Translation elongation factor P and its post-translational modification enzyme EpmA

Protein translation is a non-uniform process, whereby especially proline-containing motifs lead to ribosome stalling events. Elongation factor P (EF P) rescues the ribosome by stimulation of peptidyl transfer. The four known subgroups of bacterial EF-Ps either require post-translational modification (PTM) established by EpmABC/EarP/YmfI or are functional without PTM. In Escherichia coli, the aminomutase EpmB converts (S)-α-lysine into (R)-β-lysine, which is subject to a two-step reaction catalyzed by lysyl-tRNA synthetase paralog EpmA. First, (R)-β-lysyl-adenylate is formed, from which the β-lysyl moiety is subsequently transferred to the ε-amino group of lysine 34 of EF P. This thesis focuses on the interplay of EF-P and EpmA which ensures EF P functionality in vivo and was used to modify EF-P with seven unnatural substrates in vitro. To detect PTM status two universal peptide antibodies, nonselective for the bacterial origin of EF-P, were generated and three subtypes of one-dimensional isoelectric focusing were established (native/denaturating horizontal, vertical). EpmA and EF-P protein copy numbers indicated balanced coordination to ensure outright modification status of EF-P during all growth phases, but no mutual regulation. EpmA’s donor substrate promiscuity was pinpointed to permit C6 scaffolds with at least an amino group at α- ((R/S)-α-lysine, 5-hydroxy-(S)-α-lysine), β- ((R/S)-β-lysine, (R)-3-aminocapronic acid) or ε-position (6-aminocapronic acid). In addition, EpmA variant A298G enabled modification of EF-P with (S)-α-ornithine. For the first time, known natural PTMs of EF P were expanded by seven synthetic PTMs. In vitro transcription translation assay demonstrated superiority of (R)-β-lysylation in ribosome rescue, explaining its evolutionary selection. Modification of EF-P with (S)-α-lysine was successfully achieved in vivo, when (R)-β-lysine synthesis was impeded (E. coli ΔepmB) and epmA(_A298G) overexpressed. In Bacillus subtilis, the ratio of unmodified-to-modified EF P varied over time. Out of 13 tested aminotransferase genes dat, epsN, gsaB, ilvK and yhdR are potentially involved in the yet unsolved modification pathway. In summary, the present work not only provides new biochemical insights into the functionalization of EF-P, but also paves the way to modify proteins post-translationally using EpmA.Die Translation von Proteinen ist kein gleichförmiger Prozess. Besonders bei Polyprolinmotiven treten Verzögerungen des Ribosoms auf. Hier übernimmt Elongationsfaktor P (EF-P) eine helfende Funktion und stimuliert den Peptidyltransfer. Die vier bekannten Gruppen von bakteriellen EF-P benötigen entweder posttranslationale Modifikation (PTM) durch EpmABC/EarP/YmfI, oder sind ohne PTM funktional. In Escherichia coli wandelt die Aminomutase EpmB (S)-α-Lysin in (R)-β-Lysin um. Ein Paralog der Lysyl-tRNA-Synthetase, EpmA, katalysiert die folgende zweistufige Reaktion. Erst wird (R)-β-Lysyladenylat gebildet, dessen β-Lysylgruppe dann auf die ε-Aminogruppe von Lysin 34 von EF-P übertragen wird. Diese Dissertation widmet sich dem Zusammenspiel von EF-P und EpmA. Dieses stellt in vivo die Funktion von EF-P sicher, in vitro erlaubt es die Modifikation von EF P mit sieben unnatürlichen Substraten. Zur Detektion des PTM Status wurden zwei universelle Peptidantikörper etabliert, die EF-P unabhängig von dessen bakterieller Herkunft detektieren, sowie drei Formen der eindimensionalen Isoelektrischen Fokussierung (nativ/denaturierend horizontal, vertikal). Die Kopienzahlen von EpmA und EF-P sind aufeinander abgestimmt, um vollständige Modifikation von EF-P in allen Wachstumsphasen sicherzustellen. Sie regulieren sich aber nicht gegenseitig. Die Promiskuität von EpmA erlaubt Donorsubstrate mit C6-Ketten, die zumindest eine Aminogruppe in α- ((R/S)-α-Lysin, 5-Hydroxy-(S)-α-lysin), β- ((R/S)-β-Lysin, (R)-3-Aminohexansäure) oder ε-Position (6-Aminohexansäure) haben. Zusätzlich ermöglicht die Enzymvariante EpmA_A298G (S)-α-Ornithylierung von EF-P. Erstmals konnten so die natürlichen PTMs von EF-P um sieben synthetische PTMs erweitert werden. In vitro Transkriptions/Translations-Assays zeigten die wirkungsvollste Ribosomen-rettung bei (R)-β-lysyliertem EF-P, was dessen evolutionäre Auswahl erklärt. In vivo gelang die Modifikation von EF-P mit (S)-α-Lysin, wenn die Fähigkeit zur (R)-β-Lysinsynthese fehlte (E. coli ΔepmB) und epmA(_A298G) überexprimiert wurde. In Bacillus subtilis variiert das Verhältnis von unmodifiziertem zu modifiziertem EF-P. Von 13 untersuchten Genen, die Aminotransferasen kodieren, sind dat, epsN, gsaB, ilvK und yhdR möglicherweise am ungeklärten Modifikationsweg beteiligt. Zusammengefasst liefert die vorliegende Arbeit nicht nur neue biochemische Einsichten in die Funktionalisierung von EF-P, sondern eröffnet auch einen neuen Weg, Proteine mittels EpmA posttranslational zu modifizieren

