Este trabalho teve o objetivo de avaliar o efeito da adição da transglutaminase microbiana (MTGase) na fabricação de pão de forma, através do desenvolvimento de formulação ideal, com combinações de aditivos e enzima, e da avaliação do efeito da enzima nas proteínas, na massa crua, na massa após a fermentação e no produto final. Para comparar a qualidade dos pães produzidos com ou sem enzima, foram testadas três formulações: a básica, livre de aditivos (pão Zero); com a adição de emulsificante e ácido ascórbico (pão Controle); e a preparada com a formulação básica adicionada de enzima (pão MTGase). A avaliação da qualidade dos pães foi feita por meio de medidas físicas e instrumentais. A análise de textura foi realizada pelo método TPA (Texture Profíle Analysis), cujas respostas de firmeza, elasticidade, mastigabilidade e gomosidade podem ser correlacionadas com análises sensoriais. Paralelamente, de amostras de farinha, de massa e de pão foram obtidas as frações protéicas de gliadinas, gluteninas e os resíduos de extração. As gliadinas e as gluteninas foram analisadas por cromatografia líquida de alta eficiência em fase reversa e por eletroforese em gel de poliacrilamida contendo SDS. Os resultados de volume e de firmeza dos diferentes pães apresentaram diferenças significativas a nível de 5%, em que as respostas do pão MTGase foram melhores que as do pão Zero, porém ainda inferiores às do Controle. A melhor formulação foi obtida por meio de um planejamento composto central, com variações nas concentrações de emulsificante, ácido ascórbico e enzima, com os resultados avaliados pela metodologia de superfície de resposta. Exceto para a coesividade, todos os outros parâmetros de volume, dureza (TA), firmeza, elasticidade, mastigabilidade e gomosidade (TPA) apresentaram resultados positivos pela ação da transglutaminase a 0,6%, combinada com 0,2 % de emulsificante e 70 ppm de ácido ascórbico. Os resultados sugerem que a enzima foi capaz de modificar as propriedades químicas das proteínas, o comportamento reológico da massa e as propriedades funcionais do pão, melhorando a força da massa, a textura e o volume dos pães. As análises das frações gliadínicas apresentaram cerca de 3% de Nitrogênio total, em base seca, e as frações glutenínicas apresentaram entre 2 e 5% de Nitrogênio total. Os perfis cromatográficos e eletroforéticos dessas frações sugerem que as gliadinas não foram afetadas pela presença da enzima, que envolveram sobretudo as gluteninas. O conjunto de resultados indica que a aplicação de MTGase em associação com aditivos convencionais pode ser uma alternativa à panificação, embora os mecanismos de sua ação na massa não estejam completamente esclarecidos. / The application of microbial transglutaminase on weak gluten flour used in breadmaking was studied over the process. To verify the enzyme effects, three formulations were tested: Base formulation, characterized by the absence of enzyme and emulsifying agents; Control formulation, comprised by the presence of emulsifying agents and ascorbic acid and MTGase formulation, with the enzyme. Samples of flour, dough and bread were analyzed. The effect of enzyme on bread quality was estimated by parameters of Texture Analysis, Texture Profile Analysis and specific volume. The protein contents from those samples were determined by the total nitrogen in glutenin and gliadin fractions, that were also analyzed by RP-HPLC (reversed phase high performance liquid chromatography) and by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Although the MTGase bread did not reach the same quality parameters as those achieved by the Control samples, it showed as an alternative formulation to reduce the quantity of emulsifying agents and ascorbic acid as compared to the Control. The results indicate that the enzyme modified chemical and functional properties of glutenin fraction, improving dough strength and bread volume. Results of total nitrogen content, and electrophoretic and chromatographic profiles of the protein fractions suggest that while glutenin proteins were modified by enzyme, gliadin proteins were not affected.

