No description available.

No description available.

No description available.

No description available.

ABSTRACT Many enzymes require cofactors in order to carry out specific functions. Flavins, which are naturally fluorescent, compose a unique group of redox cofactors because they have the ability to transfer one or two electrons and are therefore found in three different oxidation states. A specific flavin, flavin adenine dinucleotide (FAD), is a crucial cofactor that facilitates electron transfer in many flavoproteins involved in DNA repair, photosynthesis, and regulatory pathways. One example of a FAD-containing DNA repair protein is DNA Photolyase (PL). E. coli PL is a monomeric flavoprotein that facilitates DNA repair via a photoinduced electron transfer reaction. The catalytic cofactor, FAD, transfers an electron to a thymidine dimer lesion, to cleave the cyclobutane ring and restore the DNA strand. Although the mechanism of repair has been partially elucidated by our group, it is still unclear whether or not the electron is transferred directly from the isoalloxazine moiety to the dimer or if the electron hops from the isoalloxazine moiety to the adenine moiety to the dimer. This sequential hopping mechanism should have excited state absorption features for the reduced flavin species, an adenine radical anion, and the semiquinone flavin species. To investigate the mechanistic role of adenine, E. coli PL has been reconstituted with -FAD, an FAD analogue in which the adenine was substituted via chemical means with 1,N6 – ethenoadenine dinucleotide. -FAD was selected due to its ease of synthesis and because its structure changes the thermodynamic driving force for the electron transfer reaction, by lowering the energetic gap (LUMO-LUMO) between the isoalloxazine ring and the modified adenine. In order to characterize the excited state dynamics of the mutant chromophore, the transient absorption measurements were made of each free flavin in solution. These measurements indicate the pathway of electron transfer must be mediated via superexchange rather than a hopping mechanism. This important result shows that the role of adenine in photolyase is to facilitate a superexchange electron transfer mechanism, and a modified flavin can act as a reporter under these experimental conditions. By exploiting Corynebacterium ammoniagenes FAD synthetase adenylation promiscuity, we have enzymatically-synthesized and purified a novel dually fluorescent flavin cofactor. This new flavin adenine dinucleotide (FAD) analogue, flavin 2-aminopurine (2Ap) dinucleotide (F2ApD), can be selectively excited through the 2Ap moiety at 310 nm, a wavelength at which flavins have intrinsically low extinction. The dinucleotide 2Ap emits at 370 nm with high efficiency. This emission has excellent overlap with the absorption spectra of both oxidized and reduced hydroquinone flavin (FlOX and FlHQ respectively), which emit at ~525 and ~505 nm respectively. We have characterized the optical properties of this dually fluorescent flavin, iFAD. Steady state fluorescence excitation and emission spectra were obtained and contrasted with the other flavins. Temperature- and solvent-dependent emission spectra suggest that F2ApD stacking interactions are significantly different compared to FAD and etheno-FAD (FAD). The optical absorption spectra of these dinucleotides were compared with FMN to explore electronic interactions between the flavin and nucleobase moieties. To probe the evolution of the different excited state populations, femtosecond transient absorption measurements were made on the iFADs, revealing that F2ApD exhibited unique transient spectra as compared to either FAD or FAD. The significance of these results to flavins, flavoprotein function, and bioimaging are discussed. The reconstituted -FAD in E.coli photolyase was catalytically active and actually repaired more efficiently than the FAD-reconstituted photolyase. To validate that an enzymatically synthesized iFAD could be reconstituted into a flavoprotein, this work shows a DNA repair assay using F2ApD that was reconstituted into E. coli photolyase, generating the reconstituted analogue, ApPL. Activity assays were compared between FAD-PL and ApPL. This comparison further elucidates the importance of the driving force on the electron transfer reaction in PL. A comparison of fluorescence spectroscopies between the reconstituted PLs highlights their applicability as biosensors and/or mechanistic reporters. / Chemistry

