Poderiam os pirofosfatos de inositol terem influenciado o desenvolvimento do parasitismo dentro dos cinetoplastídeos?
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2023-03-20
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Universidade Estadual Paulista (Unesp)
Resumo
Em eucariotos modelo, pirofosfatos de inositol (PP-IPs) – principalmente os chamados IP7 e IP8 – estão envolvidos em uma ampla gama de processos celulares, como regulação do comprimento dos telômeros, morte celular programada, recombinação homóloga (HR), entre outros. No entanto, o mecanismo de ação e o envolvimento destes metabólitos em outras vias espécie-específicas ainda são questões abertas. IP7 e IP8 são sintetizados através de vias complementares que envolvem a participação das quinases IP6K e PP-IP5K. Tripanosomatídeos parasitas (e.g., Leishmania spp. e Trypanosoma spp.) possuem um gene ortólogo para IP6K, mas aparentemente não possuem ortólogos para PP-IP5K, i.e., não sintetizam IP8, o que os torna excelentes modelos para o estudo de IP7. Além disso, estudos preliminares apontam que IP8 está presente em organismos de vida livre da classe Kinetoplastea. Assim, esta dissertação consistiu inicialmente em investigar se a presença de IP8 é mutuamente exclusiva em relação ao parasitismo dentro dos cinetoplastídeos. Para isso, realizamos análises evolutivas para confirmar a suposta perda da PP-IP5K em tripanosomatídeos. Reconstruímos árvores filogenéticas e obtivemos evidências robustas que confirmam a ausência de PP-IP5K em todos os tripanosomatídeos, exceto Paratrypanosoma confusum. As previsões das estruturas terciárias apontam que o domínio catalítico da PP-IP5K de P. confusum é naturalmente desestruturado, o que coloca em xeque sua função. Além disso, realizamos ensaios de dinâmica molecular com a PP-IP5K de Bodo saltans (BsPP-IP5K) e IP7, que é utilizado como substrato (junto com cofator ATP) para síntese de IP8. Nossas simulações sugerem que o BsPP-IP5K é totalmente capaz de sintetizar IP8. Dessa forma, amplificamos o gene BsPP-IP5K e adicionamos a ele as regiões 3' e 5' UTR da Gliceraldeído 3-Fosfato Desidrogenase (GAPDH). Posteriormente, clonamos essa construção no vetor pSP72-αHYGα (Marc Ouellette lab), gerando um plasmídeo que fornece expressão constitutiva epissomal de BsPP-IP5K em Leishmania spp. (episomal knock-in). Paralelamente, realizamos mutagênese sítio-dirigida para confirmar os resíduos de aminoácidos essenciais de BsPP-IP5K. A construção contendo a BsPP-IP5K mutada foi usada como controle nos ensaios knock-in. Com a inserção das construções em Leishmania braziliensis, realizamos a caracterização fenotípica das linhagens geradas, no qual, aparentemente, a presença da BsPP-IP5K não altera a proliferação e o ciclo celular. Futuramente, essas linhagens knock-in para PP-IP5-K serão utilizadas em ensaios de infecção para verificação da virulência e patogenicidade. Nossos achados até o momento sugerem que a transição de um estilo de vida livre para um estilo de vida parasitário resultou na perda de PP-IP5K. Os resultados deste trabalho poderão contribuir para a elucidação de novas rotas para o desenvolvimento de terapias antiparasitárias.
