Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Sequence analysis showed that the essential glnA1 gene of Mycobacterium tuberculosis might be closely related to an actinomycetes progenitor sequence and that this sequence may have undergone duplication into other non-essential GS encoding genes in some Actinobateria, notably the mycobacteria. Also, the M. tuberculosis glnA1 sequence remains conserved throughout the evolution of M. tuberculosis. It was also shown that glnA1 is widely expressed in M. tuberculosis infected human pulmonary tissue, where M. tuberculosis might be present in altered phenotypes. These results imply that glnA1 is under selective pressure against evolutionary change. At transcriptional level it was shown that M. tuberculosis glnA1 might be expressed from two alternate promoter sites, but that these promoter sites may not be controlled by environmental nitrogen (in the form of ammonium) variation. We also showed that M. tuberculosis GS is effectively exported by M. smegmatis to the cell wall, but that GS secretion into the exogenous environment does not occur. Also, evidence has been presented which suggested that M. tuberculosis GS might be modified at the C-terminus, probably as part of a mechanism that retains GS in the cytosol. This hypothesis was further substantiated where it was demonstrated that two GS isoforms of different size (short isoform in cytosol, longer isoform in cell wall) are present in M. bovis BCG. It is unknown what causes this modification, since it couldn’t be observed in M. smegmatis, but it was suggested that it might be through the action of a cis- or trans-acting element present in proximity of the M. tuberculosis glnA1 gene. It was also shown that a cluster of genes found immediately downstream of the M. tuberculosis glnA1 sequence might be regulated in an operonic fashion under conditions of elevated environmental nitrogen concentrations. Two of the genes (glnE and glnA2) in this operon arrangement have been previously shown to be involved in nitrogen and glutamine metabolism. The function of the other gene, Rv2223c, is unknown. It was shown that Rv2223c homologs are mostly found in the mycobacteria and that it may encode an exported protease. It was hypothesised that this sequence and its adjacently located progenitor sequence, Rv2224c, might be involved in M. tuberculosis GS mediated metabolism. It was showed that over-expression of Rv2223c and Rv2224c may be toxic to E. coli and mycobacterial hosts, such as M. smegmatis, but that inhibition of transcription of these genes may be fatal to M. bovis BCG and M. tuberculosis H37Rv. It was also shown that Rv2223c is widely expressed in M. tuberculosis infected human tissue, which was comparable to that of glnA1. The results presented in this study shed more light on the distribution and transcriptional regulation of GS in mycobacteria and has identified new genes that might be involved in GS regulation. These results may present new approaches to tuberculosis control and thereby contribute to alleviate the burden of the disease. / AFRIKAANSE OPSOMMING: Genetiese en proteien volgorde analise het aangedui dat die glnA1 (kodeer vir glutamien sintetase (GS), ‘n essentiele protein) geen van Mycobacterium tuberculosis die naaste verwant is aan ‘n actinomycetes voorloper volgorde wat duplikasie ondergaan het om die ander nieessensiele GS koderende gene in sommige Actinobakterieë te vorm, veral in die mikobakterieë. Voords het die glnA1 geen geneties gekonserveerd gebly gedurende die evolusie van M. tuberculosis. Dit is ook aangetoon dat volop transkribasie van die glnA1 geen voorkom in die M. tuberculosis geïnfekteerde pulmonêre weefsel waar M. tuberculosis moontlik mag voorkom. Op transkriptionele vlak is dit aangetoon dat die M. tuberculosis glnA1 geen vanaf twee onderskeie promotors uitgedruk mag word, maar dat hierdie twee promotors nie deur variasies in die konsentrasie van stikstof (in die vorm van ammonium) in die omgewing beïnvloed word nie. Daar is ook aangedui dat M. tuberculosis GS effektief deur M. smegmatis oor die selmembraan na die selwand vervoer word, maar dat daar nie GS sekresie na die ekstrasellulêre omgewing geskied nie. Ook is bewyse gevind dat M. tuberculosis GS modifikasie aan die C-terminus mag ondergaan wat waarskynlik dien om GS beweging uit die sitosol te verhoed. Hierdie hipotese is versterk deurdat twee isoforms van verskillende grootte (klein in sitosol en groter in die selwand) gevind is in M. bovis BCG. Dit modifikasie meganisme is onbekend, maar vind moontlik nie plaas in nie-patogeniese mikobakterieë soos M. smegmatis nie en mag moontlik deur cis- of trans-werkende elemente gefasiliteer word. Daar is aangedui dat ‘n groepering van vier gene lanksaan die glnA1 lokus in ‘n operoniese meganisme gereguleer mag word onder variërende konsentrasies van stikstof in die omgewing. Dit is bekend dat twee van die gene in die operon (glnE en glnA2) betrokke in stikstof en gultamien metabolisme is. Die funksie van die ander twee gene (Rv2223c en Rv2224c) is onbekend. Daar is aangetoon dat volgordes soortgelyk aan Rv2223c beperk is tot die mikobakterieë en dat die geen ‘n protease, wat moontlik gesekreteer word vanuit die sitosol, kodeer. Daar is aangetoon dat die oor-produksie van die Rv2223c en Rv2224c proteine toksies is vir E. coli en mikobakterieë, soos M. smegmatis, maar dat transkripsie inhibisie hierdie gene dodelik is vir M. tuberculosis H37Rv en M. bovis BCG. Daar is ook angedui dat die ekspresie van hierdie gene volop verspreid is in M. tuberculosis geïnfekteerde menslike weefsel, soortgelyk aan diè van glnA1. Die resultate vervat in hierdie studie werp meer lig op die verspreiding en transkiptionele regulasie van GS in mikobakteriee en nuwe gene is ontdek wat betrokke by GS regulasie mag wees. Hierdie resultate mag bydra tot nuwe maniere om tuberkulose te bekamp en daardeur die voorkoms van die siekte te beperk.