L’activité de croissance des plantes se trouve souvent limitée par les conditions contraignantes de l’environnement. La salinité du sol est l’une des majeures contraintes abiotiques qui ne cesse d’envahir les surfaces cultivés chaque année. Elle entraine chez les espèces glycophytes des perturbations d’ordre osmotique, nutritionnel et métaboliques. La nutrition et le métabolisme de l’azote minéral constituent des étapes primordiales dans la synthèse des acides aminés et des composés azotés indispensables chez les plantes. Par conséquent, l’étude de l’expression des enzymes impliquées dans l’assimilation d’azote telle que l’asparagine synthétase (AS, EC 6.3.5.4) chez l’arabette des dames (Arabidopsis thaliana) permet d’avancer nos connaissances sur la régulation transcriptionnelle du métabolisme azoté sous stress salin. Au cours des travaux de recherche entamés dans le cadre de cette thèse, un intérêt particulier est accordé au gène ASN2 chez Arabidopsis. Les résultats obtenus ont montré que la mutation ASN2 a accentué les effets du NaCl sur l’assimilation de l’ammonium. Le mutant asn2-1 se montre plus sensible au stress salin que le sauvage malgré que l’absence des transcrits du gène ASN2 est associé à une expression importante du gène ASN1. L’inhibition de l’activité glutamine synthétase (GS, EC 6.3.1.2), la faible activité aminatrice de la GDH (NADH-GDH, EC 1.4.1.2) sous stress salin ainsi que l’absence des transcrits ASN2 seraient à l’origine de l’accumulation de l’ammonium chez le mutant asn2-1. Toutefois, l’application exogène de l’ammonium nous a montré que l’action du NaCl sur l’expression de l’asparagine synthétase n’est pas directement liée à l’accumulation endogène d’ammonium. L’accumulation d’autres métabolites tels que l’asparagine, la glutamine et la glutamate pourrait être à l’origine des effets du sel sur l’expression des gènes ASN. / Plant growth activity is often limited by constraint environment conditions. Soil salinity is one of major abiotic stress which is becoming more problematic every year. In glycophytes species, it induced osmotic, nutritional and metabolic disturbances. The nitrogen nutrition and metabolism constitute an essential step in amino acid and nitrogen compounds synthesis in plants. Therefore, studying the expression of enzymes involved in nitrogen assimilation such as asparagine synthetase (AS, EC 6.3.5.4) in Arabidopsis thaliana will improve our knowledge on the transcriptional regulation of nitrogen metabolism under salt stress. In the present work of this thesis, a special attention was taken on AS gene (ASN2) wild type and mutants. Obtained results showed that ASN2 mutation accentuated the salt-induced effects on ammonium assimilation. The asn2-1 mutant was more sensitive to salt stress than the wild type, while the ASN2 transcript absence was associated with an important ASN1expression. The observed inhibition of glutamine synthetase (GS, EC 6.3.1.2) activity, the low aminatrice GDH (NADH-GDH, EC 1.4.1.2) activity under salt stress as well as the ASN2 transcript loss brought to an ammonium accumulation in asn2-1mutant. However, exogenous ammonium application showed that NaCl effect on asparagine synthetase expression was not directly related to the endogenous ammonium accumulation. Other metabolites accumulation such as asparagine, glutamine and glutamate could be involved in the obtained salt-effects on ASN expression in Arabidopsis.

Succinat und ihre Derivate werden in vielfältiger Weise in den Bereichen Tenside, Lebensmittel, Pharmazeutika und Polymere angewendet. Aufgrund der derzeit kostenintensiven petrochemischen Synthese ist die aerobe nicht-konventionelle Hefe Yarrowia (Y.) lipolytica für die biotechnologische Succinatsynthese von großem Interesse. In der vorliegenden Arbeit wurde das Potential dieser Hefe für eine industrielle Succinatproduktion unter Betrachtung des Einflusses der enzymatischen Aktivitäten von Succinyl-CoA Synthetase und Pyruvat-Carboxylase auf die Succinatsynthese untersucht. Es wurde eine Steigerung der Succinatausbeute um 40 % durch die Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 7-8 gemeinsam mit der Deletion des Gens der β Untereinheit der Succinyl-CoA Synthetase im genetisch veränderten Y. lipolytica Stamm H222-AK10 (mcPYC Δscs2) erzielt. Unter Verwendung von Glycerol als C-Quelle wurde eine Erhöhung der Succinatbildung der Transformande H222 AK10 im Vergleich zum Wildtyp von 5,1 ± 0,7 g/l auf 8,7 ± 1,6 g/l nachgewiesen. Die Raum-Zeit-Ausbeute dieses Hefestammes verdoppelte sich von 11,9 ± 1,3 mg/l*h auf 21,9 ± 2,5 mg/l*h. Eine Erhöhung der Sekretion organischer Säuren gelang hingegen nicht durch den alleinigen Verlust der Succinyl-CoA Synthetase Aktivität in den Stämmen H222-AK4 (scs1::URA3), H222-AK8 (scs2:.URA3) und H222-AK9 (scs1::URA3 Δscs2) oder durch die alleinige Aktivitätserhöhung der Pyruvat-Carboxylase in H222-AK1 (mcPYC). Des Weiteren wurde ein Y. lipolytica Stamm erzeugt, der durch die Überexpression der für die Succinyl-CoA Synthetase kodierenden Gene SCS1 und SCS2 charakterisiert ist. Die Transformande H222 AK2 (mcSCS1 mcSCS2) bildete unter den gleichen Kultivierungsbedingungen durchschnittlich 2 g/l weniger Succinat als der Wildtyp (5,1 ± 0,7 g/l). Auch die zusätzliche Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 4 in der Transformande H222 AK3 (mcPYC mcSCS1 mcSCS2) konnte den negativen Effekt der erhöhten Gen-Dosen von SCS1 und SCS2 auf die Succinatsynthese nicht aufheben. Dementsprechend wurden für H222-AK3 eine Succinatausbeute von 3,1 ± 0,3 g/l bestimmt.

