Caracterização funcional da quinase inositol polifosfato (IP6K) de Leishmania braziliensis
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Data
2023-03-06
Autores
Ferri, Yete Gambarini
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Editor
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 e recombinação homóloga (HR). No entanto, as proteínas-alvo dos PP-IPs, assim como seu mecanismo de ação, ainda não estão bem estabelecidas. IP7 e IP8 são sintetizados através de vias complementares que envolvem a participação das quinases IP6K e PP-IP5K. Tripanossomatídeos (e.g., Leishmania spp. e Trypanosoma spp.) possuem um gene ortólogo para IP6K, mas aparentemente não possuem ortólogos para PP-IP5K, i.e., teoricamente não sintetizam IP8, o que os torna excelentes modelos para o estudo de IP7. Este projeto consistiu em caracterizar funcionalmente a quinase IP6K de Leishmania braziliensis (LbrIP6K). Além disso, também realizamos a caracterização da quinase IP5K, a qual será utilizada como controle em ensaios futuros. Para isso, realizamos análises in silico utilizando o banco de dados TritrypDB. Encontramos os genes IP6K (LbrM.14.0340) e IP5K (LbrM.20.3290) e desenhamos primers para amplificação dos mesmos a partir do DNA genônico de L. braziliensis. No caso de LbrIP6K, amplificamos seu sítio catalítico (LbrIP6Kdomain). Adicionamos sítios para enzimas de restrição nas extremidades 5’ de cada primer para permitir posterior subclonagem direcional. Os produtos de PCR (amplicons) foram purificados, clonados em vetor de clonagem, inseridos por transformação em bactérias competentes E. coli, selecionados na presença de X-gal/IPTG, multiplicados e recuperados através de extração plasmidial. Realizamos a confirmação da clonagem por PCR e digestões enzimáticas. Em seguida, LbrIP6Kdomain e LbrIP5K foram excisados do plasmídeo utilizando enzimas de restrição específicas e subclonados direcionalmente no vetor de expressão pET28a+. O produto desta ligação foi transformado em bactérias competentes E. coli (cepas BL21-CódonPlus (DE3)-RP e Arctic Express RIL) para posterior expressão induzida por IPTG. As expressões de LbrIP6Kdomain e LbrIP5K foram analisadas em SDS-PAGE e confirmada por Western Blot utilizando anticorpos α-His-Tag. Os resultados demostraram que essas quinases são expressas na fração insolúvel do extrato bacteriano quando lisadas utilizando sonicação. Porém, ao lisar as células por alta pressão (French-press), conseguimos solubilizá-las parcialmente. Após isso, realizamos a purificação das proteínas pelo método de cromatografia de alta afinidade por metal imobilizado (IMAC) e confirmamos a eluição de ambas proteínas recombinantes por SDS-PAGE e Western Blot.
In model eukaryotes, inositol pyrophosphates (PP-IPs) – mainly the so-called IP7 and IP8 – are involved in a wide range of cellular processes, such as regulation of telomere length and homologous recombination (HR). However, the target proteins of PP-IPs and their mechanism of action are not yet well established. IP7 and IP8 are synthesized through complementary pathways involving the participation of IP6K and PP-IP5K kinases. Trypanosomatids (e.g., Leishmania spp. and Trypanosoma spp.) have an orthologous gene for IP6K, but apparently do not have orthologs for PP-IP5K, i.e., they do not synthesize IP8, which makes them excellent models for studying IP7. This project consisted of functionally characterizing the recombinant IP6K kinase from Leishmania braziliensis (LbrIP6K). Also, we performed the characterization of the IP5K kinase, which will be used as a control in future assays. For this, we performed in silico analyzes using the TriTrypDB database. We found the IP6K (LbrM.14.0340) and IP5K (LbrM.20.3290) genes and designed primers to amplify them from the genomic DNA of L. braziliensis. In the case of LbrIP6K, we amplified its catalytic site (LbrIP6Kdomain). Restriction enzyme sites were added at the 5' ends of each primer to allow further directional subcloning. The amplicons were purified, cloned into a cloning vector, transformed into competent E. coli cells, selected in the presence of X-gal/IPTG, and recovered by plasmid extraction. The cloning process was confirmed by PCR and enzymatic digestion. Then, LbrIP6Kdomain and LbrIP5K were excised from the plasmid using specific restriction enzymes and directionally subcloned into the pET28a+ expression vector. The product of this ligation was transformed into competent E. coli cells (strains BL21-CodonPlus (DE3)-RP and Arctic Express RIL) for further induced expression. LbrIP6Kdomain and LbrIP5K expression levels were analyzed by SDS-PAGE and Western Blot using α-His-Tag antibody. The results obtained showed that these kinases are expressed in the insoluble fraction of the bacterial extract when the cells are lysed using sonication. However, when using a high-pressure method of lysis (French-press), it was possible to partially solubilize them. Finally, we purified the proteins by the immobilized metal high-affinity chromatography (IMAC) method and confirmed the elution of both recombinant proteins by SDS-PAGE and Western Blot.
In model eukaryotes, inositol pyrophosphates (PP-IPs) – mainly the so-called IP7 and IP8 – are involved in a wide range of cellular processes, such as regulation of telomere length and homologous recombination (HR). However, the target proteins of PP-IPs and their mechanism of action are not yet well established. IP7 and IP8 are synthesized through complementary pathways involving the participation of IP6K and PP-IP5K kinases. Trypanosomatids (e.g., Leishmania spp. and Trypanosoma spp.) have an orthologous gene for IP6K, but apparently do not have orthologs for PP-IP5K, i.e., they do not synthesize IP8, which makes them excellent models for studying IP7. This project consisted of functionally characterizing the recombinant IP6K kinase from Leishmania braziliensis (LbrIP6K). Also, we performed the characterization of the IP5K kinase, which will be used as a control in future assays. For this, we performed in silico analyzes using the TriTrypDB database. We found the IP6K (LbrM.14.0340) and IP5K (LbrM.20.3290) genes and designed primers to amplify them from the genomic DNA of L. braziliensis. In the case of LbrIP6K, we amplified its catalytic site (LbrIP6Kdomain). Restriction enzyme sites were added at the 5' ends of each primer to allow further directional subcloning. The amplicons were purified, cloned into a cloning vector, transformed into competent E. coli cells, selected in the presence of X-gal/IPTG, and recovered by plasmid extraction. The cloning process was confirmed by PCR and enzymatic digestion. Then, LbrIP6Kdomain and LbrIP5K were excised from the plasmid using specific restriction enzymes and directionally subcloned into the pET28a+ expression vector. The product of this ligation was transformed into competent E. coli cells (strains BL21-CodonPlus (DE3)-RP and Arctic Express RIL) for further induced expression. LbrIP6Kdomain and LbrIP5K expression levels were analyzed by SDS-PAGE and Western Blot using α-His-Tag antibody. The results obtained showed that these kinases are expressed in the insoluble fraction of the bacterial extract when the cells are lysed using sonication. However, when using a high-pressure method of lysis (French-press), it was possible to partially solubilize them. Finally, we purified the proteins by the immobilized metal high-affinity chromatography (IMAC) method and confirmed the elution of both recombinant proteins by SDS-PAGE and Western Blot.
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Palavras-chave
Pirofosfatos de inositol, Pirofosforilação, Proteínas recombinantes, Leishmania braziliensis, Tripanossomatídeos, Difosfatos, Fosfatos de Inositol