In addition to its canonical role in aminoacylation, threonyl-tRNA synthetase (TARS) possesses pro-angiogenic activity that is susceptible to the TARS-specific antibiotic borrelidin. However, the therapeutic benefit of borrelidin is offset by its strong toxicity to living cells. The removal of a single methylene group from the parent borrelidin generates BC194, a modified compound with significantly reduced toxicity but comparable anti-angiogenic potential. Biochemical analyses revealed that the difference in toxicities was due to borrelidin's stimulation of amino acid starvation at ten-fold lower concentrations than BC194. However, both compounds were found to inhibit in vitro and in vivo models of angiogenesis at sub-toxic concentrations, suggesting a similar mechanism that is distinct from the toxic responses. Crystal structures of TARS in complex with each compound indicated that the decreased contacts in the BC194 structure may render it more susceptible to competition with the canonical substrates and permit sufficient aminoacylation activity over a wider concentration of inhibitor. Conversely, both borrelidin and BC194 induce identical conformational changes in TARS, providing a rationale for their comparable effects on angiogenesis. The mechanisms of TARS and borrelidin-based compounds on angiogenesis were subsequently tested using zebrafish and cell-based models. These data revealed ectopic branching, non-functional vessels, and increased cell-cell contracts following BC194-treatment or knockdown of TARS expression, suggesting a role for the enzyme in the maturation and guidance of nascent vasculature. Using various TARS constructs this function was found to be dependent on two interactions or activities associated with the TARS enzyme that are distinct from its canonical aminoacylation activity. Furthermore, observations that TARS may influence VEGF expression and purinergic signaling suggest the possibility for a receptor-mediated response. Taken together, the results presented here demonstrate a clear role for TARS in angiogenesis, independent of its primary function in translation. Although the exact molecular mechanisms through which TARS and borrelidin regulate this activity remain to be determined, these data provide a foundation for future investigations of TARS's function in vascular biology and its use as a target for angiogenesis-based therapy.

Cinco membros da família das Acil-CoA sintetases de cadeia longa (ACSL) são responsáveis por ativar ácidos graxos, produzindo acil-CoA, e distribuí-los entre diversas vias metabólicas no interior da célula, tais como a síntese de triacilglicerol (TAG) e ?- oxidação mitocondrial. Apesar das disfunções nas ACSLs contribuirem para muitas doenças metabólicasa função de algumas isoformas de ACSL em tecidos específicos permanece ainda sem descrição na literatura. Aqui mostramos pela primeira vez a presença de mRNA da ACSL6 no músculo esquelético de seres humanos. Além disso, indivíduos obesos apresentaram menores níveis de mRNA de ACSL6 quando comparados à indivíduos magros. Após refeição hiperlipídica aguda (high fat meal, HFM, 90% de gordura) a expressão ACSL6 aumentou 2,5 vezes em relação aos níveis de jejum. Nós também verificamos as condições metabólicas que controlam a expressão ACSL6 em ratos: o jejum de 48h modulou negativamente a expressão gênica de ACSL6 e de outros genes de síntese de lipídeos tais como SREBP-1c e DGAT1, enquanto que a ingestão aguda de HFM (80% de gordura saturada , 10 mL/kg) teve o efeito oposto; Após o treinamento aeróbio (6 semanas, 5 dias /semana, uma vez por dia, 60 min a 70% da capacidade aeróbica máxima) o mRNA da ACSL6 foi reduzido em 35%. Em células primárias de músculo esquelético de ratos, a transfecção com siRNA de ACSL6 diminuiu a expressão de ACSL6, DGAT1 e SREBP-1c e o acúmulo de TAGs e gotas lipídicas. O silenciamento gênico da ACSL6 também aumentou o conteúdo dos ácidos graxos C16:0 e C18:0, AMPK-fosforilada, capacidade respiratória mitocondrial, a oxidação de palmitato e mRNA de PGC-1?, UCP2 e UCP3, mas diminuiu a produção de espécies 11 reativas de oxigênio. Em células primárias de músculo esquelético de seres humanos, a superexpressão da ACSL6 não alterou o conteúdo de TAG e da proteína DGAT1, mas aumentou as espécies lipídicas esfingomielina e fosfatidilcolinas, e reduziu a oxidação de 1-14C-palmitato e a expressão do PGC1?. Em conclusão, ACSL6 está envolvida na síntese e distribuição de acil-CoA para a síntese de lipídeos. A inibição gênica da ACSL6 melhora a capacidade de respiração mitocondrial e oxidação lipídica, através da ativação da via AMPK/PGC1?. / Five members of long-chain acyl-CoA synthetase (ACSL) family activate fatty acids providing acyl-CoA for several metabolic pathways within the cell, such as synthesis of triacylglycerol (TAG) and mitochondrial ?