Sunkieji metalai yra vieni didžiausių ekologinių nuodų. Kadangi apsinuodijimų sunkiaisiais metalais dažnis tebėra didelis, prevencinės strategijos bei veiksmingo gydymo poreikis išlieka aktualūs. Savo eksperimentais mes siekėme įvertinti apsauginį cinko jonų poveikį baltymų biosintezės sistemai bei svarbiausiems jos komponentams (tRNR ir aminoacil-tRNR-sintetazėms) esant slopinančiam kadmio jonų poveikiui. Eksperimentai atlikti su baltosiomis laboratorinėmis pelėmis. Cinko apsauginio poveikio įvertinimui, baltymų biosintezės intensyvumas pelių kepenyse vertintas po 0,5 LD50 CdCl2 (1,6 mg Cd2+ vienam kg kūno masės) ir/arba 0,3 LD50 ZnSO4 (3,1 mg Zn2+ vienam kg kūno masės) tirpalų sušvirkštimo į laboratorinių pelių pilvo ertmę. Baltymų biosintezės intensyvumas pelių organuose nustatytas praėjus 2, 8 ir 24 val. po metalų sušvirkštimo, pagal radioaktyviai žymėto [14C]-leucino įjungimą į naujai susintetintus peptidus ir baltymus. tRNRLeu ir leucyl-tRNR sintetazių aktyvumas nustatytas vykstant reakcijai su [14C]-leucinu. Gauti rezultatai parodė, jog 2 val. po CdCl2 sušvirkštimo, cinko jonai apsaugojo baltymus sintezuojančią sistemą nuo toksinio kadmio poveikio. Praėjus 8 val. po šių abiejų metalų sušvirkštimo, cinko jonai iš dalies normalizavo baltymų biosintezę, tačiau praėjus 24 val., baltymų biosintezės intensyvumas išliko tokio paties aktyvumo, kaip ir kadmiu paveiktose pelėse. Vadinasi, praėjus ilgesniam laikui (24 val.), cinko jonai neapsaugo kepenų transliacijos... [toliau žr. visą tekstą] / The aim of this study was to evaluate protective effects of zinc ions on the total protein synthesis in mouse liver and key components of liver translation machinery (tRNR ir aminoacil-tRNR synthetases) in the present of toxic cadmium ions effects. Experiments were done on white mice using intraperitoneal injections of 0,5 LD50 CdCl2 solution (1,6 mg Cd2+ per 1 kg of body mass) and/or 0,3 LD50 ZnSO4 (3,1 mg Zn2+ per 1 kg of body mass). Protein synthesis was evaluated by incorporation of 14C-labelled leucine into newly synthesized peptides and proteins after 2, 8 and 24 hours of intoxication. Activities of tRNALeu and leucyl-tRNA synthetase were measured by an aminoacylation reaction using 14C-labelled leucine. The data showed that at the 2nd h after CdCl2 injection, Zn2+ abolished deleterious effect of Cd2+ on the protein synthesis in the liver. Although pronounced activation of the protein synthesis was observed after 8 h of intoxication with either Cd2+ or Zn2+, this effect was lower in the presence of both ions. At the 24th h the protein synthesis was as active as in the liver of Cd-treated mice. Thus, Zn2+ can counteract Cd-induced inhibition of protein synthesis in mice liver only at the early stage of Cd2+ intoxication (at the 2nd h). Zn2+ abolished deleterious effect of Cd2+ on the activity of leucyl-tRNA synthetase within 24 h of mice intoxication with CdCl2. In vitro conditions, Zn2+ increased the acceptor activity of leucyl-tRNA synthetase only in low (1... [to full text]

