Regulação transcricional de genes de hidrolases em fungos filamentosos : sistemas PacC/CreA em humicola grisea var. thermoidea e sistema Xyr1 em trichoderma reesei

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular.O pH ambiental é um sinal importante para fisiologia de fungos, intervindo na regulação da transcrição de vários genes. Em fungos filamentosos e leveduras, PacC é um importante fator transcricional que regula a expressão de genes tanto em pH ácido como alcalino. A produção de enzimas envolvidas na bioconversão de biomassa vegetal é regulada principalmente no nível da transcrição. No entanto, o envolvimento da via de regulação por pH na expressão de enzimas lignocelulolíticos não tem sido extensivamente estudado. Nosso grupo havia demonstrado anteriormente que o deuteromiceto termofílico Humicola grisea var. thermoidea é um potente produtor de celulases, apresentando um considerável potencial biotecnológico para bioconversão de resíduos agrícolas. Nossos resultados sustentam a existência de uma via de regulação por pH para as glicosil hidrolases em H. grisea. Neste trabalho, foram realizadas análises quantitativas por RT-PCR em tempo real para vários transcritos de H. grisea. O perfil transcricional de oito genes codificadores de enzimas lignocelulolíticas (cbh1.1, cbh1.2, egl1, egl2, egl3, egl4, bgl4 e xyn1) e de dois fatores de transcrição (pacC e creA) foi avaliado na presença de glicose ou de bagaço de cana-de-açúcar em condições de pH ácido e alcalino. A análise por qRT-PCR revelou uma indução forte e precoce de quase todos os genes de enzimas lignocelulolíticas, de uma maneira sinérgica quando o fungo foi cultivado com a fonte de carbono complexa e em condições alcalinas (pH 8,0). A única exceção foi egl4, que foi induzido por pH ácido. Um padrão oposto para a expressão dos dois fatores transcricionais foi observado. Enquanto pacC foi induzido em condições alcalinas e fortemente reprimido na presença de glicose, creA foi induzido por glicose e reprimido em condições alcalinas. Os dados de perfil de transcrição, combinados com a análise da ligação in vitro de PacC e CreA aos promotores dos genes analisados, apóiam a repressão por carbono, mediada por CreA, e a via de regulação por pH, mediada por PacC em H. grisea. Além disso, ensaios de mobilidade eletroforética (EMSAs) com o promotor de pacC mostraram que, in vitro, PacC e CreA competem pelo mesmo sítio de ligação. Tomados em conjunto, estes dados corroboram as hipóteses de que PacC possa ser o mediador da regulação influenciada pelo pH e de que a repressão por glicose, possivelmente atribuída a CreA, é capaz de sobrepujar a ação indutória do pH alcalino. Temos ainda evidências de que CreA também esteja envolvido na repressão de PacC, estabelecendo uma interação entre os dois sistemas regulatórios. O foco do segundo capítulo desse trabalho foi a produção e caracterização de Xyr1 (regulador de xilanases 1), que é o principal fator transcricional que atua na regulação da expressão dos genes codificadores de xilanases em Trichoderma reesei. Os resultados apresentados mostraram a bem sucedida produção heteróloga da versão completa da proteína Xyr1, algo até então inédito entre reguladores de xilanases em fungos filamentosos. Nossas análises empregando técnicas de interação DNA-proteína e géis nativos indicaram que Xyr1 pode naturalmente formar homodímeros em solução. Contudo, a dimerização não é fundamental para a interação com o DNA, o que implica em uma dinâmica complexa de ligação ao promotor do gene xyn1. São possíveis as formas de monômero, dímero e interação com ambos os motivos do sítio palindrômico. A capacidade de dimerização do fator fortalece a proposição de modulação adicional da atividade de Xyr1 por meio de possíveis interações proteína-proteína com outros fatores transcricionais, sendo candidatas as proteínas Ace1 e Ace2. O papel de modificações pós-traducionais tem sido apontado como crucial na regulação mediada por Xyr1. Mostramos que a fosforilação possivelmente está envolvida na regulação da atividade de Xyr1, sendo necessária para a interação DNA-proteína in vitro, mas sem afetar a formação de dímeros. Apresentamos ainda indícios de modulação alostérica de Xyr1 que depende tanto da interação com o DNA