Translation elongation factor P and its post-translational modification enzyme EpmA

Protein translation is a non-uniform process, whereby especially proline-containing motifs lead to ribosome stalling events. Elongation factor P (EF P) rescues the ribosome by stimulation of peptidyl transfer. The four known subgroups of bacterial EF-Ps either require post-translational modification (PTM) established by EpmABC/EarP/YmfI or are functional without PTM. In Escherichia coli, the aminomutase EpmB converts (S)-α-lysine into (R)-β-lysine, which is subject to a two-step reaction catalyzed by lysyl-tRNA synthetase paralog EpmA. First, (R)-β-lysyl-adenylate is formed, from which the β-lysyl moiety is subsequently transferred to the ε-amino group of lysine 34 of EF P. This thesis focuses on the interplay of EF-P and EpmA which ensures EF P functionality in vivo and was used to modify EF-P with seven unnatural substrates in vitro. To detect PTM status two universal peptide antibodies, nonselective for the bacterial origin of EF-P, were generated and three subtypes of one-dimensional isoelectric focusing were established (native/denaturating horizontal, vertical). EpmA and EF-P protein copy numbers indicated balanced coordination to ensure outright modification status of EF-P during all growth phases, but no mutual regulation. EpmA’s donor substrate promiscuity was pinpointed to permit C6 scaffolds with at least an amino group at α- ((R/S)-α-lysine, 5-hydroxy-(S)-α-lysine), β- ((R/S)-β-lysine, (R)-3-aminocapronic acid) or ε-position (6-aminocapronic acid). In addition, EpmA variant A298G enabled modification of EF-P with (S)-α-ornithine. For the first time, known natural PTMs of EF P were expanded by seven synthetic PTMs. In vitro transcription translation assay demonstrated superiority of (R)-β-lysylation in ribosome rescue, explaining its evolutionary selection. Modification of EF-P with (S)-α-lysine was successfully achieved in vivo, when (R)-β-lysine synthesis was impeded (E. coli ΔepmB) and epmA(_A298G) overexpressed. In Bacillus subtilis, the ratio of unmodified-to-modified EF P varied over time. Out of 13 tested aminotransferase genes dat, epsN, gsaB, ilvK and yhdR are potentially involved in the yet unsolved modification pathway. In summary, the present work not only provides new biochemical insights into the functionalization of EF-P, but also paves the way to modify proteins post-translationally using EpmA.Die Translation von Proteinen ist kein gleichförmiger Prozess. Besonders bei Polyprolinmotiven treten Verzögerungen des Ribosoms auf. Hier übernimmt Elongationsfaktor P (EF-P) eine helfende Funktion und stimuliert den Peptidyltransfer. Die vier bekannten Gruppen von bakteriellen EF-P benötigen entweder posttranslationale Modifikation (PTM) durch EpmABC/EarP/YmfI, oder sind ohne PTM funktional. In Escherichia coli wandelt die Aminomutase EpmB (S)-α-Lysin in (R)-β-Lysin um. Ein Paralog der Lysyl-tRNA-Synthetase, EpmA, katalysiert die folgende zweistufige Reaktion. Erst wird (R)-β-Lysyladenylat gebildet, dessen β-Lysylgruppe dann auf die ε-Aminogruppe von Lysin 34 von EF-P übertragen wird. Diese Dissertation widmet sich dem Zusammenspiel von EF-P und EpmA. Dieses stellt in vivo die Funktion von EF-P sicher, in vitro erlaubt es die Modifikation von EF P mit sieben unnatürlichen Substraten. Zur Detektion des PTM Status wurden zwei universelle Peptidantikörper etabliert, die EF-P unabhängig von dessen bakterieller Herkunft detektieren, sowie drei Formen der eindimensionalen Isoelektrischen Fokussierung (nativ/denaturierend horizontal, vertikal). Die Kopienzahlen von EpmA und EF-P sind aufeinander abgestimmt, um vollständige Modifikation von EF-P in allen Wachstumsphasen sicherzustellen. Sie regulieren sich aber nicht gegenseitig. Die Promiskuität von EpmA erlaubt Donorsubstrate mit C6-Ketten, die zumindest eine Aminogruppe in α- ((R/S)-α-Lysin, 5-Hydroxy-(S)-α-lysin), β- ((R/S)-β-Lysin, (R)-3-Aminohexansäure) oder ε-Position (6-Aminohexansäure) haben. Zusätzlich ermöglicht die Enzymvariante EpmA_A298G (S)-α-Ornithylierung von EF-P. Erstmals konnten so die natürlichen PTMs von EF-P um sieben synthetische PTMs erweitert werden. In vitro Transkriptions/Translations-Assays zeigten die wirkungsvollste Ribosomen-rettung bei (R)-β-lysyliertem EF-P, was dessen evolutionäre Auswahl erklärt. In vivo gelang die Modifikation von EF-P mit (S)-α-Lysin, wenn die Fähigkeit zur (R)-β-Lysinsynthese fehlte (E. coli ΔepmB) und epmA(_A298G) überexprimiert wurde. In Bacillus subtilis variiert das Verhältnis von unmodifiziertem zu modifiziertem EF-P. Von 13 untersuchten Genen, die Aminotransferasen kodieren, sind dat, epsN, gsaB, ilvK und yhdR möglicherweise am ungeklärten Modifikationsweg beteiligt. Zusammengefasst liefert die vorliegende Arbeit nicht nur neue biochemische Einsichten in die Funktionalisierung von EF-P, sondern eröffnet auch einen neuen Weg, Proteine mittels EpmA posttranslational zu modifizieren

