Les bactéries sont en permanence confrontées à des changements d'environnements. La régulation transcriptionnelle joue alors un rôle majeur dans l'adaptation des bactéries. En particulier, la bactérie phytopathogène D. dadantii s'est récemment adaptée à l'hôte végétal. Elle produit en particulier des pectate lyases (Pel) qui dégradent la pectine, ciment des parois végétales, et jouent un rôle majeur dans le développement de la maladie. Les gènes pelD et pelE, malgré la forte divergence dans leur expression, sont issus d'un transfert horizontal suivi d'une duplication récente. La question de l'intégration de ces gènes avec les régulations préexistantes s'est alors posée.Dans un premier temps, les mécanismes moléculaires détaillés de la régulation de pelD ont été étudiés. Il a été montré que cette régulation s'appuie sur un promoteur divergent de forte affinité pour l'ARN polymérase mais de faible efficacité pour la transcription et sur un arrangement stratégique de quatre sites de fixation de répresseur FIS et deux sites de l'activateur CRP. Tous ces éléments interagissent entre eux pour produire une régulation fine de l'expression de pelD. L'origine de la divergence régulatrice entre les paralogues pelD et pelE a par la suite été explorée. De manière surprenante, la divergence entre ces deux gènes et leur sélection s'appuie presque exclusivement sur un décalage de la position du promoteur de pelE (« TSS turnover ») qui l'a transformé en initiateur de la dégradation de la pectine. Ce mécanisme très fréquent chez les eucaryotes pluricellulaires (homme, drosophile, souris…) n'avait jamais encore été décrit chez les bactéries.A travers l'étude des promoteurs pelD et pelE de D. dadantii, de nouveaux mécanismes renforçant l'importance de la régulation transcriptionnelle dans les processus adaptatifs ont ainsi été découverts / Bacteria face frequent environmental changes. Transcriptional regulation plays a major role in the adaptation to these changes. In particular, the phytopathogen bacteria Dickeya have recently adapted to vegetal hosts. They produce Pecate lyases (Pel), among others, to degrade pectin in plant cell walls, which is necessary for disease development. The pelD and pelE genes, despite the strong divergence in their expression, originate from a horizontal gene transfer followed by a recent duplication. This raises the question of their integration into the preexisting regulatory networks.Detailed molecular mechanisms of the transcriptional regulation of pelD were studied first. It was shown that this regulation relies on a high-affinity but low transcription efficiency divergent promoter and a strategic arrangement of four FIS repressor binding sites and two CRP activator binding sites. These elements interact together to fine-tune the expression of pelD. Next, the origin of the regulatory divergence between the paralogous genes pelD and pelE was explored. Surprisingly, their divergence and selection relies mostly on a TSS turnover which happened on the pelE regulatory region and transformed pelE into an initiator of pectin degradation. This widespread phenomenon in multicellular eukaryotes (human, fly, mouse…) had not yet been seen in bacteria. To conclude, through the study of D. dadantii pelD and pelE promoters, new mechanisms highlighting the relevance of transcriptional regulation in adaptation were discovered in this work

Most of the chemical reactions of living cells are catalyzed by protein enzymes. These enzymes are very efficient and display a high degree of specificity with respect to the reaction catalyzed. Cellular activities depend critically on the precise three-dimensional structure and function of thousands of enzymes. Many enzymes require binding of metal ions or small organic molecules for their function. The organic molecules that are indispensible components of catalysis by proteins are called coenzymes. Pyridoxal 5ʹ-phosphate (PLP) is a versatile coenzyme found in all living cells. PLP-dependent enzymes play a key role in the function of most of the enzymes catalyzing reactions in the metabolic pathways of amino acid synthesis and degradation. The enzyme pyridoxal kinase serves to make available the co-enzyme PLP to apo-PLP dependent enzymes. Because of their key role in cellular function and their medical importance, the structure and function of PLP-dependent enzymes have been extensively investigated. In the past decade, detailed investigations on the structure and function of several PLP-dependent enzymes have been carried out in our laboratory. The enzymes studied are B. subtilis serinehydroxymethyl transferase (SHMT), S. typhimurium acetylornithine aminotransferase (AcOAT), S. typhimurium and E. coli diaminopropionate ammonia lyase (DAPAL), S. typhimurium D-serine dehydratase (DSD), S. typhimurium D-cysteine desulfhydrase (DCyD) and S. typhimurium arginine decarboxylase (ArgD). The extensive studies conducted on PLP-dependent enzymes in our laboratory during the past decade has not only resulted in deeper understanding of their structure and function but also raised several new questions regarding substrate recognition, reaction specificity, role of active site residues in the catalytic reaction, mechanism of catalysis and potential applications of these enzymes. This thesis is an attempt to answer some of these questions. The thesis also presents the structure and function of a new protein, Salmonella typhimurium pyridoxal kinase, the enzyme that provides PLP