In model eukaryotes, inositol pyrophosphates (PP-IPs) – mainly IP7 and IP8 – are involved in a wide range of cellular processes, such as telomere length regulation, programmed cell death, homologous recombination (HR), among others. However, the mechanism of action and the involvement of these metabolites in other species-specific pathways are still open questions. IP7 and IP8 are synthesized through complementary pathways that involve the participation of IP6K and PP-IP5K kinases, respectively. Parasitic trypanosomatids (e.g., Leishmania spp. and Trypanosoma spp.) have an ortholog gene for IP6K, but apparently lack orthologs for PP-IP5K, i.e., they do not synthesize IP8, which makes them excellent models for the study of IP7. Also, preliminary studies indicate that IP8 is present in free-living organisms of the Kinetoplastea class. Thus, this project consists of investigating whether the presence of IP8 is mutually exclusive in relation to parasitism within kinetoplastids. For this, we performed evolutionary analyzes to confirm the supposed loss of PP-IP5K in trypanosomatids. We reconstructed phylogenetic trees and obtained robust evidence confirming the absence of PP-IP5K in all trypanosomatids but Paratrypanosoma confusum. The predictions of tertiary structures indicate that the catalytic domain of P. confusum PP-IP5K is naturally unstructured, which calls into question its function. Also, we performed molecular dynamics assays with B. saltans PP-IP5K (BsPP-IP5K) and IP7, which was used as a substrate (along with ATP) to synthesize IP8. Our simulations suggested that the BsPP-IP5K is fully capable of synthesizing IP8. Thus, we amplified the BsPP-IP5K gene and added to it the 3' and 5' UTR regions of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). Subsequently, we cloned this construct into the pSP72-αHYGα vector (Marc Ouellette lab), generating a plasmid that provides episomal constitutive expression of BsPP-IP5K in Leishmania spp. (knock-in episomal). In parallel, we performed site-directed mutagenesis to confirm the essential amino acid residues of BsPP-IP5K. The construct containing the BsPP-IP5K was used as a control in the knock-in assays. With the insertion of constructs in Leishmania braziliensis, we performed the phenotypic characterization of the generated lineages, in which, apparently, the presence of BsPP-IP5K does not alter proliferation and cell cycle. In the future, these knock-in strains for PP-IP5-K will be used in infection assays to verify virulence and pathogenicity. To date, our findings suggest that the transition from a free to a parasitic lifestyle resulted in the loss of PP-IP5K. The results of this work may contribute to the elucidation of new routes for the development of antiparasitic therapies.
In model eukaryotes, inositol pyrophosphates (PP-IPs) – mainly IP7 and IP8 – are involved in a wide range of cellular processes, such as telomere length regulation, programmed cell death, homologous recombination (HR), among others. However, the mechanism of action and the involvement of these metabolites in other species-specific pathways are still open questions. IP7 and IP8 are synthesized through complementary pathways that involve the participation of IP6K and PP-IP5K kinases, respectively. Parasitic trypanosomatids (e.g., Leishmania spp. and Trypanosoma spp.) have an ortholog gene for IP6K, but apparently lack orthologs for PP-IP5K, i.e., they do not synthesize IP8, which makes them excellent models for the study of IP7. Also, preliminary studies indicate that IP8 is present in free-living organisms of the Kinetoplastea class. Thus, this project consists of investigating whether the presence of IP8 is mutually exclusive in relation to parasitism within kinetoplastids. For this, we performed evolutionary analyzes to confirm the supposed loss of PP-IP5K in trypanosomatids. We reconstructed phylogenetic trees and obtained robust evidence confirming the absence of PP-IP5K in all trypanosomatids but Paratrypanosoma confusum. The predictions of tertiary structures indicate that the catalytic domain of P. confusum PP-IP5K is naturally unstructured, which calls into question its function. Also, we performed molecular dynamics assays with B. saltans PP-IP5K (BsPP-IP5K) and IP7, which was used as a substrate (along with ATP) to synthesize IP8. Our simulations suggested that the BsPP-IP5K is fully capable of synthesizing IP8. Thus, we amplified the BsPP-IP5K gene and added to it the 3' and 5' UTR regions of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). Subsequently, we cloned this construct into the pSP72-αHYGα vector (Marc Ouellette lab), generating a plasmid that provides episomal constitutive expression of BsPP-IP5K in Leishmania spp. (knock-in episomal). In parallel, we performed site-directed mutagenesis to confirm the essential amino acid residues of BsPP-IP5K. The construct containing the BsPP-IP5K was used as a control in the knock-in assays. With the insertion of constructs in Leishmania braziliensis, we performed the phenotypic characterization of the generated lineages, in which, apparently, the presence of BsPP-IP5K does not alter proliferation and cell cycle. In the future, these knock-in strains for PP-IP5-K will be used in infection assays to verify virulence and pathogenicity. To date, our findings suggest that the transition from a free to a parasitic lifestyle resulted in the loss of PP-IP5K. The results of this work may contribute to the elucidation of new routes for the development of antiparasitic therapies.
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Palavras-chave
Parasitismo, Tripanosomatídeos, Inositol pirofosfatos, Proteínas de quinases, Leishmania, Fosfatos de inositol