quanto, possivelmente, com carboidratos, o que torna mais complexo este modelo de regulação. _______________________________________________________________________________________ ABSTRACTEnvironmental pH is an important signal in fungal physiology, acting at transcriptional regulation of several genes. In filamentous fungi and yeasts, the PacC zinc-finger transcription factor regulates gene expression in response to changes of external pH. The production of enzymes involved in plant cell wall breakdown is regulated mainly at the transcriptional level. Nonetheless, the involvement of the pH-related regulatory pathway in the lignocellulolytic enzymes expression has not been extensively studied. We have demonstrated that the thermophilic deuteromycete Humicola grisea var. thermoidea is a potent cellulases producer, presenting a considerable potential for agricultural wastes bioconversion processes. Previous results of our group support the existence of a pH regulatory pathway in H. grisea var. thermoidea. In this work, we have performed a time course transcription analysis of several H. grisea genes by quantitative real time RT-PCR. Eight enzyme genes (cbh1.1, cbh1.2, egl1, egl2, egl3, egl4, bgl4 and xyn1), and two transcription factors genes (pacC and creA) were analyzed in the presence of simple (glucose) or complex (sugarcane bagasse) carbon source, in acid and alkaline medium conditions. The qRT-PCR analyses revealed an early and strong induction of almost all glycoside hydrolase genes transcription, in a synergistic way, when the mycelia were grown in the presence of the complex carbon source, under alkaline conditions (pH 8.0). The only exception was egl4, which was acid-induced. An opposite pattern was observed for the expression of the two transcription factors. While pacC was induced in alkaline conditions and strongly repressed in presence of glucose, creA was induced by glucose and repressed in alkaline conditions. The transcriptional profile data combined with the analysis of the in vitro binding of PacC and CreA transcription factors to the promoters support the CreA-mediated carbon repression and the PacC-related pH regulation of H. grisea cellulase and xylanase encoding genes. Moreover, electrophoretic mobility shift assays (EMSAs) employing the upstream regulatory sequences of pacC showed that an in vitro interaction occurs between the proteins PacC and CreA on the pacC upstream regulatory sequence, with both factors competing for the same binding site. Taken together, this data corroborate our previous evidences, supporting the existence of a pH transcriptional regulatory pathway in H. grisea, mediated by PacC. Moreover, PacC is probably transcriptionally auto-regulated and may be subject to the carbon repression mechanism mediated by CreA. The second chapter of this work describes the production and characterization of Xyr1 (xylanase regulator 1), which is the main transcription factor that acts regulating the expression of xylanase encoding genes in Trichoderma reesei. The results showed the successful heterologous production of the full version of Xyr1, something never previously described for xylanases regulators in filamentous fungi. Our analyses employing techniques of DNA-protein xviii interactions and native gels indicated that Xyr1 can form homodimers in solution. Nevertheless, dimerization is not essential for the interaction with DNA, suggesting a complex binding dynamics to the xyn1 promoter region (as monomer, dimer and interacting with both palindromic binding sites). The dimerization capacity of Xyr1f strengthens the proposition of an additional modulation for this factor activity, probably by protein-protein interactions with other transcription factors, such as Ace1 and Ace2. A crucial role for post-translational modifications was proposed for gene regulation mediated by Xyr1. We initially showed that phosphorylation has a possible role in the regulation of Xyr1 activity, being necessary for the DNA-protein interactions in vitro, but not affecting the dimer formation. Additionally, we present evidence suggesting an allosteric modulation of Xyr1, dependent on interactions, both with DNA and carbohydrates, which adds an additional degree of complexity to this regulatory model