for PLP-dependent enzymes. Single crystal X-ray diffraction technique is the most powerful tool currently available for the elucidation of the three-dimensional structures of proteins and other biological macromolecules and for revealing the relationship between their structure and function. X-ray diffraction studies have provided in depth understanding of the topology of secondary structural elements in the three-dimensional structures of proteins, the hierarchical organization of protein domains, structural basis for the substrate specificity of enzymes, intricate details of mechanisms of enzyme catalyzed reactions, allosteric regulation of enzyme activity, mechanisms of feed-back inhibition, structural basis of protein stability, symmetry of oligomeric proteins and their possible biological implications and a myriad of other biochemical and biophysical properties of proteins. The work reported in this thesis is primarily based on X-ray diffraction studies. X-ray crystal structure investigations are complemented by spectral and biochemical studies on the catalyzed reactions. The thesis begins with an introduction to PLP-dependent enzymes and presentation of a brief summary of the earlier work carried out in our laboratory on PLP-dependent enzymes (Chapter 1). A brief description of earlier functional classification of PLP-dependent enzymes and the more recent classification of these enzymes into the four groups based on their three-dimensional structure is provided. Although enzymes belonging to these four structural classes have evolved from independent evolutionary lineages, they share some common features near their active sites and in the mode of PLP binding. Earlier work carried out elsewhere on pyridoxal kinase and its key role in maintaining PLP at a low concentration in the cytosol is presented. Different mechanisms that have been proposed for the transfer of PLP from pyridoxal kinase to other apo PLP-dependent enzymes are briefly described. The experimental procedures and computational methods used during the course of these investigations to obtain the results reported in chapters 3-6 are presented in Chapter 2. Most of these methods are applicable to the isolation of plasmids, cloning, over expression, protein purification, mutant construction, crystallization, X-ray diffraction data collection and processing, structure elucidation and refinement, validation and structural analysis presented in the next three chapters. Various programs and protocols used for data processing, structure determination, refinement, model building, structure validation and analysis are also briefly described. In chapter 3, the role of a number of active site residues in the reaction catalyzed by EcDAPAL, a fold type II PLP-dependent enzyme, the structure of which was determined earlier in the laboratory is explored by mutational, biochemical and structural analyses. Earlier studies had established the probable role of Asp120 and Lys77 in the reaction leading to the breakdown of D-DAP and L-DAP, respectively (Bisht et al., 2012). To further validate the earlier observations, a number of active site mutants were generated for Asp 120 (D120N, D120C, D120S and D120T), Asp 189 (D189N, D189C, D189S and D189T), Lys77 (K77T, K77H, K77R and K77A), His 123 (H123L) and Tyr 168 (Y168F). The structure of D120N mutant crystal obtained after soaking in crystallization cocktail containing D-DAP revealed the presence of an intact external aldimine complex at the active site supporting the earlier proposal that Asp120 is the base abstracting the Cα proton from the D-isomer of DAP. Biochemical and structural observations suggested that none of the Asp189 mutants may bind PLP and were catalytically inactive suggesting an essential role for Asp189 in catalysis. In contrast to type I PLP-dependent enzymes, none of the Lys 77 mutants of EcDAPAL could bind PLP either covalently or non-covalently and were inactive with both the isomers of DAP. Thus, Lys77 appears to be important for both PLP binding and catalysis. H123L mutant formed an external aldimine with D-DAP and a gem-diamine complex with L-DAP indicating that this residue is also crucial for catalysis. These studies have provided additional support to the catalytic mechanism of EcDAPAL proposed earlier. The next Chapter 4 explores the structure, function and catalytic mechanism of Salmonella typhimurium DAPAL (StDAPAL). The protein was purified from a construct carrying a hexa-histidine tag at the C-terminus by Ni-NTA chromatography. The purified protein was demonstrated to be homogeneous by SDS-PAGE and MALDI-TOF. Crystals of StDAPAL belonging to the C-centred monoclinic space group (C121) with four molecules in the asymmetric unit were obtained by the micro batch method and used for collecting X-ray diffracting data. The crystal structure was determined by molecular replacement using the homologous enzyme from E. coli (PDB code 4D9M, Bisht et al., 2012), which shares a sequence identity of 50% with the S. typhimurium enzyme as the phasing model in the program Phaser (McCoy et al., 2007) of the CCP4 suite. The model was refined with Refmac5 of CCP4 suite to R and Rfree values of 25.5% and 30.9%, respectively. A superposition of the structure so obtained over EcDAPAL revealed that the two structures are very similar. A sulfate molecule bound to the active site of StDAPAL could be located. The position of the sulfate corresponds to that of the carboxyl group of aminoacrylate