PDI ("potato cathepsin D inhibitor ") and NID ("novel inhibitor of cathepsin D ") from potato (Solanum tuberosum) belong to the protein family of Kunitz inhibitors (I3 family, Merops database). These 20 kDa isoinhibitors with the typical β-trefoil architecture inhibit aspartic and serine peptidases. In this thesis, the constructs for recombinant expression of PDI and NID in the yeast Pichia pastoris system were prepared and high-producing colonies were selected. Both proteins were identified in the cultivation media by mass spectrometry and N-terminal sequencing. A purification protocol for PDI with three chromatographic steps was designed. Analogous functional properties were demonstrated for the purified recombinant PDI and the native PDI isolated from a natural source. Analysis of the inhibitory specificity showed that PDI is a potent inhibitor of selected aspartic peptidases from the A1 family and serine peptidases from the S1 family, including a relevant enzyme of insect origin. This finding supports the hypothesis that Kunitz inhibitors are involved in plant defense against herbivorous insects. The inhibitors prepared within the project will be used for analysis of the reactive centers against target peptidases by protein crystallography. (In Czech) Key words: proteolytic enzymes, activity...

Indiana University-Purdue University Indianapolis (IUPUI) / Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.

Indiana University-Purdue University Indianapolis (IUPUI) / Oxalate decarboxylase (EC 4. 1. 1. 2 OxDC) from Bacillus subtilis is a manganese-dependent enzyme that catalyzes the cleavage of the chemically inactive C-C bond in oxalate to yield formate and carbon dioxide. A mechanism involving Mn(III) has been proposed for OxDC, however no clear spectroscopic evidence to support this mechanism has yet been obtained. In addition, a recent study has shown that N-terminal metal binding site loop variants of OxDC were able to catalyze the oxidation of oxalate to yield hydrogen peroxide and carbon dioxide, which makes OxDc function as another oxalate degradation protein in the cupin superfamily, oxalate oxidase (EC 1.2.3.4 OxOx). In this work, wild-type (WT) Bacillus subtilis OxDC and a series of variants with mutations on conserved residues were characterized to investigate the catalytic mechanism of OxDC. The application of membrane inlet mass spectrometry (MIMS), electronic paramagnetic resonance (EPR) spectroscopy and kinetic isotope effects (KIEs) provided information about the mechanism. The Mn(III) was identified and characterized under acidic conditions in the presence of dioxygen and oxalate. Mutations on the second shell residues in the N-terminal metal binding site affected the enzyme activity properties of the metal. In the N-terminal domain, the functional importance of the residues in the active site loop region, especially Glu162, was confirmed, and evidence for the previously proposed mechanism in which OxDC and the OxDC/OxOx chimeric variant share the initial steps has been found. In addition, the mono-dentate coordination of oxalate in the N-terminal metal binding site was confirmed by X-ray crystallography. A proteinase cleavable OxDC was constructed and characterized, revealing the interaction between the N-terminal and C-terminal domains.