-oxidation, and their dysfunctions contribute to many metabolic diseases. Despite this, the existence and function of some ACSL isoforms in specific tissues remains unclear. Here we show for the first time the presence of ACSL6 mRNA and protein in skeletal muscle (SM) of humans. Obese subjects had lower levels of ACSL6 mRNA when compared to leans, and acute high fat meal (HFM, 90% fat) increased ACSL6 expression 2.5 times over fasted levels in both. We also verify the metabolic conditions that control ACSL6 expression in rats: fasting (48h) negatively modulated the ACSL6 mRNA and the expression of other genes of lipid synthesis SREBP-1c and DGAT1 in rat SM, while acute ingestion of HFM (80% saturated fat, 10 mL/Kg) had the opposite effect; After aerobic training (6 weeks, 5 days/week, once a day, 60 min at 70% of maximal aerobic capacity) ACSL6 mRNA was reduced 35%. In primary skeletal muscle cells (PSMC) of rats, ACSL6-specific siRNA oligo transfection (20 nM) decreased ACSL6, DGAT1 and SREBP-1c mRNA and the accumulation of TAGs and lipid droplets (LD). The knockdown also increased the content of C16:0 and C18:0 fatty acids, AMPK-Phosphorylated, mitochondrial content and respiratory rates, palmitate oxidation and PGC-1?, UCP2 and UCP3 mRNA, but decreased reactive oxygen species production. In PSMC of humans, ACSL6 overexpression did not change the contents of TAG or DGAT1 mRNA, but increased sphingomyelin and phosphatidylcholines and reduced 14C-palmitate oxidation and PGC1? mRNA expression. In conclusion, ACSL6 drives acyl-CoA toward lipid synthesis and its 13 downregulation improves mitochondrial capacity of respiration, lipid oxidation and biogenesis, which involves the activation of AMPK/PGC1-? pathway.

A principal forma biológica do selênio em vários organismos é o aminoácido Selenocisteína (Sec, U), que é incorporado em um polipeptídio emergente em códons UGA específicos. Em Escherichia coli, esta incorporação requer os genes que codificam para Seril-tRNA Sintetase (SerRS), Selenocisteína Sintase (SELA), um tRNASec específico (SELC), Selenofosfato Sintetase (SELD) e um fator de elongação de transcrição específico (SELB). A proteína Selenofosfato Sintetase (EC 2.7.9.3) pertence à família AIRS, de proteínas que têm o ATP como substrato, e produz o composto biologicamente ativo doador de selênio, o monoselenofosfato, a partir de ATP e seleneto. O gene selD em E. coli tem 1041 pares de bases e codifica uma proteína com 347 aminoácidos e massa molecular de 37 kDa. A fase aberta de leitura do gene selD foi amplificada do DNA genômico de E. coli e clonada em vetor de expressão pet28a(+) (Novagen). A proteína recombinante foi superexpressa em E. coli por indução com IPTG e purificada por cromatografia de afinidade por ligação a metal e a fração eluída foi concentrada por ultrafiltração. Em seguida, o produto foi submetido à clivagem da cauda de histidinas com Trombina. Para purificar o produto de reação de clivagem com protease e para estimar sua massa molecular e estado oligomérico, empregou-se cromatografia de exclusão molecular. A proteína pura foi utilizada em experimentos de Gel Nativo e em estudos das suas propriedades hidrodinâmicas realizados por meio de Espalhamento Dinâmico de Luz (DLS), Espalhamento de Raios-X a Baixo Ângulo (SAXS) e Ultracentrifugação Analítica (AUC). Os resultados obtidos revelam uma mistura de oligômeros em solução, em um equilíbrio dímero-tetrâmero e tetrâmero-octâmero. Um modelo tridimensional para o homodímero de SELD de E. coli foi obtido por Modelagem Molecular e suas propriedades hidrodinâmicas preditas concordam com aquelas obtidas experimentalmente. Adicionalmente, triagens de condições de cristalização da proteína revelaram condições em que a proteína cristaliza na forma de pequenas agulhas e ensaios de otimização por variação da concentração de agente precipitante e pH não resultaram em monocristais adequados para difração de raios-X. A análise do papel da SELD na via de biossíntese de Selenocisteínas levanta a hipótese de que esta proteína deve entregar o monoselenofosfato para o complexo SELA-SELC de modo que o selênio seja incorporado para formação do aminoácido Selenocisteína, já que os compostos de selênio são tóxicos quando estão livres na célula. Portanto, a investigação da interação da SELD com o complexo SELA-SELC foi observada pelo monitoramento da anisotropia de fluorescência do complexo SELA-SELC mediante titulação de SELD. A análise local da interação para manutenção do complexo SELD-SELA-SEC foi feita por meio de espectrometria de massas com troca H/D, que revelou possíveis sítios de interação na superfície da SELD. Os resultados mostrados neste trabalho ampliam o conhecimento sobre a via de biossíntese de Selenocisteína, revelando detalhes da interação da SELD com o complexo SELA-SELC. / The main biological form of selenium in several organisms is the amino acid Selenocysteine (Sec, U), which is incorporated into selenoproteins in specific UGA codons. In Escherichia coli, it requires the genes that codify to Seryl-tRNA Synthetase (SerRS), Selenocysteine Synthase (SELA), a specific tRNASec (SELC), Selenophosphate Synthetase (SELD) and a specific translation elongation factor (SELB). Selenophosphate Synthetase (EC 2.7.9.3) belongs to AIRS superfamily of proteins that have ATP as a substrate and this protein produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide. The selD gene from E. coli is 1041 base pairs long and codifies a protein with 347 amino acids and molecular mass of 37 kDa. The open reading frame of selD gene was amplified from E. coli genomic DNA and cloned into pET28a(+) expression vector (Novagen). The recombinant protein was overexpressed in E. coli by IPTG induction and purified by metal affinity chromatography, and the eluted fraction was concentrated by ultrafiltration. The product was used for Thrombin protease cleavage of the 6-His tag. In order to purify the product of proteolysis and to estimate its molecular mass and oligomeric state, we used size exclusion chromatography. The pure protein sample was used for