intermediate of EcDAPAL (4D9M). The PLP was bound to Lys78 as an internal aldimine. Since the active sites of the two protomers in fold type II PLP-dependent enzymes are independent, it might be possible to obtain functional monomers of EcDAPAL. With this view, mutation of a conserved Trp (Trp399) present in the dimeric interface resulted in the destabilization of the dimeric interface and partial conversion of the dimeric protein to a monomeric protein. However, the monomeric species of EcDAPALW399R was unable to bind PLP and hence did not possess any catalytic activity. This highlights the importance of dimeric organization for efficient binding of PLP as well as for the activity of the enzyme. A remarkable difference between EcDAPAL and StDAPAL is the absence of a disulfide bond between residues Cys271 and Cys299 in StDAPAL equivalent to the bond formed between Cys265 and Cys291 in EcDAPAL. Mutation of Cys265 and Cys291 of EcDAPAL to Ser did not affect the activity of the enzyme towards either of the isomers of the substrate indicating that the disulfide bond is not crucial for enzyme activity. The stability of the loop corresponding residues 261-295 of EcDAPAL was believed to be promoted by the disulfide bond. However, the equivalent loop was found to be ordered in StDAPAL even though the disulfide bond is absent. In contrast to StDAPAL, EcDAPAL did not show any metal dependent activity. The previous two chapters dealt with fold type II PLP-dependent enzymes. In contrast, Chapter 5 deals with revisiting the structure and function of a fold type I PLP-dependent enzyme, Salmonella typhimurium arginine decarboxylase (StADC). ADC is a very large polypeptide in comparison with other fold type I enzymes. It is induced when the bacterium is subjected to low pH and plays a major role in protecting the cells from acid stress. The structure of StADC was determined but not satisfactorily refined by Dr. S. R. Bharat earlier. The X-ray diffraction data collected by Bharat needed to be improved and the structure needed to be further refined and compared with the homologous E. coli enzyme. Therefore, the entire process of data processing, structure solution and refinement was repeated. The refined structure of StADC was found to correspond to the apo form of the enzyme with only a phosphate molecule occupying the position equivalent to that of 5’ phosphate of PLP observed in EcADC holo enzyme structure. This allowed examination of structural changes that accompany PLP binding and formation of an internal aldimine. The apo to holo transition in StADC involves the movement and ordering of two loops consisting of residues 151-164 and 191-196 which are in the linker and PLP binding domains of the protein, respectively. Phosphate binding by itself appears to be insufficient for these structural changes. These two loops are close to the PLP binding site of the other protomer of the dimer. Hence, these movements are probably important for the catalytic function of the enzyme. Holo ADC has been found as a decamer in other studies. The decameric form of the apo-StADC suggests that PLP binding may not be essential for the oligomeric state of the protein. ADC appears to reduce proton concentration inside the cell in two ways; (i) by surface charge neutralization and (ii) by arginine decarboxylation by extracting a proton from the cytoplasm. The resulting product agmatine is exchanged for extra cellular arginine by arginine-agmatine antiporter. The low sequence identity and lack of structural similarity of the inducible and constitutive forms of ADC from S. typhimurium shows that these are unlikely to be products of divergent evolution. The final chapter 6 of the thesis presents the work carried out on S. typhimurium pyridoxal kinase (PLK). In the salvage pathway of pyridoxal 5’phosphate (PLP), PLP is produced as the product of the reaction catalyzed by PLK using PL, PN and PM as substrates. Thus, PLK plays the critical role of ensuring availability of PLP to the large number of PLP-dependent enzymes. S. typhimurium PLK was purified to homogeneity, crystallized in its native as well as ligand bound forms. It was necessary to circumvent an unusual problem caused by spots arising from a contaminant crystal to obtain the structure of the native crystals of PLK that belonged to the P212121 space group with two protomers in the crystal asymmetric unit. It was then straight forward to determine the ligand bound structures of StPLK (space group P43212) obtained by co-crystallization with ATP, PL and Mg2+ by molecular replacement using the wild type structure as the phasing model. The structures obtained by co-crystallization revealed the presence of ADP, Mg2+ and a PL bound to the active site Lys233 via a Schiff base (internal aldimine). This is the first structure in which the presence of an internal aldimine in the active site of PLK has been observed. Formation of the internal aldimine might be one way to prevent the release of excess PLP and protecting the cell from PLP induced toxicity. The enzyme was shown to be inhibited by the product which will also help in maintaining PLP concentration at low levels. It was also demonstrated that PLK interacts with apo-PLP-dependent enzymes. This observation supports possible direct transfer of PLP from PLK to PLP-dependent enzymes. The thesis ends with an appendix where the work carried out during the course of the thesis work but not as part of the thesis is briefly described.

Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-10-11T18:07:04Z No. of bitstreams: 1 DissCDSJ.pdf: 5508281 bytes, checksum: 494e75225dc01ef99c7d2a4685a0f5a7 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-17T18:15:48Z (GMT) No. of bitstreams: 1 DissCDSJ.pdf: 5508281 bytes, checksum: 494e75225dc01ef99c7d2a4685a0f5a7 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-17T18:15:57Z (GMT) No. of bitstreams: 1 DissCDSJ.pdf: 5508281 bytes, checksum: 494e75225dc01ef99c7d2a4685a0f5a7 (MD5) / Made available in DSpace on 2016-10-17T18:23:05Z (GMT). No. of bitstreams: 1 DissCDSJ.pdf: 5508281 bytes, checksum: 494e75225dc01ef99c7d2a4685a0f5a7 (MD5) Previous issue date: 2016-02-24 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Phenylalanine ammonia lyase (PAL) catalyzes the non-oxidative deamination of L-Phe. It is an important enzyme in the production of enantio-pure mixtures of phenylalanine. Besides being important in plant secondary metabolism, PAL has also been used both for industrial applications as in the treatment of leukemia and phenylketonuria. The PAL have high similarity with histidine ammonia lyase (HAL) and it is believed that the PAL originated from HAL. Use of HAL termini sequences, previously obtained from the metagenome of Poraquê Lake (03°57' S, 63°10' W) to produce an engineered enzyme. The HAL assembled from them had its catalytic core exchanged by one which was designed from the previously described PAL sequences. Thus, We aim to shift the HAL activity making it active in the presence of phenylalanine through an easy protocol. Our phylogenetic analysis shows a clear division between plant and fungal PALs, and showed that recombination played a key role in the separation of these groups. In addition, We also observed that PAL catalytic core is conserved despite positive selection operating in protein termini. The recombinant enzyme, mPAL_c1, was produced in insoluble form and was refolded in vitro. mPAL_c1, when using L-Phe as substrate, has a TOPT. of 30°C (at pH 7.5 with 2mM MnCl2), KM of 55μM and Kcat/KM of 0.01633 mM-1s-1. The activity of mPAL_c1 showed about 30% of activity of commercial PAL from Rhodotorula toruloides at 2 mM L-Phe. Activation of mPAL_c1 was tested through substrate concentration rising from 2 mM to 5 mM of L-Phe, and its activity was almost 5 times higher. The results still suggest a histidine ammonia lyase remnant activity. The low catalytic rates are justified due to protein aggregation and misfolding, detected by size exclusion chromatography and by circular dichroism. In summary, our protocol has proved to be useful in protein design. / A fenilalanina amônia liase (PAL) catalisa a desaminação não oxidativa de L-Phe. Ela é uma enzima importante na produção de misturas enântio-puras de fenilalanina. Além de ser importante no metabolismo secundário vegetal, a PAL também tem sido utilizada tanto para aplicações industriais quanto no tratamento de leucemia e fenilcetonúria. As PAL apresentam alta similaridade com as histidina amônia liases (HAL) e acredita-se que as PAL se originaram das HAL. Utilizamos sequências das extremidades de uma HAL, previamente obtidas do metagenoma do Lago Poraquê (03°57' S e 63°10' W) para produção de uma enzima engenheirada. A HAL montada a partir delas teve seu núcleo catalítico trocado por um que foi desenhado a partir de sequências de PAL previamente descritas. Desta forma visamos deslocar as atividades da HAL tornando-a ativa em presença de fenilalanina por meio de um protocolo simplificado. As nossas análises filogenéticas revelam uma divisão clara entre PALs vegetais e fúngicas, e mostram que a recombinação teve um papel fundamental na separação destes grupos. Além disto, também observamos que o núcleo catalítico da enzima é conservado, com relação às extremidades. A enzima recombinante, mPAL_c1, foi produzida sob forma insolúvel e reenovelada in vitro. A mPAL_c1 tendo como substrato a L-Phe, possui uma Topt. de 30°C num pH de 7,5 com 2mM de MnCl2 e um KM de 55μM, VMáx de 15mU e Kcat/KM de 0,01633 mM-1s-1. A atividade da mPAL_c1 se mostrou cerca de 30% da atividade da PAL comercial de Rhodotorula toruloides a 2 mM de L-Phe. A ativação pelo substrato foi de quase 5 vezes quando a concentração de L-Phe foi elevada de 2mM até 5mM. Os resultados sugerem um resquício de atividade de histidina amônia-liase. As baixas taxas catalíticas se justificam devido à agregação e misfolding proteicos, detectados pela cromatografia de exclusão molecular e pelo dicroísmo circular. Por fim, o protocolo estabelecido neste estudo se mostrou útil para a engenharia de proteínas.