La détermination de la structure des protéines à une résolution atomique est cruciale pour la compréhension de leur fonction cellulaire. Actuellement, la cristallographie aux rayons X est la méthode la plus efficace pour la détermination à haute résolution de la structure de protéines monomériques allant 40 et 100 kDa. Par contre, elle est limitée par la croissance de cristaux de bonne qualité, qui est problématique pour nombreuses cibles. La cryo-microscopie électronique (cryoME) permet la détermination structurale à résolution quasi-atomique de larges structures protéiques, de préférence symétrique et en solution. Cependant, les images de cryoME sont très bruitées, car une faible dose d'électrons est appliquée de manière à limiter les dommages d'irradiation. En moyennant des dizaines d'images correspondant à la même orientation moléculaire, le rapport signal sur bruit est amélioré. La combinaison des images moyennées de plusieurs orientations permet l'obtention d'une carte de densité électronique 3D de la molécule d'intérêt. Si la taille et la symétrie de la molécule diminuent, l'analyse cryoME devient de moins en moins précise, il est alors impossible d'analyser des protéines monomériques de taille inférieure à 100 kDa. Le but de ce travail a été de développer une nouvelle approche pour réduire cette limite de poids moléculaire. Elle consiste à fusionner la protéine d'intérêt (cible) à une matrice homo-oligomérique, générant une particule symétrique et de taille importante adaptée à l'analyse par cryoME. Dans cette thèse, nous avons cherché à tester et démontrer la faisabilité de cette approche de symétrisation en utilisant des protéines cibles de structure connue.Pour mettre en place notre étude pilote, nous avons choisi différentes combinaisons de cibles et de matrices connectées par des peptides de liaison (linker) de longueur différentes. Nous avons caractérisé les fusions exprimées en bactéries par microscopie électronique après coloration négative et par plusieurs techniques biophysiques. Grace à ces techniques, nous avons trouvé que la meilleure combinaison est la fusion entre la protéine matrice glutamine synthétase (GS), un 12-mer de symétrie D6 et la cible maltose binding protein (Mbp), connectées par un linker contenant trois alanines, que nous avons appelée « Mag ». En jouant sur la longueur du linker nous avons ensuite sélectionné la fusion la plus compacte pour l'analyse cryoME: MagΔ5. Nous avons obtenu la carte cryoME à 10 Å de MagΔ5, qui présente une bonne corrélation avec les modèles atomiques de Mbp et GS. Plus particulièrement, le site catalytique et quelques hélices α sont identifiables. Ces résultats sont confirmés par l'étude cristallographique que nous avons conduite sur MagΔ5. L'ensemble de ce travail souligne que la présence d'une grande interface d'interactions cible-matrice stabilise la fusion et améliore la résolution en cryoME. Pour la symétrisation d'une cible inconnue, nous envisageons la même procédure expérimentale que celle développée pour MagΔ5. La matrice et le linker les plus adaptés devront être identifiés en utilisant les mêmes méthodes biophysiques.En conclusion, ce travail établit la preuve de concept que la méthode de symétrisation des protéines permet la détermination de la structure de protéines de poids moléculaire inférieur à 100 kDa par cryoME. Cette méthode a le potentiel d'être un nouvel outil prometteur, qui faciliterait l'analyse de cibles résistantes à l'analyse structurale conventionnelle. / Structural determination of proteins at atomic level resolution is crucial for unravelling their function. X-ray crystallography has successfully been used to determine macromolecular structures with sizes ranging from kDa to MDa, and currently remains the most efficient method for the high-resolution structure determination of monomeric proteins within the 40-100 kDa range. However, this method is limited by the ability to grow well diffracting crystals, which is problematic for several targets, such as membrane proteins. Single particle cryo electron microscopy (cryoEM) allows near atomic (3-4Å) resolution structural determination of large, preferably symmetric, assemblies in solution. Biological molecules scatter electrons weakly and, to avoid radiation damage, only low electron doses can be used during imaging. Consequently, raw cryoEM images are extremely noisy. However, averaging many molecular images aligned in the same orientation permits one to increase the signal-to-noise ratio, ultimately allowing the achievement of a 3D density map of the molecule of interest. Nevertheless, as the molecular size and degree of symmetry decrease, the individual images loose adequate features for accurate alignment. Currently, cryoEM analysis is practically impossible for monomeric proteins below ~100 kDa in mass. We propose to circumvent this obstacle by fusing such monomeric target proteins to a homo-oligomeric protein (template), thereby generating a self-assembling particle whose large size and symmetry should facilitate cryoEM analysis. In the present thesis we seek to test and demonstrate the feasibility of this ‘protein symmetrization' approach and to evaluate its usefulness for protein structure determination. To set up the pilot study we combined selected targets of known structure with two templates: Glutamine Synthetase (GS), a 12-mer with D6 symmetry and a helical N-terminus, and the E2 subunit of the pyruvate dehydrogenase complex, a 60-mer with icosahedral symmetry and an unstructured N-terminus. After recombinant production in E.coli we identified by negative stain EM a promising dodecameric chimera for structural analysis, comprising maltose binding protein (Mbp) connected to GS by a tri-alanine linker (denoted “Mag”). In order to optimize sample homogeneity we produced a panel of Mag deletion constructs by sequentially truncating the 17 residues between the Mbp and GS domains. A combination of biophysical techniques (thermal shift assay, dynamic light scattering, size exclusion chromatography) and negative stain EM allowed us to select the best candidate for cryoEM analysis, MagΔ5. By enforcing D6 symmetry we obtained a cryoEM map with a resolution of 10Å (FSC 0.5 criterion). The density of the symmetrized 40 kDa Mbp presents shape and features corresponding to the known atomic structure. In particular, the catalytic pocket and specific α-helical elements are distinguishable. The cryoEM map is additionally validated by a 7Å crystal structure of the MagΔ5 oligomer. The presence of a continuous helical connection between target (Mbp) and template (GS) likely contributed to the conformational homogeneity of MagΔ5. Moreover, comparing MagΔ5 with other chimeras studied in this work suggests that a large buried surface area and favorable interactions between the target and template limit the flexibility of the chimera and improve its resolution by cryoEM. For the symmetrization of a target of unknown structure, we envisage proceeding by a trial and error approach by fusing it to a panel of templates with helical termini and different surface properties, and subsequently selecting the best ones using biophysical assays. In conclusion, the present work establishes the proof-of-concept that protein symmetrization can be used for the structure determination of monomeric proteins below 100 kDa by cryoEM, thereby providing a promising new tool for analyzing targets resistant to conventional structural analysis.