Native Gel Electrophoresis. Hydrodynamic properties of the protein were studied by Dynamic Light Scattering (DLS), Small angle X-ray scattering (SAXS) and Analytical Ultracentrifugation (AUC). The results show an equilibrium between SELD oligomeric forms, as dimer-tetramer and tetramer-octamer association in solution. A tridimensional model of E. coli SELD was obtained by Molecular Modelling and its predicted hydrodynamic properties agree with those observed experimentally. In addition, crystal screening revealed crystallization conditions suitable for protein crystallization as small needles, but optimization of these conditions by precipitant agent and pH variation did not result in monocrystals reliable for X-ray diffraction. An analysis of SELD´s role in the Selenocysteine biosynthesis pathway indicates that SELD must deliver monoselenophosphate to the SELA-SELC complex so that the selenium is incorporated to the amino acid to form selenocysteyl-SEC, since selenium compounds are toxic when they are freely available in the cell. This interaction was observed by fluorescence anisotropy. The local analysis of complex formation was monitored by mass spectrometry after H/D exchange and revealed possible sites for this interaction on SELD surface. The results improve our knowledge about the Selenocysteine pathway in the cell, showing details of the interaction between SELD and the SELA-SELC complex.

Fosforribosil pirofosfato (PRPP) sintetases são enzimas de central importância em diversas vias metabólicas em todas as células. A enzima PRPP sintetase humana é constituída por um complexo composto de três subunidades catalíticas (PRSI, PRSII e PRSIII) e por proteínas homólogas de 39 e 41 kDa denominadas PRS Associated Proteins (PAPs) cuja função é desconhecida. A importância da PRPP sintetase em humanos, tem sido documentada pela identificação de uma desordem associada ao cromossomo X, resultando em uma atividade aumentada da PRPP sintetase.Em conseqüência se percebem concentrações elevadas na dosagem de ácido úrico, purino nucleotídeos levando ao desenvolvimento de patogenias como a gota e problemas neurológicos. Nesse sentido, realizaram-se estudos moleculares com o complexo enzimático que compõem as PRPP sintetases. Foram obtidos clones do gene hprsI em vetor de expressão pET29a(+) e a enzima foi expressa em bactérias Escherichia coli cepa BL21 (DE3). A proteína recombinante hPRSI foi purificada depois de fracionamento com sulfato de estreptomicina, sulfato de amônio e em coluna cromatográfica de troca iônica. O raio hidrodinâmico e o pI da enzima foram determinados usando respectivamente a técnica de espalhamento dinâmico de luz e o sistema Fast de eletroforese. A enzima foi caracterizada quanto ao seu enovelamento através da técnica de Dicroísmo Circular, apresentando um espectro característico de estrutura enovelada, composta predominantemente por a-hélices. Os genes hprsII e hpap4l-1 que codificam respectivamente para as proteínas hPRSI1 e HPAP41-1 foram clonados no vetor de clonagem pCR4-TOPO. A proteína recombinante hPAP39 foi clonada em vetor pMAL-c2X em fusão com a proteína Maltose Binding Protein (MBP) e expressa em E. coli. A proteína hPAP39 está em fase de purificação e foi submetida ao experimento de Imunoblotting. Investigações estruturais destas enzimas poderão trazer informações fundamentais para o conhecimento da via biossintética, assim como para o desenvolvimento de inibidores específicos visando o tratamento das desordens a elas associadas. / Phosphoribosyl pyrophosphate (PRPP) synthetases are enzymes of central importance in several metabolic pathways in all cells. The enzyme PRPP synthetase complex is composed of three catalytic subunitis (PRSI, PRSII and PRSIII) and homologous 39 and 41 kDa proteins termed PRPP synthetase-Associated Proteins (PAPs) which function is unknown. The importance of PRPP synthetase function in humans has been documented by the identification of an X chromosome-linked disorder associated with super activity of PRPP synthetase. As a consequence uric acid overproduction, purine nucleotide are observed resulting in the development of diseases such as gout and neurodevelopment impairment. In this line, molecular studies were done with the enzyme complex that constitutes the PRPP synthetases. Clones were obtained from the hprsI gene in the pET29a(+) expression vector and the enzyme was expressed in Escherichia coli BL21(DE3) bacterial. The recombinant human PRSI enzyme was purified, after streptomicine and ammonium sulfate fractionation and by anion exchange chromatography. The hPRSI hydrodynamic radius and pI were determined using, respectively, measures of Dynamic Light Scattering (DLS) and isoeletrophocusing electrophoresis. In addition, according to circular dichroism spectroscopy, hPRSI prevalent secondary structure is a-helix. The hprsí and hpap41-1 genes that codify, respectively, to hPRSII and hPAP41-1 proteins were cloned in pCR4-TOPO cloning vector. The recombinant protein hPAP39 was cloned in the pMAl-c2X expression vector in fusion with the Maltose Binding Protein (MBP) and expressed in E.coli. A purification protocol is been establish for the hPAP39 protein and is submitted by imunoblotting technique. Structural investigation of these enzymes will provide information about the biosynthetic pathway de novo of purine nucleotides, as well as to development of specific inhibitors aiming at the treatment of the associated disorders.