Die Bildung von Amyloidablagerungen ist ein Kennzeichen der Alzheimerschen Erkrankung. Hauptbestandteil dieser senilen Plaques sind sogenannte A beta Peptide, die durch proteolytische Prozessierung aus einem Vorläufermolekül (APP) gebildet werden. Die vorliegende Arbeit beschreibt die Klonierung des Meerschweinchen - APP. Diese cDNA-Sequenz zeigt auf DNA-Ebene eine Homologie zum Human-APP von ca. 90%, auf Proteinebene beträgt die Identität ca. 97 %. Damit wird ein weiterer experimenteller Beweis für die evolutionäre Konservierung des Amyloidvorläuferproteins in Säugetieren erbracht. APP mRNA wird in Meerschweinchen-Geweben ubiquitär exprimiert. Durch alternatives Spleißen wird ein zum Human-APP im wesentlichen ähnliches Isoformenmuster gebildet: Isoformen, welche eine Proteaseinhibitordomäne enthalten, werden dominierend in peripheren Organen exprimiert, dagegen ist im Zentralnervensystem das APP 695 mit über 60 % der Gesamttranskripte die bevorzugt exprimierte Isoform. Die klonierte cDNA des Meerschweinchen-APP wurde in prokaryontischen wie auch eukaryontischen Zellsystemen exprimiert. Dabei wurde die Eignung einer Anzahl von gegen Human-APP gewonnenen Antikörpern zur Detektion des Meerschweinchen-APP und seiner Prozessierungsprodukte gezeigt. Die Expression der neuronal dominierend exprimierten Isoform APP 695 des Meerschweinchen-APP in humanen Neuroblastom-Zellen zeigte keine Unterschiede hinsichtlich der APP-Prozessierung und A beta-Bildung im direkten Vergleich zu Human-APP 695. Die proteolytische Prozessierung des Proteins wurde durch Detektion der typischen Spaltprodukte in vivo (im Liquor) als auch in einem neu etablierten in vitro-Modell primär kultivierter neuronaler Zellen untersucht. Diese Zellkulturen wurden zunächst immunhistochemisch und biochemisch charakterisiert und als "mixed brain"-Typ mit einem hohen neuronalen Anteil beschrieben. Die Prozessierung des endogenen Meerschweinchen-APP in kultivierten Zellen führt dabei zur Bildung und Akkumulation aggregationsfähiger A beta - Peptide. Zur Detektion dieser Peptide wurde ein sensitiver Nachweis durch Western-Blot etabliert. Es wird damit ein Modellsystem für in vitro-Untersuchungen vorgeschlagen, welches ein Studium der Expression und Prozessierung des Amyloidvorläuferproteins unter angenähert physiologischen Bedingungen ermöglicht. / A beta peptides, the major component of neuritic plaques found in the brains of patients with Alzheimer’s disease, are derived by proteolytic processing from a larger precursor molecule (amyloid precursor protein - APP). A combination of PCR methods was used to clone and sequence APP cDNA from guinea pig (Cavia porcellus). Guinea pig APP exhibits extensive similarities to human APP in terms of primary structure, mRNA expression of differentially spliced isoforms as shown by Northern blot and RT-PCR analysis as well as proteolytic processing to amyloidogenic A beta peptides. In contrast to rat and mouse APP, guinea pig APP - recombinantly expressed in human neuroblastoma-cells - was processed indistinguishable from human APP thus excluding intrinsic sequence-specific factors influencing processing. Further studies were performed using newly established primary cell cultures of guinea pig neurons. Refined methods have been used to detect and characterize major proteolytic processing products of APP in vitro and in vivo. In conclusion, guinea pigs provide a model to study expression and processing of APP that closely resembles the physiological situation in humans and should, therefore, be important in elucidating potential strategies to prevent amyloid formation in Alzheimers Disease.

This dissertation primarily covers the impact of bulky ,-dehydroamino acids on the proteolytic stability and enhanced folding of -hairpins. It partly describes the progress towards the total synthesis of yakuamide A, a potent anticancer peptide with an IC50 value of 14 ng/mL against leukemia cells. Proteins and peptides are a very attractive source of potential medicinal agents as they can target various protein“protein interactions that are implicated in several diseases and disorders. The global sales of peptide drugs in 2013 were estimated to be about $28 billion and are constantly rising at an appreciable rate. However, peptide drugs have a short plasma half-life because of their susceptibility to proteolysis. Multiple approaches have been discovered to overcome this shortcoming, but there is still an urgent need for better peptidomimetics to increase the stream of peptides entering the pharmaceutical market. Here, it has been demonstrated that the incorporation of a bulky ,-dehydroamino acid in the turn regions of -hairpins can substantially increase their proteolytic stability and folding. Insertion of a dehydrovaline (ΔVal) residue at the i+1 position imparted ca. 7-fold increase in proteolytic resistance and ca. 15% increase in folding when compared to the parent peptide. Since the insertion of a bulky ,-dehydroamino acid into the turn regions of -hairpins can promote proteolytic stability without perturbing the secondary structures, it is believed that this novel approach is very promising in stabilizing bioactive turn-containing peptides for therapeutic use.Yakuamide A is a medium-sized peptide that contains several bulky dehydroamino acids, -hydroxyamino acids and unique N- and C-termini. It has an unprecedented anticancer profile, and potent bioactivity, hence it was imperative to accomplish its total synthesis to elicit its unique mode of action and biological target. More efficient methods were developed to synthesize bulky dehydroamino acids and -hydroxyamino acids. A regioselective base-free aminohydroxylation was developed for the synthesis of -hydroxyamino acids. The major focus was the three-step synthesis of the N-terminal acyl group from a known compound by a one-pot indium-catalyzed cross-Claisen condensation/reduction and the synthesis of (2S,3R)--hydroxyisoleucine, and racemic -hydroxyisoleucine, which are the precursors of E- and Z-dehydroisoleucine.