Le récepteur hépatique aux lipoprotéines LSR est impliqué dans la clairance des lipoprotéines riches en triglycérides telles que les résidus de chylomicrons pendant la phase postprandiale. La réduction de l’expression du LSR chez la souris (LSR+/-) est associée à une dyslipidémie et une lipémie postprandiale élevée. Afin de mieux comprendre la régulation de la distribution des lipides alimentaires, nous avons cherché quels étaient les facteurs pouvant affecter le niveau protéique de LSR. La leptine, hormone sécrétée par le tissu adipeux et connue pour son action d’hormone de satiété au niveau du système nerveux central, a été démontrée dans cette thèse comme modulant l’expression de LSR par la régulation de la transcription du gène lsr. La leptine est impliquée dans la régulation de la lipogénèse à travers SREBP-1. Grâce à l’utilisation d’un extrait de Garcinia cambogia contenant un inhibiteur de l’ATP citrate lyase, nous avons démontré une interaction importante entre les enzymes lipogéniques, l’expression de LSR et d’autres récepteurs lipoprotéiques, afin de maintenir un équilibre entre la synthèse de lipides endogènes et l’apport alimentaire de lipides exogènes. Soumises à un régime hyperlipidique, les souris sauvages montrent une diminution de l’expression des enzymes lipogéniques hépatiques, aggravée chez les souris LSR+/-. Ces résultats indiquent qu’il existe un mécanisme de maintien de l’équilibre entre la lipogénèse (synthèse endogène de lipides), la lipolyse (utilisation lipidique comme substrat énergétique) et le stockage de lipides à travers une forte interaction entre les enzymes lipogéniques et LSR. / The hepatic lipoprotein receptor LSR is involved in the clearance of triglyceride-rich lipoproteins including chylomicrons remnants during the post-prandial phase. Reduced LSR protein expression in mice (LSR+/-) is associated with dyslipidemia and increased postprandial lipemia; these mice exhibit increased weight gain with aging or when placed under a high-fat diet. In order to better understand the regulation of the distribution of dietary lipids, we looked for factors that could regulate LSR protein levels. Leptin is a hormone secreted by the adipose tissue that is a centrally-acting satiety factor, and was demonstrated to modulate LSR mRNA and protein expression through the modulation of transcription of the gene lsr. Leptin has been reported be involved in the control of lipogenesis through SREBP-1c. Using Garcinia cambogia extract containing an inhibitor of ATP citrate lyase, we demonstrated that there is an important link between lipogenic enzymes and LSR protein levels and with other lipoprotein receptors that provides the means to maintain a balance between endogenous lipid synthesis and dietary intake of exogenous lipids. When exogenous lipid intake is increased in the form of a high-fat diet, mice exhibited a decrease in hepatic lipogenic enzymes expression, but a deficiency of LSR led to increased lipid content in the peripheral tissues. These results suggest the presence of mechanisms for the maintenance for the balance between lipogenesis (de novo endogenous lipid synthesis), lipolysis (lipids used as energy substrate), and lipid storage through an important link between lipogenic enzymes and LSR.

No description available.