Nos organismos eucariotos, aproximadamente 50% das proteínas traduzidas no citoplasma são transportadas para as organelas, onde irão desempenhar suas funções. Com isso, surgiu um intricado sistema de transporte intracelular de proteínas. Nas plantas, a presença de uma segunda organela endossimbionte, o plastídio, tornou este sistema mais complexo e gerou demanda adicional por transporte. Ainda, grande maioria das proteínas mitocondriais e plastidiais são codificadas por genes nucleares e importadas do citosol. O dogma uma proteína-uma localização foi associado ao conceito de um gene-uma proteína na biologia celular. Entretanto, proteínas individuais podem ter mais de uma função, e mais recentemente, proteínas codificadas por um único gene foram identificadas em mais de um compartimento subcelular, o que deu origem ao conceito de duplo direcionamento (DD). Um exemplo bem estudado de DD vem das proteínas da família das aminoacil-tRNA sintetases (aaRS), que participam da síntese protéica ao acoplar o aminoácido ao seu tRNA cognato. Dentre as aaRSs, a glutamil-tRNA sintetase citosólica (GluRS), através de sua extensão N-terminal, parece estar envolvida com outras funções além da tradução. Em Arabidopsis thaliana, há dois genes nucleares que codificam a GluRS, um para uma proteína de duplo direcionamento (DD) e outro para uma proteína citosólica. Resultados recentes em nosso laboratório mostraram que a GluRS citosólica pode estar relacionada ao controle da localização subcelular de proteínas organelares em Arabidopsis. Para verificar um eventual papel desta proteína na localização subcelular de outras proteínas, foram realizados ensaios de duplo-híbrido em levedura, os quais mostraram interação entre a GluRS e a glutamina sintetase (GS) de Arabidopsis thaliana, proteína de DD para mitocôndrias e cloroplastos Esta interação foi confirmada in planta, sendo a sequência da GluRS responsável pela interação localizada na região N-terminal, do resíduo 207 ao 316. Análises filogenéticas apontam que esta região encontra-se ausente nas bactérias e que originou-se provavelmente em Archea, entre 2,6 e 1,8 bilhões de anos. Além disso, observa-se que esta sequência é conservada em fungos, musgos e plantas vaculares, tendo originado-se em Arabidopsis há cerca de 2 bilhões de anos. / In eukaryotic organisms, about 50% of cytoplasmic translated proteins are transported to the organelles, where they can play their roles. Thus, a complex system for intracellular transport was established. In plants, the presence of a second endosymbiont organelle, the plastid, turned this system still more intricated and required an additional transport mechanism. Besides, most of organellar proteins are coded by nuclear genes and imported from the cytosol. The one protein-one localization was associated to the idea of one gene-one protein, which has long been established in molecular biology. However, individual proteins can show more than one function, and recently, proteins coded by one single gene were identified in more than one subcellular compartment, which has originated the concept of dual targeting. One of the most studied example of dual targeted proteins is the aminoacyl-tRNA synthetase (aaRS) family, which are related to protein synthesis by attaching the correct amino acid onto the cognate tRNA molecule. Among the aaRSs, cytosolic glutamyl-tRNA synthetase (GluRS), through its N-terminal extension, seems to be involved in other cellular role beyond translation. In Arabidopsis thaliana, there are two genes encoding GluRS, one for a dual-targeted protein and other for a cytosolic protein. Recent results in our laboratory showed that GluRS interacts with proteins destinated to other organelles, which suggest that this protein might have a role in interfering on protein localization in Arabidopsis. In order to gain some information on the role of this protein in subcellular localization, yeast two-hybrid assays were performed. These studies showed the interaction between GluRS and glutamine synthetase (GS), a mitochondrial and chloroplastic dual-targeted protein. This interaction was confirmed in planta. In addition, the GluRS sequence associated to protein interaction was localized at its N-terminal portion, between the residues 207 316. Phylogenetic analysis revealed that this region is absent in bacteria and it probably arose from Archea between 2.6 and 1.8 billion years ago. Also, this sequence is conserved in fungi, moss and all the green plants investigated. Finally, datation analysis showed that this sequence arose in Arabidopsis between 2 and 1.7 billion years ago.