Critical review of supercritical carbon dioxide extraction of selected oil seeds

Supercritical carbon dioxide extraction, as a relatively new separation technique, can be used as a very efficient process in the production of essential oils and oleoresins from many of plant materials. The extracts from these materials are a good basis for the new pharmaceutical products and ingredients in the functional foods. This paper deals with supercritical carbon dioxide extraction of selected oil seeds which are of little interest in classical extraction in the food industry. In this article the process parameters in the supercritical carbon dioxide extraction, such as pressure, temperature, solvent flow rate, diameter of gound materials, and moisture of oil seed were presented for the following seeds: almond fruits, borage seed, corn germ, grape seed, evening primrose, hazelnut, linseed, pumpkin seed, walnut, and wheat germ. The values of investigated parameters in supercritical extraction were: pressure from 100 to 600 bar, temperature from 10 to 70oC, diameter of grinding material from 0.16 to 2.0 mm, solvent flow used from 0.06 to 30.0 kg/h, amount of oil in the feed from 10.0 to 74.0%, and moisture of oil seed from 1.1 to 7.5%. The yield and quality of the extracts of all the oil seeds as well as the possibility of their application in the pharmaceutical and food, industries were analyzed

Molecules as Sources for Indistinguishable Single Photons

We report on the triggered generation of indistinguishable photons by solid-state single-photon sources in two separate cryogenic laser scanning microscopes. Organic fluorescent molecules were used as emitters and investigated by means of high resolution laser spectroscopy. Continuous-wave photon correlation measurements on individual molecules proved the isolation of single quantum systems. By using frequency selective pulsed excitation of the molecule and efficient spectral filtering of its emission, we produced triggered Fourier-limited single photons. In a further step, local electric fields were applied to match the emission wavelengths of two different molecules via Stark effect. Identical single photons are indispensible for the realization of various quantum information processing schemes proposed. The solid-state approach presented here prepares the way towards the integration of multiple bright sources of single photons on a single chip.Comment: Accepted for publication in J. Mod. Opt. This is the original submitted versio

Realization of two Fourier-limited solid-state single-photon sources

We demonstrate two solid-state sources of indistinguishable single photons. High resolution laser spectroscopy and optical microscopy were combined at T = 1.4 K to identify individual molecules in two independent microscopes. The Stark effect was exploited to shift the transition frequency of a given molecule and thus obtain single photon sources with perfect spectral overlap. Our experimental arrangement sets the ground for the realization of various quantum interference and information processing experiments.Comment: 6 page

Molecular and functional studies on the transcript elongation factor FACT and the SAGA-DUBm subunit ENY2 in Arabidopsis thaliana

