Estudo da acetilação na regulação da enzima glicolítica aldolase de Trypanosoma brucei
Data
2019-06-18
Autores
Leite, Ariely Barbosa 
Orientadores
Moretti, Nilmar Silvio
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Modificações pós-traducionais reversíveis em proteínas são um meio relativamente rápido e econômico para que as células respondam a mudanças ambientais como temperatura e disponibilidade de nutrientes. A acetilação é uma dessas modificações pós-traducionais e ocorre através da transferência de um grupamento acetil do Acetil-CoA para cadeia Nε-amino dos resíduos de lisina, eliminando a carga positiva desse aminoácido. Recentemente, nosso laboratório descreveu o acetiloma das formas procíclicas e sanguíneas do parasito protozoário causador da Tripanossomíase Africana, Trypanosoma brucei, e verificamos que o perfil de acetilação das duas formas é muito distinto. Uma das diferenças observadas foi para as enzimas da via glicolítica, onde níveis maiores de acetilação foram detectados na forma procíclica, que utiliza principalmente fosforilação oxidativa para obtenção de energia, do que na forma sanguínea, cuja principal fonte produtora de energia é através da glicólise. Esses dados sugeriam que a acetilação poderia atuar na regulação da atividade dessas enzimas da via glicolítica em T. brucei, como já verificado para aldolase e glicerol-3-fosfato desidrogenase, em mamíferos. Assim, neste trabalho buscamos investigar o papel da acetilação na regulação da atividade da fructose 1,6-bisfosfato aldolase de T. brucei. Inicialmente, avaliamos o efeito da glicose na regulação dos níveis de acetilação proteica das proteínas totais, mitocôndriais e glicossomais, e verificamos alterações dependendo da disponibilidade ou não de glicose. Em seguida, utilizando extratos totais de procíclicos cultivados na presença ou ausência de glicose dosamos a atividade da aldolase, e detectamos redução significativa da atividade dos extratos de parasitas cultivados na ausência de glicose. A atividade aumenta quando submetemos previamente os extratos a desacetilação in vitro. Uma redução de mais de 50% é observada nos extratos proteicos obtidos de parasitas crescidos em meio contendo glicose após acetilação in vitro. Estes dados foram reforçados através de ensaios de imunoprecipitação de proteínas que mostram um aumento na acetilação da aldolase de procíclicos cultivados na ausência de glicose, comparado com parasitas cultivados na presença de glicose. Através de mutações sítio-dirigidas demonstramos que acetilação na lisina 157 (K157) é capaz de extinguir completamente a atividade in vitro da aldolase do parasita. Além disso, através de análises in silico, de docking e de dinâmica molecular demonstramos que acetilação na K157 afeta o perfil eletrostático do sítio catalítico da enzima, a maneira como o substrato interage com a enzima e afeta a estrutura do sítio catalítico, fazendo com que ele assuma um estado de volume reduzido. Em conjunto, esses dados demonstraram pela primeira vez o papel da acetilação na regulação da atividade de enzimas da via glicolítica de T. brucei, abrindo a possibilidade de explorar o amplo espectro regulatório que esta modificação pós-traducional pode ter neste parasita.