A regulação da localização de proteínas é um dos aspectos fundamentais na biologia celular vegetal. Os cloroplastos importam mais de 90% de suas proteínas do citosol, portanto, é importante caracterizar os fatores citosólicos que podem estar envolvidos no direcionamento de proteínas para as organelas. Um ensaio de duplohíbrido em leveduras com as proteínas cloroplastidiais HMPPK/TMPPase (TH1) e Glutamina Sintetase (GS) II usados como iscas revelou que a forma citosólica da glutamil-tRNA sintetase - GluRS (At5g26710) de Arabidopsis thaliana interagiu com ambas as proteínas. Estudos de Complementação da Fluorescência Bimolecular (BiFC) confirmaram tais interações in planta. Estudos com deleções na região Nterminal da GluRS mostraram que esta região é responsável pelas interações com HMPPK/TMPPase e GSII. Além disso, seis resíduos de aminoácidos parecem ser cruciais para a interação entre as proteínas. Curiosamente, foi mostrado que a GluRS está envolvida na localização de proteínas em leveduras. A fim de obter mais informações sobre o envolvimento da GluRS ns localização de proteínas nos cloroplastos, foram produzidos plantas de tabaco transgênicas expressando uma proteína quimérica, feita pela fusão do gene codificador da HMPPK/TMPPase, TH1- GFP, e GSII-GFP e posteriormente usados em ensaios de agroinfiltração com RNA de interferência (RNAi) para GluRS. Análises em microscópio confocal mostraram que TH1-GFP e GSII-GFP acumulam no citosol em vez de serem direcionados aos cloroplastos. Neste trabalho, mostramos pela primeira vez que a GluRS está envolvida na localização de proteínas cloroplastidiais em plantas e esse mecanismo é também conservado em Saccharomyces cerevisiae. / Regulation of protein localization is one of the key aspects in plant cell biology. Chloroplasts import more than 90% of their proteins from the cytosol, therefore, it is important to identify and characterize cytosolic factors that might be involved in protein delivery to the organelar envelope. A yeast two-hybrid screen with a chloroplastlocalized HMPPK/TMPPase protein and glutamine synthetase (GS), used as baits, revealed that the cytosolic form of the glutamyl-tRNA synthetase (GluRS) (At5g26710) from Arabidopsis thaliana interacted with both proteins. Bimolecular Fluorescence Complementation (BiFC) studies confirmed such interactions in planta. Deletion studies of GluRS showed that the N-terminal region of the protein is responsible for proteinprotein interactions (PPI) with TH1 and GS. In addition, six amino acid residues appeared to be crucial for PPI. Interestingly, GluRS has been also shown to be involved in regulating protein localization in yeast. In order to gain more information about the involvement of GluRS on protein localization in chloroplasts, we produced transgenic tobacco plants expressing a chimeric protein made by the fusion of TH1- GFP and GSIIGFP and agroinfiltrated with a RNA interference (RNAi) construct against GluRS. Confocal analysis showed that TH1-GFP and GSII-GFP accumulated in the cytosol instead of being targeted to chloroplasts. Here, we show for the same time that GluRS is involved in protein localization in plants and this mechanism is also conserved in Saccharomyces cerevisiae.

O espaço intergênico (IGS) juntamente com suas subunidades flanqueadoras (cpcB) e (cpcA) do operon do ficocianina foi usado para identificar linhagens de cianobactérias. Dentro do domínio Bacteria somente as cianobactérias possuem o operon da ficocianina e a região cpcBA-IGS é suficientemente variável para diferenciar linhagens desses microrganismos. No presente estudo 25 linhagens de cianobactérias isoladas de diversos locais brasileiros foram caracterizadas usando a seqüência cpcBA-IGS. DNA genômico foi extraído das ordens Chroococcales (oito linhagens), Oscillatoriales (duas linhagens), Nostocales (onze linhagens) e Stigonematales (quatro linhagens). Os oligonucleotídeos iniciadores PCβF/PCαR, específicos para a seqüência cpcBA-IGS, foram usados para amplificar fragmentos de DNA de aproximadamente 685 pb. Os produtos da PCR foram clonados, seqüenciados e as seqüências foram comparadas pela análise BLAST. Todas as seqüências de Microcystis e também as seqüências de Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 e Gloeotrichia UFV-B2 mostraram identidades com seqüências do GenBank. Entretanto, nenhuma identidade foi encontrada para as seqüências restantes. As relações filogenéticas das seqüências de cpcBA-IGS foram investigadas junto com outras seqüências de cianobactéria do Genbank usando a análise “Neighbour Joining". A topologia da árvore foi congruente com outras árvores de cianobactérias, com exceção de todas as seqüências sem identidades no GenBank, as quais formaram um agrupamento separado. Os dados das seqüências de cpcBA-IGS analisadas confirmam que as cianobactéria heterocitadas formam um grupo monofilético. Estudos anteriores realizados com linhagens de cianobactéria mostraram