Le régulateur transcriptionnel BAP1 est une déubiquitinase nucléaire (DUB) dont le substrat est l’histone H2A modifiée par monoubiquitination au niveau des residus lysines 118 et 119 (K118/K119). Depuis les dernières années, BAP1 emerge comme un gene suppresseur de tumeur majeur. En effet, BAP1 est inactivé dans un plethore de maladies humaines héréditaires et sporadiques. Cependant, malgré l’accumulation significative des connaissances concernant l’occurrence, la pénétrance et l’impact des défauts de BAP1 sur le développement de cancers, ses mécanismes d’action et de régulation restent très peu compris. Cette étude est dédiée à la caractérisation moléculaire et fonctionnelle du complexe multi-protéique de BAP1 et se présente parmi les premiers travaux décrivant sa régulation par des modifications post-traductionnelles. D’abord, nous avons défini la composition du corps du complexe BAP1 ainsi que ses principaux partenaires d’interaction. Ensuite, nous nous sommes spécifiquement intéressés a investiguer d’avantage deux principaux aspects de la régulation de BAP1. Nous avons d’abord décrit l’inter-régulation entre deux composantes majeures du complexe BAP1, soit HCF-1 et OGT. D’une manière très intéressante, nous avons trouvé que le cofacteur HCF-1 est un important régulateur des niveaux protéiques d’OGT. En retour, OGT est requise pour la maturation protéolytique de HCF-1 en promouvant sa protéolyse par O-GlcNAcylation, un processus de régulation très important pour le bon fonctionnement de HCF-1. D’autre part, nous avons découvert un mécanisme unique de régulation de BAP1 médiée par l’ubiquitine ligase atypique UBE2O. en effet, UBE2O se caractérise par le fait qu’il s’agit aussi bien d’une ubiquitine conjuratrice et d’une ubiquitine ligase. UBE2O, multi-monoubiquitine BAP1 au niveau de son domaine NLS et promeut son exclusion du noyau, le séquestrant ainsi dans le cytoplasme. De façon importante, nos travaux ont permis de mettre de l’emphase sur le rôle de l’activité auto-catalytique de chacune de ces enzymes, soit l’activité d’auto-déubiquitination de BAP1 qui est requise pour la maintenance de sa localisation nucléaire ainsi que l’activité d’auto-ubiquitination d’UBE2O impliquée dans son transport nucléo-cytoplasmique. De manière significative, nous avons trouvé que des défauts au niveau de l’auto-déubiquitination de BAP1 due à des mutations associées à certains cancers indiquent l’importance d’une propre regulation de cette déubiquitinase pour les processus associés à la suppression de tumeurs. / BAP1 is a nuclear deubiquitinating enzyme (DUB) that acts as a transcription regulator and a DUB of nucleosomal histone H2AK119. In the recent years, it has become clear that BAP1 is a major tumor suppressor, inactivated in a plethora of hereditary and sporadic human malignancies. Although, we now accumulated a significant body of knowledge in respect to the occurrence, penetrance and impact of BAP1 disruption in cancer, its mechanism of action and regulation remained poorly defined. This work is dedicated to the biochemical and functional characterization of the BAP1 multiprotein complex and presents one of the first cases regarding its regulation by post-translational modifications. First, we defined the initial composition of the BAP1 complex and its main interacting components. Second, we specifically focused on two aspects of BAP1 regulation. We described the cross regulation between the two major components of the complex namely HCF-1 and OGT. We found that HCF-1 is important for the maintenance of the cellular levels of OGT. OGT, in turn, is required for the proper maturation of HCF-1 by promoting O-GlcNAcylation-mediated limited proteolysis of its precursor. Third, we discovered an intricate regulatory mechanism of BAP1 mediated by the atypical ubiquitin ligase UBE2O. UBE2O multi-monoubiquitinates BAP1 on its NLS and promotes its exclusion from the nucleus. Importantly, our work emphasises the role of the autocatalytic activity of both enzymes namely the auto-deubiquitination activity of BAP1, required for the maintenance of nuclear BAP1 and the auto-ubiquitination of UBE2O implicated in its nucleocytoplasmic transport. Significantly, we found that auto-deubiquitination of BAP1 is disrupted by cancer-associated mutations, indicating the involvement of this process in tumor suppression.

