protein species. The attention is also focused on cleavage strategies other than trypsin, as 100% sequence coverage in bottom-up experiments is only obtainable with a combination of cleavage reagents.

• A proteomic approach to investigate the differential antigenic profile of two Coxiella burnetii strains.

  Authors: Anastasia Papadioti, Stavroula Markoutsa, Iosif Vranakis, Yiannis Tselentis, Michael Karas, Anna Psaroulaki, Georgios Tsiotis

  Journal of proteomics. 06/2011; 74(7):1150-9.

  Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Current diagnostics of Q fever is based on serological testing of patient serum.Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Current diagnostics of Q fever is based on serological testing of patient serum. Biological distinction among C. burnetii strains has been referred at the genetic level as well as in virulence in animal models of Q fever. Disclosure of strain specific antigens might show insight into the biology and pathogenesis of this query pathogen, as well as it can provide the literature with potential serodiagnostic markers. In the present study, we sought to obtain an outer membrane enriched fraction of two C. burnetii reference strains, which originate from different sources, in order to investigate the way in which their antigenic profile is differentiated against a patient serum. We systematically analyzed the sarcosyl-insoluble fraction, enriched in outer membrane proteins, of the two C. burnetii strains using doubled SDS-PAGE combined with MS/MS analysis. In total, twenty-two outer membrane proteins were identified, representing 26% of the overall 86 identified proteins. The sarcosyl-insoluble fraction was then separated on 2DE IEF/SDS-PAGE and probed with serum from an infected patient. Different immuno-reactive proteins between the two C. burnetii strains were identified and included 2 outer membrane proteins, a hypothetical protein (CBU_0937) with unknown function and OmpH (CBU_0612), a previously identified marker for Q fever endocarditis. This approach can be used to reveal strain-specific proteins involved in pathogenesis and new serodiagnostic markers.

• Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes.

  Authors: Dominic Baeumlisberger, Marion Rohmer, Tabiwang N Arrey, Benjamin F Mueller, Tobias Beckhaus, Ute Bahr, Guenes Barka, Michael Karas

  Journal of proteome research. 06/2011; 10(6):2889-94.

  The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramaticallyThe beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.

• Compelling evidence for Lucky Survivor and gas phase protonation: the unified MALDI analyte protonation mechanism.

  Authors: Thorsten W Jaskolla, Michael Karas

  Journal of the American Society for Mass Spectrometry. 06/2011; 22(6):976-88.

  This work experimentally verifies and proves the two long since postulated matrix-assisted laser desorption/ionization (MALDI) analyte protonation pathways known as the Lucky Survivor and the gasThis work experimentally verifies and proves the two long since postulated matrix-assisted laser desorption/ionization (MALDI) analyte protonation pathways known as the Lucky Survivor and the gas phase protonation model. Experimental differentiation between the predicted mechanisms becomes possible by the use of deuterated matrix esters as MALDI matrices, which are stable under typical sample preparation conditions and generate deuteronated reagent ions, including the deuterated and deuteronated free matrix acid, only upon laser irradiation in the MALDI process. While the generation of deuteronated analyte ions proves the gas phase protonation model, the detection of protonated analytes by application of deuterated matrix compounds without acidic hydrogens proves the survival of analytes precharged from solution in accordance with the predictions from the Lucky Survivor model. The observed ratio of the two analyte ionization processes depends on the applied experimental parameters as well as the nature of analyte and matrix. Increasing laser fluences and lower matrix proton affinities favor gas phase protonation, whereas more quantitative analyte protonation in solution and intramolecular ion stabilization leads to more Lucky Survivors. The presented results allow for a deeper understanding of the fundamental processes causing analyte ionization in MALDI and may alleviate future efforts for increasing the analyte ion yield.

• Structure elucidation and biosynthesis of lysine-rich cyclic peptides in Xenorhabdus nematophila.

  Authors: Sebastian W Fuchs, Anna Proschak, Thorsten W Jaskolla, Michael Karas, Helge B Bode

  Organic & biomolecular chemistry. 03/2011; 9(9):3130-2.

  Thirteen novel PAX (peptide-antimicrobial-Xenorhabdus) peptides were identified in Xenorhabdus nematophila HGB081. Their structures including the absolute configuration were elucidated using aThirteen novel PAX (peptide-antimicrobial-Xenorhabdus) peptides were identified in Xenorhabdus nematophila HGB081. Their structures including the absolute configuration were elucidated using a combination of labeling experiments, detailed MS/MS experiments, the advanced Marfey's method, and a detailed analysis of the biosynthesis gene cluster, which was identified as well.

• A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes.

  Authors: Roman Ladig, Maik S Sommer, Alexander Hahn, Matthias S Leisegang, Dimitrios G Papasotiriou, Mohamed Ibrahim, Rajae Elkehal, Michael Karas, Volker Zickermann, Michael Gutensohn, Ulrich Brandt, Ralf Bernd Klösgen, Enrico Schleiff

  The Plant journal : for cell and molecular biology. 03/2011; 67(1):181-94.

  Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and highNative polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.

• Premature transcript termination, trans-splicing and DNA repair: a vicious path to cancer.

  Authors: Eric Kowarz, Jennifer Merkens, Michael Karas, Theo Dingermann, Rolf Marschalek

  American journal of blood research. 01/2011; 1(1):1-12.

  So far, about 800 different chromosomal translocations have been characterized in hemato-malignant and solid tumors. Chromosomal translocations mostly result in the expression of chimeric fusionSo far, about 800 different chromosomal translocations have been characterized in hemato-malignant and solid tumors. Chromosomal translocations mostly result in the expression of chimeric fusion proteins associated with enhanced proliferation and/or malignant transformation. Here, we demonstrate that genes frequently involved in such genetic rearrangements exhibit a unique feature: premature transcriptional termination. These early-terminated RNA molecules have an abundance of 10-20% when compared to their cognate full-length transcripts. They exhibit an unsaturated splice donor site that gives rise to trans-splicing events, leading to RNAs displaying exon repetitions or chimeric fusion RNAs. These arbitrary fusion RNAs mimic the presence of a chromosomal translocation in genetically unaffected cells. Based on our and published data, we propose the hypothesis that these artificial "chimeric fusion transcripts" may influence DNA repair processes, resulting in the generation of de novo chromosomal translocations. This idea provides a rational explanation why different individuals suffer from nearly identical genetic rearrangements.

• Entianin, a novel subtilin-like lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029T with high antimicrobial activity.

  Authors: Sebastian W Fuchs, Thorsten W Jaskolla, Sophie Bochmann, Peter Kötter, Thomas Wichelhaus, Michael Karas, Torsten Stein, Karl-Dieter Entian

  Applied and environmental microbiology. 01/2011; 77(5):1698-707.

  Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for eachLantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029(T). Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029(T) secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.

• Nerve injury evoked loss of latexin expression in spinal cord neurons contributes to the development of neuropathic pain.

  Authors: Hilmar Nils Kühlein, Irmgard Tegeder, Christine Möser, Hee-Young Lim, Annett Häussler, Katharina Spieth, Ingo Jennes, Rolf Marschalek, Tobias Beckhaus, Michael Karas, Markus Fauth, Corina Ehnert, Gerd Geisslinger, Ellen Niederberger

  PloS one. 01/2011; 6(4):e19270.

  Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess proteinNerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.

• Hypoxia and reoxygenation of primary human hepatocytes induce proteome changes of glucose metabolism, oxidative protection and peroxisomal function.

  Authors: Christoph W Strey, Johannes Gestrich, Tobias Beckhaus, Rosa Maria Marquez-Pinilla, Elsie Oppermann, Christian Mönch, John D Lambris, Michael Karas, Wolf O Bechstein

  International journal of molecular medicine. 10/2010; 26(4):577-84.

  Protective hepatocellular responses to a hypoxic challenge are crucial to preserve liver function. The knowledge of affected metabolic functions could help assess and enhance hepatic ischemicProtective hepatocellular responses to a hypoxic challenge are crucial to preserve liver function. The knowledge of affected metabolic functions could help assess and enhance hepatic ischemic tolerance. Here we studied adaptive mechanisms in human hepatocytes after hypoxia and reoxygenation using a proteomic approach. Proteins from primary hepatocytes were extracted after 6 h of hypoxia and 24 h of reoxygenation. The proteome was analyzed by 2D-electrophoresis. Densitometry and mass spectrometry (MALDI-TOF-MS) were used for protein identification. Two hundred and sixty-two spots were differentially analyzed and 33 spots displayed significant differences between hypoxic and normoxic cells. Seventeen proteins were identified by mass spectrometry. After hypoxia and reoxygenation the UTP-glucose-1-phosphate uridyltransferase, phosphoglycerate kinase1, fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, thiosulfat-sulfurtransferase, thioredoxin peroxidase, peroxiredoxin III, and annexin A2 proteins were down-regulated. An increased expression was found for carbamoyl phosphate synthetase I, heat shock 70 kDa protein5, phosphoenolpyruvate carboxy-kinase, catalase isoform2, peroxiredoxin II, glutathione S-transferase, hydroxyacid oxidase1, and F1-ATP synthase, alpha subunit1. Hepatocellular adaptation to hypoxia and reoxygenation involve glucose metabolism, peroxisomal functions, and oxidative stress protection. The identified proteins can serve as possible diagnostic targets to monitor hepatic hypoxic tolerance e.g. in the context of liver surgery and transplantation.

• The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA.

  Authors: Britta Meyer, Jan Philip Wurm, Peter Kötter, Matthias S Leisegang, Valeska Schilling, Markus Buchhaupt, Martin Held, Ute Bahr, Michael Karas, Alexander Heckel, Markus T Bohnsack, Jens Wöhnert, Karl-Dieter Entian

  Nucleic acids research. 10/2010; 39(4):1526-37.

  The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed inThe Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.