Hydrogen sulfide (H2S) is receiving increasing interest, as much as nitric oxide (NO) and carbon monoxide have received previously, to understand its physiological functions as it meets all the criteria to define as a third gasotransmitter. Endogenous synthesis from L-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) and the function of H2S as an inhibitor of smooth muscle contraction in gastrointestinal tract are known. However, the loci of generation and action of H2S, and the mechanism of inhibition of contraction are unknown. Hence, my aims in the present study are to: i) identify the expression of enzymes in smooth muscle, ii) determine the effects of endogenously released and exogenously applied H2S on smooth muscle function; and iii) identify the targets and mechanism involved in mediating the effects of H2S using isolated smooth muscle cells from rabbit colon. I have identified the expression of CSE, but not CBS, in smooth muscle and demonstrated that L-cysteine (an activator of CSE) and NaHS (H2S donor): 1) inhibited carbachol-induced contraction in muscle strips and isolated muscle cells that was independent of KATP channels, a known S-sulfhydration target of H2S; 2) induced S-sulfhydration of small G protein, RhoA leading to inhibition of RhoA and Rho kinase activities, a key pathway in the sustained smooth muscle contraction; and 3) inhibited PDE5 activity leading to augmentation NO-induced cGMP formation and muscle relaxation. Sodium nitroprusside (an NO donor) induced an increase in H2S production via PKG-dependent phosphorylation and activation of CSE. We conclude that smooth muscle cells selectively express CSE, and endogenous generation of H2S via activation of CSE inhibits muscle contraction and augments muscle relaxation. Inhibition of contraction is mediated via S-sulfhydration of RhoA and suppression of RhoA/Rho kinase pathway. Augmentation of relaxation is mediated via inhibition of PDE5 activity and stimulation of cGMP/PKG pathway, which in addition initiates generation of H2S via PKG-mediated phosphorylation and activation of CSE. The findings are important in providing the underlying mechanisms involved in the regulation of smooth muscle function by H2S and could offer insights for the development of therapeutic agents that may act on smooth muscle in the gut to treat motility disorders.

Thesis advisor: Thomas C. Chiles / Normal B lymphocytes require extrinsic factors to grow and proliferate. Surface receptors (e.g., B-cell antigen receptor, BCR) function, in part, to link growth factors to signal transduction/metabolic pathways and the cell cycle machinery. Accumulating evidence indicates that signal transduction-dependent changes in both glucose energy metabolism and de novo transcription of the D-type cyclin-cdk4/6 pathway are necessary for quiescent B-lymphocytes to enter G1-phase of the cell cycle and grow. B cell growth represents a critical checkpoint for subsequent proliferation and clonal expansion of antigen-specific lymphocytes. On the former, we have shown earlier that acquisition of extracellular glucose and metabolism via the glycolytic pathway is required for conventional splenic B-2 lymphocytes to grow (i.e., increase cell size and mass) in response to antigen challenge; however, the metabolic fate and biological significance of glucose-derived carbons are unknown. Here, we show that in response to BCR ligation, glucose carbon flow is directed into a de novo lipogenic pathway that is regulated, in part, via phosphoinositide-3 kinase (PI-3K)-dependent activation of ATP citrate lyase (ACL), a key rate-limiting enzyme in de novo lipogenesis. Inhibition of ACL results in a loss of B-cell growth and cell viability. Regarding the latter point, the B-1a lymphocyte subset expresses cyclins D2 and D3 that are transiently expressed in a non-overlapping manner, notably cyclin D3 expression immediately precedes the G1/S phase transition, suggesting distinct functions for these D-type cyclins in B-1a lymphocyte G0-to-S phase progression. We show herein that murine B-1a cells deficient in cyclin D3 proliferate normally in response to extracellular stimuli, in part, due to a compensatory sustained up-regulation of cyclin D2. In keeping with this, human diffuse large B-cell lymphoma (DLBCL) represents a malignant clonal expansion of B cells characterized by several subsets, including germinal center (GC) and activated B-cell (ABC) types. Here, we show that the GC-type LY18 human DLBCL exhibits constitutive expression of cyclin D3, but not cyclins D1 and D2. Targeting of cyclin D3-holoenzyme complexes with cell permeable chemical- and peptide-based cdk4 inhibitors results in G1-phase arrest and apoptosis via a pathway that involves inhibition of pRb phosphorylation. By contrast, endogenous knock down of cyclin D3 with siRNA did not induce growth arrest or apoptosis, in part, due to redundancy with cyclin E. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