L’ubiquitination, une modification post-traductionnelle importante pour le contrôle de nombreux processus cellulaires, est une réaction réversible. La réaction inverse, nommée déubiquitination est catalysée par les déubiquitinases (DUB). Nous nous sommes intéressés dans nos travaux à étudier l’ubiquitination de l’histone H2A (H2Aub), au niveau des résidus lysines 118 et 119 (K118/K119), une marque épigénétique impliquée dans la régulation de la prolifération cellulaire et la réparation de l’ADN. Le régulateur transcriptionnel BAP1, une déubiquitinase nucléaire, a été initialement identifié pour sa capacité à promouvoir la fonction suppressive de tumeurs de BRCA1. BAP1 forme un complexe multi-protéique avec plusieurs facteurs transcriptionnels et sa fonction principale est la déubiquitination de H2Aub. Plusieurs études ont démontré que BAP1 est un gène suppresseur de tumeurs majeur et qu’il est largement muté et inactivé dans une multitude de cancers. En effet, BAP1 émerge comme étant la DUB la plus mutée au niveau des cancers. Cependant, le ou les mécanismes d’action et de régulation du complexe BAP1 restent très peu connus. Dans cette étude nous nous sommes intéressés à la caractérisation moléculaire et fonctionnelle des partenaires protéiques de BAP1. De manière significative nous avons caractérisé un mécanisme unique de régulation entre deux composants majeurs du complexe BAP1 à savoir, HCF-1 et OGT. En effet, nous avons démontré que HCF-1 est requis pour maintenir le niveau protéique de OGT et que cette dernière est indispensable pour la maturation protéolytique de HCF-1 en promouvant son clivage par O-GlcNAcylation, une signalisation cellulaire nécessaire au bon fonctionnement de HCF-1. Également, nous avons découvert un nouveau mécanisme de régulation de BAP1 par l’ubiquitine ligase atypique UBE2O. En effet, UBE2O agit comme un régulateur négatif de BAP1 puisque l’ubiquitination de ce dernier induit sa séquestration dans le cytoplasme et l’inhibition de sa fonction suppressive de tumeurs. D’autre part nous nous sommes penchés sur la caractérisation de l’association de BAP1 avec deux facteurs de la famille des protéines Polycombes nommés ASXL1 et ASXL2 (ASXL1/2). Nous avons investigué le rôle de BAP1/ASXL1/2, particulièrement dans les mécanismes de déubiquitination et suppression de tumeurs. Nous avons démontré que BAP1 interagit directement iii via son domaine C-terminale avec le même domaine ASXM de ASXL1/2 formant ainsi deux complexes mutuellement exclusifs indispensables pour induire l’activité déubiquitinase de BAP1. De manière significative, ASXM s’associe avec BAP1 pour créer un nouveau domaine composite de liaison à l’ubiquitine. Ces interactions BAP1/ASXL1/2 régulent la progression harmonieuse du cycle cellulaire. De plus, la surexpression de BAP1 et de ASXL2 au niveau des fibroblastes induit la sénescence de manière dépendante de leurs interactions. D’autre part, nous avons identifié des mutations de cancers au niveau de BAP1 le rendant incapable de lier ASXL1/2, d’exercer sa fonction d’autodéubiquitination et de ce fait d’agir comme suppresseur de tumeurs. Ainsi nous avons révélé un lien étroit entre le gène suppresseur de tumeurs BAP1, son activité déubiquitinase et le contrôle de la prolifération cellulaire. / The reverse reaction of ubiquitination, a crucial post-translational modification, is catalyzed by deubiquitinases (DUBs). BAP1 is an ubiquitously