Reversible post-translational modifications in proteins are a relatively quick and economical way for cells to respond to environmental changes, such as temperature and nutrient availability. Acetylation is one of these modifications and occurs by the addition of acetyl group from acetyl-CoA to Nε-amino chain of lysine residues, eliminating the positive charge of that amino acid. Recently, our laboratory has described the acetylome of the procyclic and bloodstream forms of the protozoan parasite that causes African Trypanosomiasis, Trypanosoma brucei, and we found that the acetylation profile of the two forms is very different. One of the differences observed was for the glycolytic pathway enzymes, where higher levels of acetylation were detected in the procyclic form, which mainly uses oxidative phosphorylation to obtain energy, than in the bloodstream form, whose main source of energy is through glycolysis. These data suggest that acetylation could act to regulate the activity of these glycolytic enzymes in T. brucei, as already verified for aldolase and glycerol-3-phosphate dehydrogenase, in mammals. Thus, in this work we investigated the role of acetylation in regulating fructose 1,6-bisphosphate aldolase activity of T. brucei. Initially, we evaluated the effect of glucose on the regulation of protein acetylation levels of total, mitochondrial and glycosomal proteins, and verified changes depending on the availability of glucose. Then, using total procyclic extracts grown in the presence or absence of glucose, we measured aldolase activity and detected a significant reduction in the activity of parasite extracts grown in the absence of glucose. This activity increases when we pre-submit the extracts to deacetylation in vitro. A reduction of more than 50% is observed in protein extracts obtained from parasites grown in glucose-containing medium after in vitro acetylation. These data were reinforced by protein immunoprecipitation assays, which show an increase in aldolase acetylation of procyclic cultures grown in the absence of glucose, compared to parasites cultured in the presence of glucose. Through site-directed mutations we have shown that acetylation at lysine 157 (K157) is able to completely eliminates the in vitro aldolase activity of the parasite. In addition, through in silico, docking and molecular dynamics analyzes we have demonstrated that K157 acetylation affects the electrostatic profile of the enzyme catalytic site, the way the substrate interacts with the enzyme and changes the structure of the catalytic site, which assumes a reduced volume state. Taken together, these data demonstrated for the first time the role of acetylation in regulating the activity of enzymes in the glycolytic pathway of T. brucei, opening the possibility of exploring the broad regulatory spectrum that this post-translational modification can have in this parasite.
Reversible post-translational modifications in proteins are a relatively quick and economical way for cells to respond to environmental changes, such as temperature and nutrient availability. Acetylation is one of these modifications and occurs by the addition of acetyl group from acetyl-CoA to Nε-amino chain of lysine residues, eliminating the positive charge of that amino acid. Recently, our laboratory has described the acetylome of the procyclic and bloodstream forms of the protozoan parasite that causes African Trypanosomiasis, Trypanosoma brucei, and we found that the acetylation profile of the two forms is very different. One of the differences observed was for the glycolytic pathway enzymes, where higher levels of acetylation were detected in the procyclic form, which mainly uses oxidative phosphorylation to obtain energy, than in the bloodstream form, whose main source of energy is through glycolysis. These data suggest that acetylation could act to regulate the activity of these glycolytic enzymes in T. brucei, as already verified for aldolase and glycerol-3-phosphate dehydrogenase, in mammals. Thus, in this work we investigated the role of acetylation in regulating fructose 1,6-bisphosphate aldolase activity of T. brucei. Initially, we evaluated the effect of glucose on the regulation of protein acetylation levels of total, mitochondrial and glycosomal proteins, and verified changes depending on the availability of glucose. Then, using total procyclic extracts grown in the presence or absence of glucose, we measured aldolase activity and detected a significant reduction in the activity of parasite extracts grown in the absence of glucose. This activity increases when we pre-submit the extracts to deacetylation in vitro. A reduction of more than 50% is observed in protein extracts obtained from parasites grown in glucose-containing medium after in vitro acetylation. These data were reinforced by protein immunoprecipitation assays, which show an increase in aldolase acetylation of procyclic cultures grown in the absence of glucose, compared to parasites cultured in the presence of glucose. Through site-directed mutations we have shown that acetylation at lysine 157 (K157) is able to completely eliminates the in vitro aldolase activity of the parasite. In addition, through in silico, docking and molecular dynamics analyzes we have demonstrated that K157 acetylation affects the electrostatic profile of the enzyme catalytic site, the way the substrate interacts with the enzyme and changes the structure of the catalytic site, which assumes a reduced volume state. Taken together, these data demonstrated for the first time the role of acetylation in regulating the activity of enzymes in the glycolytic pathway of T. brucei, opening the possibility of exploring the broad regulatory spectrum that this post-translational modification can have in this parasite.