An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50 ∘ C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55 ∘ C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as = 27.5 ± 4.33 mg/mL, max = 1.185 ± 0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Produção de proteínas de interesse terapêutico em células de mamíferos em cultura

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2006.As proteínas recombinantes de interesse terapêutico vêm ganhando cada vez mais espaço na indústria farmacêutica e atualmente já movimentam um mercado anual de cerca de 50 a 60 bilhões de dólares em todo o mundo. As células de mamíferos são as hospedeiras de expressão preferencialmente escolhidas no caso de proteínas que requerem um grau sofisticado de processamento pós-traducional, sendo crescente a iniciativa de identificação de novas linhagens de células, especialmente humanas, como sistemas alternativos de expressão às células utilizadas. Nosso grupo de pesquisa tem interesse na produção de antígenos para seleção de anticorpos com potencial neutralizante, especialmente os antígenos de superfície do envelope viral de HIV-1, agente etiológico da pandemia mundial de AIDS, que atualmente apresenta mais de 40 milhões de infectados. O presente trabalho teve por objetivo a avaliação preliminar das células de ducto de glândula submandibular humana (HSG) como sistema de expressão heteróloga alternativo às células de ovário de hamster chinês (CHO-K1). Comparativamente, foi avaliada a eficiência de transfecção, assim como a de expressão transiente do anticorpo quimérico anti-Z-DNA Z22, na forma recombinante de fragmento FvFc pelas duas linhagens celulares. Outro objetivo foi a produção de versões recombinantes das glicoproteínas virais de HIV-1. Os resultados apontaram as células HSG como um bom sistema alternativo para a produção de proteínas heterólogas secretadas, especialmente quando transfectadas por co-precipitação com fosfato de cálcio, sendo ainda necessários alguns ajustes, uma vez que os choques osmóticos com glicerol e DMSO, considerados pontencializadores da transfecção, mostraram-se tóxicos da forma como foram executados. Foram amplificados e clonados em vetor de expressão para células de mamíferos os segmentos gênicos correspondentes a quatro versões recombinantes das glicoproteínas do envelope viral de HIV-1 (gp160, gp140, gp120 e gp41+PS), subtipo C que, de acordo com as nossas análises, utiliza CCR5 como co-receptor. Até o presente momento, não foi possível a detecção das glicoproteínas recombinantes, expressas de forma transiente em células CHO-K1, sendo necessários ajustes, principalmente na etapa de transfecção. _______________________________________________________________________________ ABSTRACTRecombinant therapeutic proteins have become more and more important in the pharmaceutical industry, and nowadays they are responsible for an injection of about 50 to 60 million dollar a year into the worldwide market. Animal cell cultures are the preferential expression systems for those proteins which require extensive posttranslational modifications. In this view, the identification of alternative expression systems is an issue of increasing concern, specially considering human cell lines. Our research group has been interested in the production of antigens to be used for the selection of neutralizing antibodies, particularly those antigens derived from the envelope surface of HIV-1, the etiologic agent of the pandemic infection of AIDS, which nowadays affects more than 40 million people. This work aimed the preliminary evaluation of the human salivary gland duct cells (HSG) as a heterologous expression system alternative to the Chinese hamster ovary cells (CHO-K1). The transfection efficiency for both cell lines was comparatively evaluated, as well as the transient expression of the anti-Z-DNA Z22 chimeric antibody, as a recombinant FvFc fragment. Another objective was the production of recombinant versions of HIV-1 glycoproteins. Our results pointed out to the HSG cells as a good alternative system for the production of secreted heterologous proteins, specially when transfected by co-precipitation with calcium phosphate. Some adjusts are still needed, considering that the glycerol and DMSO osmotic shocks, generally considered as transfection pontentializers, proved to be toxic in the employed protocol. The genic fragments corresponding to four recombinant versions of the HIV-1 envelope glycoproteins (gp160, gp140 gp120 and gp41+PS), subtype C, were amplified and cloned in a mammal cells expression vector. According to our analysis, this virus subtype uses CCR5 as co-receptor. So far, it was not possible to detect the recombinant glycoproteins expressed in a transient form in the CHO-K1 cells. In order to achieve this objective, some adjustments are still necessary, specially concerning the transfection protocol

Welchen Einfluss haben Chromatin Remodellierungen auf die Expression von Genen aus dem Xyr1 Regulon von Trichoderma reesei