expressed nuclear DUB that recently emerged as an important tumor suppressor highly mutated and inactivated in an increasing number of cancers of diverse origins. Both somatic and germline mutations with loss of heterozygosity were observed in tumors, making BAP1 the most mutated DUB in human malignancies. We previously reported that BAP1 is a component of a large multi-protein complex that includes several transcription regulators. The Drosophila homologue of BAP1, Calypso, forms the Polycomb-repressive DUB (PR-DUB) complex with Additional Sex Comb, ASX. This complex catalyzes the deubiquitination of histone H2A, an essential chromatin modification that regulates gene expression. Despite the ever increasing number of findings describing the occurrence of BAP1 mutations in cancers, few studies investigated the mechanisms of action of this DUB as a tumor suppressor. Therefore, the biological function and the mechanism of action and regulation of BAP1 remains largely uncharacterized. In the work described in this thesis, we investigated the roles of BAP1 partners in modulating its catalytic activity and tumor suppressor function. More specifically we discovered a unique mechanism of regulation between two major components of BAP1 complexes, namely HCF-1 and OGT. Indeed, HCF-1 is important for the maintenance of the cellular levels of OGT. OGT, in turn, is required for the proper proteolytic maturation of HCF-1 by promoting its O-GlcNAcylation. This signaling event is required for HCF-1 function as a cell cycle regulator. On the other hand, we deciphered an intricate mechanism of regulation of BAP1 by the atypical E2/E3 ligase, UBE2O. UBE2O, promote the multi-monoubiquitination of BAP1 on its NLS mediating its cytoplasmic sequestration and thus inhibition of its tumor suppressor function. Another aspect of modulation of BAP1 H2Aub catalysis is provided by the association of BAP1 with ASXL1 and ASXL2 (ASXL1/ASXL2), two orthologs of ASX. We investigated the role of BAP1/ASXL1/2, particularly in the mechanisms of deubiquitination and tumor suppression. We have demonstrated that BAP1 interacts directly via its C-terminal domain with the ASXM domain of ASXL1/2, thus forming two mutually exclusive complexes. Significantly, ASXM promote, through assembly with BAP1, the generation of a composite ubiquitin binding domain (CUBI), indispensable for inducing the deubiquitinase activity of BAP1 towards H2Aub. The interactions between BAP1 and ASXL1/2 regulate cell cycle progression. In addition, overexpression of BAP1 or ASXL2 in fibroblasts induces senescence in CTD- and ASXM-dependent manner. We also identified cancer-derived mutation of BAP1 that selectively abolish its interaction with ASXL1 and ASXL2 as well as its H2A deubiquitinase activity. Significantly, this mutant suppressed senescence induced by BAP1 overexpression. Thus we provided a link between the tumor suppressor BAP1, its deubiquitinase activity and the control of cell proliferation.

The theoretical part of work is focused on the issue of biomass which can be used for energy purposes, inparticular agricultural waste, as well as can serve as a substrate for biogas station. It also deals with proteomics, its goals and approaches, separation methods. The aim of this work was to measure each sample of enzyme activity of biomass, which are used as a raw materials for biogas plants and their proteomic identification.