FACT, a heterodimer of SSRP1 and SPT16, is a conserved and essential histone chaperone. FACT binds to H2A-H2B dimers to reorganizes octameric nucleosomes and to make the genomic DNA accessible. For instance, FACT facilitates the progression of the transcription machinery through the chromatin template by destabilizing nucleosomes in the path of the elongating RNAPII. By the reverse action, FACT restores the chromatin structure in the wake of the RNAPII and maintains the status quo. By in vitro EMSA experiments, this study shows that the DNA- and nucleosome binding properties of Arabidopsis SSRP1 were mediated by its C-terminal HMG-box domain. In vivo, the loss of the HMG-box domain did not alter the subcellular localization of SSRP1 or the nuclear protein dynamics/binding properties as shown by in-detail CLSM and FRAP analysis, respectively. Additionally, immunoblot analysis showed that HMG-box-deficient SSRP1 was still associated with SPT16 and the transcriptionally active RNAPII in vivo. Phenotyping of SSRP1 HMG-box deficiency mutants showed that the lack of the DNA-binding domain had no effect on Arabidopsis growth and development. The in vitro data indicate that the binding of FACT to H2A-H2B dimers in higher eukaryotes depends, in the first place, on the association of the SSRP1 HMG-box domain with nucleosomal DNA. Nevertheless, the in vivo data suggest that the loss of the SSRP1 HMG-box domain can be compensated in Arabidopsis by other unknown factors as may be HMGB proteins that provide the DNA-binding function for FACT. During transcript elongation, the histone chaperone FACT facilitates together with other TEFs efficient mRNA synthesis by RNAPII. This study contributed to reveal the composition of the Arabidopsis transcript elongation complex (TEC) by a proteomic approach using reciprocal tagging in combination with affinity purification and mass spectrometry. The TEFs FACT, PAF1-C, SPT4/5, SPT6, and TFIIS co-purified robustly with each other and the elongating RNAPII, while P-TEFb was not among the interactors. Additionally, further chromatin modifying factors including NAP1 and the Elongator were repeatedly co-purified with different TEFs. The phenotypic analysis of Arabidopsis double mutants that are defective in different combinations of TEFs revealed genetic interactions between the genes encoding subunits of FACT, PAF1-C, and TFIIS, resulting in synergistic/epistatic effects on plant growth/development Genome-wide transcriptome profiling of SSRP1- or SPT16-depleted plants in comparison to wild-type revealed that almost the identical small set of genes was differentially expressed in both mutants. Strikingly, genes encoding key anthocyanin biosynthesis enzymes were overrepresented among the genes that were downregulated in both FACT mutants. A phenotypic analysis showed that FACT-depleted plants have clear defects in the light-induced accumulation of anthocyanin in their leaves. In response to high light (HL) stress, anthocyanin biosynthesis genes were upregulated to a lesser extend in the FACT mutants compared to wild type. Additionally, the gene expression of SSRP1 and SPT16 was upregulated upon HL stress. These data suggest that FACT is novel factor required for the accumulation of anthocyanins in response to HL stress. ENY2, an evolutionary conserved adaptor protein, links transcription by RNAPII with export of the newly synthesized mRNA to the cytoplasm by being part of the transcriptional coactivator SAGA and the NPC-associated mRNA export complex TREX-2. Histochemical GUS staining revealed that the Arabidopsis ENY2 promoter is widely active during plant growth and development. In the plant cells that are expressing ENY2, the protein is forming speckle-like structures in the nucleoplasm and is highly mobile as shown by in-detail CLSM and FRAP analysis, respectively. Using reciprocal tagging of ENY2 and its putative interactors in combination with affinity purification and mass spectrometry revealed that ENY2 associates with two components /SGF11, UBP22) of the SAGA histone H2B deubiquitinase (DUB) module. Furthermore, no subunits of other SAGA modules or the TREX-2 complex were co-purified with ENY2. Additionally, several splicing complexes especially the U2, U5 and NTC/NTR were identified in the affinity purification of ENY2. In accordance with these findings, ENY2 and the NTC component MOS4 co-localized in splicing speckles, whereas no direct protein-protein (PPIs) interactions between ENY2 and NTC/NTR components were found by Y2H and FRET. Three (ENY2, SGF11, UBP22) of the four SAGA-DUB components that were identified in yeast, fruit-fly and humans were highly conserved in Arabidopsis, the two adaptor proteins ENY2 and SGF11 as well as enzymatically active UBP22. SGF73, the missing protein that links the DUB module to the remaining SAGA complex in other organisms had no homolog in plants. Direct PPIs between SGF11 and ENY2 as well as UBP22 could be detected by Y2H and FRET analysis. The composition of the Arabidopsis SAGA complex was revealed by a proteomic approach using reciprocal tagging of one representative of each bioinformatically predicted SAGA module in combination with affinity purification and mass spectrometry. In total, 17 Arabidopsis SAGA subunits were biochemically identified. The DUB module did almost not co-purify with the other SAGA modules (HAT, SPT, TAF), which suggest that the plant DUB module can act in a SAGA-independent manner. Additionally, several subunits of the spliceosome were repeatedly co-purified with the DUB, HAT, and SPT modules of SAGA. A reverse genetics approach revealed that knockdown of ENY2 by RNAi had no obvious effect on plant growth and development, while the complete knockout of ENY2 by CRISPR/Cas9 induced a late flowering phenotype. The overexpression of ENY2 did not cause any obvious phenotype. The knockdown of SGF11 by a T-DNA insertion resulted as well in a late flowering phenotype and an upregulation of the floral repressor FLC. Moreover, the global H2Bub levels were increased in ENY2- and SGF11-depleted plants. Taken together, this study revealed the composition of the Arabidopsis SAGA complex. ENY2 is part of a histone H2B de-ubiquitinating module that can exist most likely SAGAindependent (Figure 9.1). DUB-defective plants show a late flowering phenotype. Additionally, the DUB as well as other SAGA modules show a strong association to the pre-mRNA splicing machinery. Surprisingly, Arabidopsis ENY2 is no part of the mRNA export complex TREX-2 as it was shown in other eukaryotes