Aminoacyl-tRNA synthetases (ARS) are a family of enzymes that catalyze the charging of amino acids to their cognate tRNA in an aminoacylation reaction. Many members of this family have been found to have secondary functions independent of their primary aminoacylation function. Threonyl-tRNA synthetase (TARS), the ARS responsible for charging tRNA with threonine, is secreted from endothelial cells in response to both vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), and stimulates angiogenesis and cell migration. Here we show a novel experimental approach for studying TARS secretion, and for observing the role of intracellular TARS in the endoplasmic reticulum (ER) stress response and in angiogenesis. Using Western blotting, immunofluorescence microscopy and RT-qPCR we were able to investigate changes in TARS protein and transcript levels. We initially hypothesized that TARS was secreted by exosomal release, and so we treated a human ovarian cancer cell line (CaOV-3) with monensin, an ionophore that increases exosome production, and VEGF to observe changes in intracellular and extracellular TARS protein. Monensin treatment consistently increased extracellular and intracellular TARS protein, however CD63, an exosome marker protein, levels were unaffected by monensin treatment. VEGF had no effect on intracellular TARS. We therefore hypothesized that the TARS response was a result of ER stress. The unfolded protein response (UPR) is a series of signaling pathways that are activated upon ER stress. When CaOV-3 cells were treated with increasing concentrations of monensin, intracellular levels of TARS and p-eIF2α, a downstream UPR target, increased accordingly. Monensin increased intracellular TARS protein and transcript levels in CaOV-3 cells. Monensin also increased DNAJB9, an ER chaperone protein, transcript levels, further confirming ER stress. Interestingly, monensin increased VEGF transcript levels about 6-fold. Borrelidin, a natural TARS inhibitor, also increased VEGF transcript levels, and caused an increase in p-eIF2α protein. Although the mechanism of TARS secretion remains unresolved, these data indicate that intracellular TARS expression increases in response to ER stress by monensin. Given TARS and VEGF transcript expression increased accordingly, it is possible that intracellular TARS may have pro-angiogenic function. Future directions may include investigating TARS interactions with translational control machinery.