que estes microrganismos são uma fonte rica de produtos naturais. No presente estudo conduzido com 59 linhagens de cianobactérias, sendo a maioria isolada de ambientes brasileiros, isto foi confirmado. Para alcançar esse objetivo, dois conjuntos de iniciadores degenerados foram usados para produzir seqüências amplificadas por PCR das sintetases de peptídeos não-ribossômicos (NRPSs), e de sintases policetídeos (PKSs) modulares, as quais são enzimas multifuncionais envolvidas na produção de produtos naturais. O sistema híbrido NRPS/PKS também foi amplificado por PCR usando uma combinação de iniciadores de NRPS e de PKS. Essa abordagem molecular mostrou a presença de genes de NRPS e de PKS em 93% e 81% linhagens de cianobactérias, respectivamente. Genes de NRPS/PKS foram encontrados em 87% das cianobactérias examinadas. Numa tentativa de atribuir funções a oito fragmentos de PKS identificados por PCR, estas seqüências foram clonadas, seqüenciadas e analisadas filogeneticamente. As seqüências de PKSs da Microcystis aeruginosa NPCD1 e Fischerella CENA62 mostraram correlação com a síntese de sideróforo e de microcistina, respectivamente. Todas as 59 linhagens foram analisadas para a produção do microcistinas e 20 linhagens apresentaram resultados positivos. Para a maioria das linhagens potencialmente produtoras de microcistinas os produtos de PCR esperados de NRPS, PKS e NRPS/PKS foram amplificados. A produção de sideróforos foi testada em 28 linhagens e somente cinco produziram resultados positivos. Em três linhagens produtoras de sideróforos todos os três sistemas moleculares analisados estavam presentes. Estes resultados serão altamente valiosos na exploração futura de cada peptídeo dessas cianobactérias e para a elucidação da bioatividade de tais produtos naturais. / The intergenic spacer (IGS) together with its flanking subunits  (cpcB) and  (cpcA) of the phycocyanin operon has been used to identify cyanobacterial strains. Within the Bacteria domain only cyanobacteria present phycocyanin operon and the cpcBA-IGS region is variable enough to differentiate strains of these microorganisms. In the present study 25 cyanobacterial strains isolated from several Brazilian locations were characterized using the cpcBA-IGS sequence. Genomic DNA was extracted from the orders Chroococcales (eight strains), Oscillatoriales (two strains), Nostocales (eleven strains) and Stigonematales (four strains). The primers PCβF/PCαR targeting the cpcBA-IGS sequence were used to amplify DNA fragments of approximately 685 bp. The PCR products were cloned, sequenced and the sequences were compared by BLAST analysis. All Microcystis sequences and also sequences from Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 and Gloeotrichia UFV-B2 showed identities with sequences from GenBank. However, no identities were found for the remaining sequences. Phylogenetic relationships of the cpcBA-IGS sequences were investigated together with other cyanobacterial sequences from Genbank using the Neighbour Joining analysis. The tree topology was congruent with previous cyanobacterial trees, except for all sequences with no identities in the GenBank, which formed a separated cluster. The cpcBA-IGS sequences analysis data confirm that heterocyte-forming cyanobacteria are a monophyletic group. Previous studies carried out with cyanobacterial strains showed that these microorganisms are a rich source of natural products. This has been confirmed in the present study conducted with 59 cyanobacterial strains, with the majority of them isolated from Brazilian environment. To reach this goal, two sets of degenerate primers were used to generate PCR amplification sequences of nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), which are multifunctional enzymes implicated in natural products production. Also, NRPS/PKS hybrid system was PCR amplified by using a combination of NRPS and PKS primers. This molecular approach revealed the presence of NRPS and PKS genes in 93% and 81% cyanobacterial strains, respectively. NRPS/PKS genes were found in 87% of cyanobacteria examined. In an attempt to attribute functions to eight PCR identified PKS fragments, these sequences were cloned, sequenced and phylogenetically analyzed. PKSs sequences of Microcystis aeruginosa NPCD1 and Fischerella CENA62 showed correlation with the synthesis of siderophore and microcystin, respectively. All 59 strains were analyzed for microcystin production and 20 strains presented positive results. For the majority of potentially producing-microcystin strains expected PCR products of NRPS, PKS and NRPS/PKS were amplified. The siderophores production was tested in 28 strains and only five gave positive results. In three producing-siderophore strains all three molecular systems analyzed were present. These results will be highly valuable for further exploring each of these cyanobacterial peptides and for elucidating the bioactivity of such natural products.