Zusammenfassung in deutscher SpracheAbweichender Titel nach Übersetzung der Verfasserin/des VerfassersDer Ascomycet Trichoderma reesei wird für die Produktion von Zellulasen und Hemizellulasen im industriellen Bereich verwendet. Die Expression der Gene, die diese Enzyme kodieren, wurde bisher hauptsächlich auf Ebene der Transkriptionsregulation durch regulierende Proteine erforscht, insbesondere die Transaktivatoren Xyrl (Xylanase Regulator 1) und der Repressor Cre1(Carbon Katabolit Repressor 1), deren Zusammenspiel hauptsächlich die Expression dieser Enzyme reguliert. Den Auswirkungen von Chromatin-Remodellierung, d.h. der dynamischen Modifikation der Chromatin-Architektur um den Zugang der regulatorischen Proteine an die genomische DNA zu ermöglichen, auf die Genexpression in T. reesei wurde bisher kaum Aufmerksamkeit geschenkt. Einer der wichtigsten Stämme vom T. reesei ist RUT-C30 ein hyperzellulolytischer Mutant, auf dem die meisten Stämme für die industrielle Herstellung von Enzymen, insbesondere Zellulasen, basieren. Ein wichtiges genetisches Merkmal von RUT-C30 ist eine teilweise Deletion des Gens cre1, durch die der Stamm partiell von Kohlenstoff Katabolit Repression (KKR) freigesetzt wird. Diese Deletion wird als Hauptgrund für den herausragenden Phänotyp dieses Stammes angesehen. Es ist jedoch noch unklar, was genau die Zellulaseproduktion in RUT-C30 verbessert. Diese Forschungsarbeit bringt neue Erkenntnisse über die Genexpression von Zellulasen und Hemizellulasen bezogen auf den Chromatin-Zugang von Cre1 und Xyr1. Sie erforscht weiters die Verkürzung von Cre1 in RUT-C30 als eine zusätzliche verstärkende Eigenschaft dieses Stammes, die über einfache Befreiung von KKR hinausgeht. Die hier vorgestellten Ergebnisse weisen darauf hin, dass die Verkürzung von Cre1 zu Cre1-96 in Rut-C30 einen positiven regulatorischen Einfluss auf die Expression hat. Sie wirkt möglicherweise nicht nur in direkter Weise auf den Promotor von Zielgenen, sondern trägt durch die Regulierung eines Gens, das ein Protein zur Chromatin-Remodellierung kodiert, auch indirekt zu einem offeneren Chromatin-Status bei. Andererseits beeinflusst Xyr1 auch die Chromatin-Verpackung. Chromatin-Zugänglichkeit Echtzeit-PCR in Kombination mit Transkriptanalyse ergab, dass Xyr1 für die volle Induktion von Zellulase-kodierenden Genen durch Sophorose erforderlich ist und, dass höhere Genexpressionen mit Chromatin-Öffnung einhergehen. Darüber hinaus offenbart RNA-seq Analyse, dass Gen-Produkte, die an Histon-Acetylierung und ATP-abhängiger Chromatin-Remodellierung beteiligt sind, auch Chromatin Öffnung während Sophorose-Induktion beeinflussen. Schließlich wurde der Promotor von xyr1 auch als Ziel von Chromatin-Remodellierung enthüllt. Chromatin-Umlagerung erfolgt im xyr1 Promotor während der Induktion durch Sophorose. Diese Umlagerung erfolgt vor der Aktivierung von Zellulase Genexpression und die Zugänglichkeit des Promoters in einem Cre1-freien Hintergrund ist insgesamt höher, egal welche Kohlenstoffquelle vorhanden ist. Zusammenfassend ist die Regulierung der Genexpression von Zellulasen und Hemizellulasen in T. reesei nicht nur auf die Wirkung von Transkriptionsfaktoren beschränkt, sondern steht offensichtlich auch in Beziehung zu Änderungen in der Chromatin-Verpackung.The ascomycete Trichoderma reesei is used for the production of plant cell wall-degrading enzymes (PCWDEs) in industrial scale. The expression of the PCWDE-encoding genes has been so far primarily investigated on the level of transcriptional regulation by regulatory proteins, especially the transactivator Xyr1 and the repressor Cre1. The interplay between these two transcription factors mainly regulates the expression of PCWDEs. Otherwise, the impact of chromatin remodeling, i.e. the dynamic modification of chromatin architecture to allow the regulatory proteins access to genomic DNA, on gene expression in T. reesei has received hardly any attention so far. One of the most important T. reesei strains, RUT-C30, is a hypercellulolytic mutant that became the ancestor of most industry strains used in the production of enzymes, in particular cellulases. One important genetic trait of RUT-C30 is a partial deletion of the gene cre1, which releases the strain from carbon catabolite repression (CCR). This deletion has been considered the main reason for the outstanding phenotype the strain presents. However, it is still unclear, what exactly enhances cellulase production in RUT-C30. This thesis presents new insights on the regulatory mechanism behind gene expression of PCWDEs, especially concerning the role of Cre1 and Xyr1 in chromatin access. It also explores the truncation of Cre1 in RUT-C30 as an additional enhancing characteristic of this strain that goes beyond just simple CCR release. The results presented here point towards the fact that the truncated form of Cre1 of RUT-C30, Cre1-96, exerts a positive regulatory influence on the expression. It possibly acts in a direct manner on the promoter of target genes, but also contributes indirectly to a more open chromatin status by regulating a gene encoding a possible chromatin-remodeling protein. On the other hand, Xyr1 also influences the chromatin packing. Chromatin accessibility real-time PCR combined with transcript analysis showed that Xyr1 is required for the full induction of cellulase-encoding genes by sophorose and that higher gene expression overlaps with chromatin opening. Additionally, RNA-seq analysis revealed that gene products involved in histone acetylation and ATP-dependent chromatin remodeling may also influence chromatin opening during sophorose induction. Finally, the promoter region of xyr1 was also identified as a target of chromatin remodeling. Chromatin rearrangement occurs in the xyr1 promoter during induction by sophorose and it takes place prior to activation of cellulase gene expression. Also, xyr1 promoter accessibility is overall higher in a cre1-truncated background, no matter which carbon source is present. To sum up, the regulation of PCWDEs gene expression in T. reesei is not only restricted to the action of transcription factors, but is clearly related to changes in the chromatin packaging.5

Montagem de um modelo tridimensional de uma célula animal

Propõe a construção de um modelo tridimensional para visualização das estruturas de uma célula animalComponente Curricular::Ensino Médio::Biologi