O gene TrCit do fungo filamentoso Trichoderma reesei codifica a proteína citrato sintase, uma enzima chave do ciclo de Krebs. Análise da região 5´ upstream de TrCit mostra que o gene está adjacente ao gene TrIcl (que codifica a proteína isocitrato liase, uma enzima do ciclo de glioxalato), em uma orientação cabeça-cabeça. A região promotora intergênica de 647 pb rica em G + C, apresenta uma ilha CpG, seqüência INR, caixas GC, caixas CAAT, sítios de ligação para diversos fatores de transcrição e é isenta de caixa TATA. O gene TrCit de 1573 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1422 pb produz uma proteína de 474 aminoácidos, com um peso molecular estimado de 52,3 kD. O gene TrIcl de 1880 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1788 pb produz uma proteína de 596 aminoácidos, com um peso molecular estimado de 65,4 kD. A atividade transcricional da região promotora foi analisada utilizando como repórter o gene de higromicina B fosfotransferase (hph). Uma região funcional necessária à transcrição de ambos os genes foi identificada na região central do promotor e contém uma caixa GC que liga o putativo fator de transcrição Sp1 de T. reesei (TrZnFSp1). O gene do putativo fator de transcrição “zinc-finger" TrZnFSp1 de 1500 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1344 pb produz uma proteína de 448 aminoácidos, com um peso molecular estimado de 48,4 kD. Os resultados mostram que ambos os genes são transcritos de forma divergente a partir de um promotor bidirecional que compartilha na região central uma caixa GC, necessária para a transcrição de ambos os genes. / The TrCit gene from the filamentous fungus Trichoderma reesei codes for the citrate synthase protein, a key enzyme in the Krebs cycle. Analysis of TrCit 5’ upstream region showed that it is adjacent to the TrIcl gene that codes for isocitrate lyase protein, an enzyme involved in the glyoxylate cycle. Both genes, on a head-to-head orientation, are separated by an intergenic GC-rich and TATA-less promoter region of 647 base pairs. This bidirectional promoter has diverse cis regulatory elements: a CpG island, two INR sequences, GC boxes, CAAT boxes and several putative interaction sites for different transcription factors. The TrCit gene, 1,573-base pair-long, has an open reading frame of 1,422 base pairs interrupted by two introns. The gene codes for a protein with an estimated molecular weight of 52.3 kD. The TrIcl gene, 1,880-base pair-long, contains 3 exons and 2 introns and a putative coding sequence of 1,788 base pairs. The estimated molecular weight of TrICL is 65.4 kD. he transcriptional activity of the intergenic promoter region was analyzed using hygromicin B phosphotransferase (hph) as a reporter gene. A functional region required for the transcription of both genes was identified in the centre of this promoter. It has a GC box that interacts with a putative transcription factor Sp1 from T. reesei (TrZnFSp1). The results presented in this work show that both genes are divergently transcribed from a bidirectional promoter that shares an essential central GC box.

Introdução: A acidemia 3-hidroxi-3-metilglutárica é causada pela deficiência da 3-hidroxi-3-metil-glutaril-CoA-liase, uma enzima do metabolismo da leucina, levando ao acúmulo, especialmente, do ácido 3-hidroxi-3-metilglutárico nos tecidos. Estudos sugerem que o estresse oxidativo pode contribuir para os danos neurológicos observados em algumas acidúrias orgânicas. Objetivo: Avaliar parâmetros de estresse oxidativo em pacientes com acidúria 3-hidroxi-3-metilglutárica antes e após o tratamento. Materiais e Métodos: Amostras de sangue e urina foram coletadas de pacientes no momento do diagnóstico e após tratamento com dieta com restrição de proteínas e suplementação de L-carnitina (100mg/kg/dia) e de controles. O TBA, um subproduto final da peroxidação lipídica, foi medido no plasma. A determinação do teor de carbonilas e de grupos sulfidrila, marcadores de dano oxidativo a proteínas, foi realizada no plasma. Para avaliar na urina a oxidação de proteínas, os níveis de di-tirosina foram medidos por autofluorescência. O ensaio da capacidade antioxidante urinária foi realizado utilizando um kit comercial. Os níveis de carnitina livre e isovalerilcarnitina foram analisados em amostras de sangue por espectrometria de massas em tandem usando o método de monitorização de reação múltipla (MRM). A concentração de proteínas foi determinada pelo método de biureto em amostras de plasma usando um kit comercial. Resultados e Discussão: Os resultados demonstraram um aumento significativo nos níveis de isovalerilcarnitina em sangue total, das concentrações plasmáticas de malondialdeído e urinárias de di-tirosina, além de uma redução significativa da capacidade antioxidante urinária e dos níveis sanguíneos de carnitina livre nos pacientes no momento do diagnóstico em relação aos controles. Verificou-se uma diminuição nas concentrações do malondialdeído plasmático e da