Fosforribosil pirofosfato (PRPP) sintetases são enzimas de central importância em diversas vias metabólicas em todas as células. A enzima PRPP sintetase humana é constituída por um complexo composto de três subunidades catalíticas (PRSI, PRSII e PRSIII) e por proteínas homólogas de 39 e 41 kDa denominadas PRS Associated Proteins (PAPs) cuja função é desconhecida. A importância da PRPP sintetase em humanos, tem sido documentada pela identificação de uma desordem associada ao cromossomo X, resultando em uma atividade aumentada da PRPP sintetase.Em conseqüência se percebem concentrações elevadas na dosagem de ácido úrico, purino nucleotídeos levando ao desenvolvimento de patogenias como a gota e problemas neurológicos. Nesse sentido, realizaram-se estudos moleculares com o complexo enzimático que compõem as PRPP sintetases. Foram obtidos clones do gene hprsI em vetor de expressão pET29a(+) e a enzima foi expressa em bactérias Escherichia coli cepa BL21 (DE3). A proteína recombinante hPRSI foi purificada depois de fracionamento com sulfato de estreptomicina, sulfato de amônio e em coluna cromatográfica de troca iônica. O raio hidrodinâmico e o pI da enzima foram determinados usando respectivamente a técnica de espalhamento dinâmico de luz e o sistema Fast de eletroforese. A enzima foi caracterizada quanto ao seu enovelamento através da técnica de Dicroísmo Circular, apresentando um espectro característico de estrutura enovelada, composta predominantemente por a-hélices. Os genes hprsII e hpap4l-1 que codificam respectivamente para as proteínas hPRSI1 e HPAP41-1 foram clonados no vetor de clonagem pCR4-TOPO. A proteína recombinante hPAP39 foi clonada em vetor pMAL-c2X em fusão com a proteína Maltose Binding Protein (MBP) e expressa em E. coli. A proteína hPAP39 está em fase de purificação e foi submetida ao experimento de Imunoblotting. Investigações estruturais destas enzimas poderão trazer informações fundamentais para o conhecimento da via biossintética, assim como para o desenvolvimento de inibidores específicos visando o tratamento das desordens a elas associadas. / Phosphoribosyl pyrophosphate (PRPP) synthetases are enzymes of central importance in several metabolic pathways in all cells. The enzyme PRPP synthetase complex is composed of three catalytic subunitis (PRSI, PRSII and PRSIII) and homologous 39 and 41 kDa proteins termed PRPP synthetase-Associated Proteins (PAPs) which function is unknown. The importance of PRPP synthetase function in humans has been documented by the identification of an X chromosome-linked disorder associated with super activity of PRPP synthetase. As a consequence uric acid overproduction, purine nucleotide are observed resulting in the development of diseases such as gout and neurodevelopment impairment. In this line, molecular studies were done with the enzyme complex that constitutes the PRPP synthetases. Clones were obtained from the hprsI gene in the pET29a(+) expression vector and the enzyme was expressed in Escherichia coli BL21(DE3) bacterial. The recombinant human PRSI enzyme was purified, after streptomicine and ammonium sulfate fractionation and by anion exchange chromatography. The hPRSI hydrodynamic radius and pI were determined using, respectively, measures of Dynamic Light Scattering (DLS) and isoeletrophocusing electrophoresis. In addition, according to circular dichroism spectroscopy, hPRSI prevalent secondary structure is a-helix. The hprsí and hpap41-1 genes that codify, respectively, to hPRSII and hPAP41-1 proteins were cloned in pCR4-TOPO cloning vector. The recombinant protein hPAP39 was cloned in the pMAl-c2X expression vector in fusion with the Maltose Binding Protein (MBP) and expressed in E.coli. A purification protocol is been establish for the hPAP39 protein and is submitted by imunoblotting technique. Structural investigation of these enzymes will provide information about the biosynthetic pathway de novo of purine nucleotides, as well as to development of specific inhibitors aiming at the treatment of the associated disorders.

Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the oxazole-containing phthoxazolins and the bis(benzoxaozle) AJI9561, were investigated. Phthoxazolins A-D are a group of oxazole trienes produced by a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway in Streptomyces sp. KO-7888 and Streptomyces sp. OM-5714. The phthoxazolin pathway was used as a model to study 5-oxazole and primary amide formation in PKS-NRPS pathways. An unusually large gene cluster for phthoxazolin biosynthesis was identified from the complete genome sequence of the producer strains and various gene deletions were performed to define the minimal gene cluster. PhoxP was proposed to encode an ATP-dependent cyclodehydratase for 5-oxazole formation on an enzyme-bound N-formylglycylacyl-intermediate, and its deletion abolished phthoxazolin production. In vitro reconstitution of the early steps of phthoxazolin biosynthesis was attempted to validate the role of PhoxP, but was unsuccessful. Furthermore, Orf3515, a putative flavin-dependent monooxygenase coded by a remote gene, was proposed to hydroxylate glycine-extended polyketide-peptide chain(s) at the α-position to yield phthoxazolins with the primary amide moiety. On the other hand, an in vitro approach was employed to establish the enzymatic logic of the biosynthesis of AJI9561, a bis(benzoxazole) antibiotic isolated from Streptomyces sp. AJ9561. The AJI9561 pathway was reconstituted using the precursors 3-hydroxyanthranilic acid and 6-methylsalicylic acid and five purified enzymes previously identified from the pathway as key enzymes for benzoxazole formation, including two adenylation enzymes for precursor activation, an acyl carrier protein (ACP), a 3-oxoacyl-ACP synthase and an amidohydrolase-like cyclase. Intermediates and shunt products isolated from enzymatic reactions containing different enzyme and precursor combinations were assessed for their competence for various steps of AJI9561 biosynthesis. Further bioinformatic analysis and in silico modelling of the amidohydrolase-like cyclase shed light on the oxazole cyclisation that represents a novel catalytic function of the amidohydrolase superfamily.