Papel da acetilação na regulação de processos biológicos: uma abordagem de bioinformática e biologia sintética
Data
2024-07-01
Autores
Bonifácio, Bruno Souza 
Orientadores
Moretti, Nilmar Silvio
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A acetilação de lisina é uma modificação pós-traducional crucial e avanços em técnicas proteômicas permitiram investigar os acetilomas de diferentes espécies, destacando a importância da acetilação em diversos organismos. Para melhor entender o papel dessa modificação na regulação de processos celulares, na primeira parte desse trabalho, avaliamos uma gama de 50 acetilomas publicados entre 2009 e 2022 de espécies de vários grupos filogenéticos: bactérias, fungos, protozoários, vermes, plantas, insetos, peixes e mamíferos. Análises dos níveis de acetilação, mostraram que bactérias apresentam o maior número de sítios de acetilação, seguidas por fungos e protozoários. Proteínas com um único sítio de acetilação foram predominantes em todas as espécies. Além disso, analisamos domínios acetilados em todas as espécies, constatando que proteínas contendo os domínios HSP70 foram as que apresentaram maiores níveis de acetilação. Os processos metabólicos de ATP e as vias de aminoacil-tRNA sintetase mostraram-se altamente acetilados, destacando seus papéis cruciais na produção de energia e na expressão gênica, respectivamente. Nossa análise também revelou acetilação significativa em vias metabólicas relacionadas à glicose e à produção de ATP. Além disso, verificamos que o mecanismo regulatório da acetilação na proteína superóxido dismutase A (SODA) parece ser conservado entre diferentes espécies. Na segunda parte do trabalho, exploramos o sistema de biologia sintética do par ortogonal da pirrolisil-tRNA sintetase e do tRNApyl (Pylt) para o estudo da acetilação proteica de resíduos específicos de lisina. Portanto, um dos objetivos foi implementar esse método para expressar proteínas contendo resíduos de lisina acetilada usando como modelo as enzimas aldolase de T. brucei e SODA de T. cruzi. Conseguimos expressar e purificar ambas as enzimas e validar a presença da acetilação por Western blot e dot-blot. Utilizando as proteínas acetiladas purificadas confirmamos o efeito inibitório da acetilação, como observado anteriormente pelo nosso grupo. Com isso, conseguimos estabelecer a ferramenta de par-ortogonal para expressão de proteínas acetiladas, abrindo novas oportunidades para estudar modificações proteicas em tripanosomatídeos e outras espécies.
Lysine acetylation is a crucial post-translational modification and advances in proteomic techniques have allowed us to investigate the acetylomes of different species, highlighting the importance of acetylation in various organisms. To better understand the role of this modification in regulating cellular processes, in the first part of this work, we evaluated 50 acetylomes published between 2009 and 2022 from species of various phylogenetic groups: bacteria, fungi, protozoa, worms, plants, insects, fish and mammals. Analysis of acetylation levels showed that bacteria have the highest number of acetylation sites, followed by fungi and protozoa. Proteins with a single acetylation site were predominant. In addition, we analyzed acetylated domains in different species, finding that HSP70 domains were the most acetylated proteins among all species. The ATP metabolic processes and aminoacyl-tRNA synthetase pathways were shown to be highly acetylated, highlighting their crucial roles in energy production and gene expression, respectively. Our analysis also revealed significant acetylation in metabolic pathways related to glycolysis and ATP production. In addition, we found that the regulatory mechanism of acetylation in the superoxide dismutase A (SODA) protein appears to be conserved between different species. In the second part of the work, we have exploited the synthetic biology system of the orthogonal pair of pyrrolysyl-tRNA synthetase and tRNApyl (Pylt) to study protein acetylation of specific lysine residues. Therefore, one of the objectives was to implement this method to express proteins containing acetylated lysine residues using as a model the enzymes aldolase from T. brucei and SODA from T. cruzi. Using the orthogonal pair approach, we could express and purify both enzymes and validate the presence of acetylation by Western blot and dot-blot. Using the purified acetylated proteins, we confirmed the inhibitory effect of acetylation, as previously observed by our group. With this, we established the orthogonal pair tool for the expression of acetylated proteins, opening the opportunity to study protein modifications in trypanosomatids and other species.
Lysine acetylation is a crucial post-translational modification and advances in proteomic techniques have allowed us to investigate the acetylomes of different species, highlighting the importance of acetylation in various organisms. To better understand the role of this modification in regulating cellular processes, in the first part of this work, we evaluated 50 acetylomes published between 2009 and 2022 from species of various phylogenetic groups: bacteria, fungi, protozoa, worms, plants, insects, fish and mammals. Analysis of acetylation levels showed that bacteria have the highest number of acetylation sites, followed by fungi and protozoa. Proteins with a single acetylation site were predominant. In addition, we analyzed acetylated domains in different species, finding that HSP70 domains were the most acetylated proteins among all species. The ATP metabolic processes and aminoacyl-tRNA synthetase pathways were shown to be highly acetylated, highlighting their crucial roles in energy production and gene expression, respectively. Our analysis also revealed significant acetylation in metabolic pathways related to glycolysis and ATP production. In addition, we found that the regulatory mechanism of acetylation in the superoxide dismutase A (SODA) protein appears to be conserved between different species. In the second part of the work, we have exploited the synthetic biology system of the orthogonal pair of pyrrolysyl-tRNA synthetase and tRNApyl (Pylt) to study protein acetylation of specific lysine residues. Therefore, one of the objectives was to implement this method to express proteins containing acetylated lysine residues using as a model the enzymes aldolase from T. brucei and SODA from T. cruzi. Using the orthogonal pair approach, we could express and purify both enzymes and validate the presence of acetylation by Western blot and dot-blot. Using the purified acetylated proteins, we confirmed the inhibitory effect of acetylation, as previously observed by our group. With this, we established the orthogonal pair tool for the expression of acetylated proteins, opening the opportunity to study protein modifications in trypanosomatids and other species.
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Citação
BONIFÁCIO, Bruno Souza. Papel da acetilação na regulação de processos biológicos: uma abordagem de bioinformática e biologia sintética. 2024. 79 f. Tese (Doutorado em Microbiologia e Imunologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2024.