Zum Endothelinsystem im Morris-Hepatom-7777-Behandlung mit Endothelinrezptorantagonisten in vitro und in vivo

Das Peptid Endothelin-1 (ET-1) hat neben seiner vasokonstriktorischen Wirkung auch wachstumsinduzierende Eigenschaften. Um seine Bedeutung für Tumorwachstum zu untersuchen wurde das ET-System des experimentellen Lebertumors Morris-Hepatom (MH)-7777 in vitro und in vivo zunächst charakterisiert. Erstmals wurde dann versucht, Tumorwachstum auch in vivo durch Behandlung mit einem ET-Rezeptorantagonisten zu inhibieren. METHODEN: Im Kulturüberstand von MH-7777-Zellen wurde die Menge des synthetisierten irET-1 gemessen. Über Proliferationsassays erfolgte die Untersuchung des Zellwachstums bei Inkubation mit ET-1 sowie den ET-Rezeptorantagonisten BQ 123, LU 135252 und LU 302872. Zur Charakterisierung des ET-Systems im MH-7777 in vivo wurden ir(Big-)ET-1-Plasma- und Gewebekonzentrationen bestimmt. Die Messung der ET-Rezeptordichte und der Rezeptoraffinität erfolgte in Scatchard-Rezeptor-Bindungsstudien. Die Hälfte einer Gruppe von 44 hepatomtagenden Buffaloratten wurde über 28 Tage mit dem kombinierten ETA/B-Rezeptorantagonisten LU 302872 behandelt (orale Applikation). DIE ERGEBNISSE zeigen eine endogene irET-1-Produktion der MH-7777-Zellen. Exogene ET-1-Stimulation der Zellen bewirkt eine signifikante Proliferationssteigerung. Die Proliferationshemmung durch die beiden ETA-Rezeptorantagonisten ist schwächer ausgeprägt als die in zwei verschiedenen Proliferationsassays bestätigte Hemmung (pThe peptide endothelin-1 (ET-1) has growth-promoting properties besides its vasopressor characteristic. In order to gain information about its importance for tumor growth the ET system of the experimental liver tumor Morris hepatoma (MH)-7777 was characterized in vitro and in vivo. For the first time it was then tried to inhibit tumor growth in vivo by treatment with an ET receptor antagonist. METHODS: The endogenously produced immunoreactive (ir) ET-1 was quantified in the supernatant of MH-7777 cells. Cell growth at incubation with different concentrations of ET-1 and the ET receptor antagonists BQ 123, LU 135252 and LU 302872 was evaluated performing cell proliferation assays. In order to characterize the ET system in MH-7777 in vivo ir(big-)ET-1 concentrations were determined in plasma and tissue. ET receptor density and affinity in hepatoma was determined performing Scatchard receptor binding assays. Half of 44 hepatoma-bearing Buffalo rats received the combined ETA/B receptor antagonist LU 302872 for 28 days orally. THE RESULTS show an endogenous irET-1 production by MH-7777 cells. Exogenous ET-1 stimulation causes a significant increase in cell proliferation. Growth inhibitory action of ETA-receptor antagonists is not as strong as the inhibition (