di-tirosina na urina dos pacientes tratados, o que sugere um efeito de proteção do tratamento sobre a peroxidação de lípidos e do dano oxidativo a proteínas, bem como uma normalização dos níveis de L-carnitina durante o tratamento. Conclusões: Esses resultados permitem sugerir que o estresse oxidativo ocorre em pacientes com acidemia 3-hidroxi-3-metilglutárica e que o tratamento com a dieta restrita de proteína e suplementada com L-carnitina pode oferecer proteção contra o dano oxidativo a biomoléculas. / Introduction: The 3-hydroxy-3-methylglutaric acidemia is caused by the deficiency of 3-hydroxy-3-methyl-glutaryl-CoA lyase, an enzyme of leucine metabolism, leading to accumulation of 3-hydroxy-3-methylglutaric acid in tissues. Studies have suggested that oxidative stress may contribute to the neurological damage observed in some organic acidurias. Objective: Evaluate oxidative stress parameters in patients with 3-hydroxy-3-methylglutaric aciduria patiets before and after treatment. Materials and Methods: Blood and urine samples were collected from patients at diagnosis and after treatment with restricted protein diet and supplemented with L-carnitine (100mg/kg/dia) and from controls. TBA , an end subproduct of lipid peroxidation, was measured in plasma. Determination of carbonyl and sulphydryl content, biomarkers of oxidative damage to proteins, was done in plasma. To assess urine protein oxidation, levels of di-tyrosine were measured by autofluorescence. The assay of antioxidant urinary capacity was performed using a commercial kit. The levels of free carnitine and isovalerylcarnitine were analyzed in blood samples by tandem mass spectrometry using the method of multiple reaction monitoring (MRM). Protein content was determined by the biuret method for plasma samples using a commercial kit. Results and Discussion: The results demonstrated a significant increase of total blood isovalerylcarnitine, malondialdehyde plasma concentrations and di-tyrosine urinary levels and a significant reduction of the urinary antioxidant capacity and free-carnitine blood levels in pacients at diagnosis compared to controls. It was verified a decrease in plasma malondialdehyde concentrations and urinary di-tyrosine levels in treated patients, suggesting a protective effect of the treatment on lipid peroxidation and protein oxidative damage, as well as a normalization of L-carnitine levels during treatment. Conclusions: These results allow to suggest that oxidative stress occurs in 3-hydroxy-3-methyl-glutaryl-CoA lyase deficient patients and treatment with restricted protein diet and L-carnitine may offer protection against oxidative damage.

Cyanobacteria are photosynthetic prokaryotes that able to produce oxygen. They have light harvesting complexes called phycobilisomes (PBS). PBS are generally composed of an allophycocyanin core with phycocyanin and phycoerythrin rods connected to the core. PBS are able to efficiently harvest light energy from different wavelengths of visible light due to the evolution of PBP. Phycoerythrin has five chromophores that are attached to six cysteine residues and is essential for efficient green light capture and transfer of energy for use in photosynthesis. The attachment of these chromophores to PBP is facilitated by enzymes known as bilin lyases. In this study, we characterize and explore the role of enzymes that are involved in the biosynthesis of phycoerythrin in cyanobacteria. Biochemical and molecular techniques were used in the characterization of these proteins to gain a better understanding of their roles in the post-translational modification of phycobiliprotein. In F. diplosipohon, the lyase activity of CpeT was characterized and studied using a heterologous, co-expression system in E. coli. It was determined that CpeT was able to ligate PEB to Cys-165 of CpeB in the presence of CpeZ, a chaperone-like protein. Next, the roles of three proteins, MpeY from RS9916 and MpeQ and MpeW from A15-62, were analyzed using a combination of gene-interruption mutants and recombinant protein expression techniques. The absence of mpeY resulted in the reduction of PEB chromophorylation of MpeA in green light conditions, and recombinant protein coexpression confirmed that MpeY was responsible for PEB attachment to Cys-83 of MpeA. The interruption of mpeQ in A15-62 resulted in a reduced PUB phenotype in MpeA in blue light. Recombinant protein expressions revealed that MpeQ was a lyase-isomerase responsible for the attachment of PUB to Cys-83 of MpeA. Two regulatory proteins located in two conserved configurations of a genomic island present in species that are able to change their phycobilin content in response to different light environments, known as Type-IV chromatic acclimation (CA4), were investigated. FciA and FciB from RS9916 were studied using gene interruption mutants from RS9916 and they were found to be responsible for the CA4 response in CA4-A containing species of Synechococcus.