Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases
Autor(a) principal: | |
---|---|
Data de Publicação: | 2022 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/15798 |
Resumo: | Cystatins are tight binding competitive inhibitors of cysteine peptidases. In plants, they act regulating different physiological mechanisms, as well as defending from biotic and abiotic stresses, making them of potential use for biotechnological applications in agriculture. In humans, cystatins are associated with the control of cysteine-cathepsins, which, when deregulated, can trigger distinct pathologies. Then, the intervention with exogen cystatins, like the phytocystatins, have been studied as a therapy for cancer or inflamatory diseases, for example. In the face of the wide variety of cystatins applications, the search for new variants in different plant species is relevant. The maquiberry (Aristotelia chilensis) is a native plant from South America, closely related to the Chilean culture. Recently, it has been coming into light due to its high anthocyanin content, and now the species is considered a superfruit. As an understudied species, little is known about the biotechnological potential of its proteins, until then, underinvestigated. In light of this, this work consisted in the identification, recombinant production and characterization of maquicystatins. From a trascriptome of the plant cultivated in Temporary Immersion Bioreactor, six sequences were identified, from which five were obtained by cDNA amplification. These were cloned in pET-28a expression vector for production in Escherichia coli Rosetta (DE3). The purified proteins were submitted to fluorimetric assays of inhibition against cysteine peptidases. All cystatins were able to inhibit papain with Ki of 7.13, 1.42, 3.29, 2.99, 5.05 nM, for MaquiCPIs 1 to 5, respectively, proving that they were expressed in the right conformation. They were also able to inhibit human cathepsin L efficiently (Ki of 0.34, 0.33, 0.38, 0.57 and 1.25 nM), but only MaquiCPIs 1, 2 and 3 could inhibit human cathepsin B (Ki of 35.74, 20.97 e 21.94 nM, respectively) in nanomolar order, while the others inhibited the protein in micromolar order. The capacity of maquicystatins in inhibiting human cathepsin indicates a potential in controlling distinct pathologies. |
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Souza, Eduardo Pereira deSilva, Flávio Henrique dahttp://lattes.cnpq.br/1757309852446263http://lattes.cnpq.br/0850771035889805d1ffe123-a8c6-4525-af50-27555a3801642022-04-04T16:08:54Z2022-04-04T16:08:54Z2022-02-15SOUZA, Eduardo Pereira de. Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases. 2022. Dissertação (Mestrado em Genética Evolutiva e Biologia Molecular) – Universidade Federal de São Carlos, São Carlos, 2022. Disponível em: https://repositorio.ufscar.br/handle/ufscar/15798.https://repositorio.ufscar.br/handle/ufscar/15798Cystatins are tight binding competitive inhibitors of cysteine peptidases. In plants, they act regulating different physiological mechanisms, as well as defending from biotic and abiotic stresses, making them of potential use for biotechnological applications in agriculture. In humans, cystatins are associated with the control of cysteine-cathepsins, which, when deregulated, can trigger distinct pathologies. Then, the intervention with exogen cystatins, like the phytocystatins, have been studied as a therapy for cancer or inflamatory diseases, for example. In the face of the wide variety of cystatins applications, the search for new variants in different plant species is relevant. The maquiberry (Aristotelia chilensis) is a native plant from South America, closely related to the Chilean culture. Recently, it has been coming into light due to its high anthocyanin content, and now the species is considered a superfruit. As an understudied species, little is known about the biotechnological potential of its proteins, until then, underinvestigated. In light of this, this work consisted in the identification, recombinant production and characterization of maquicystatins. From a trascriptome of the plant cultivated in Temporary Immersion Bioreactor, six sequences were identified, from which five were obtained by cDNA amplification. These were cloned in pET-28a expression vector for production in Escherichia coli Rosetta (DE3). The purified proteins were submitted to fluorimetric assays of inhibition against cysteine peptidases. All cystatins were able to inhibit papain with Ki of 7.13, 1.42, 3.29, 2.99, 5.05 nM, for MaquiCPIs 1 to 5, respectively, proving that they were expressed in the right conformation. They were also able to inhibit human cathepsin L efficiently (Ki of 0.34, 0.33, 0.38, 0.57 and 1.25 nM), but only MaquiCPIs 1, 2 and 3 could inhibit human cathepsin B (Ki of 35.74, 20.97 e 21.94 nM, respectively) in nanomolar order, while the others inhibited the protein in micromolar order. The capacity of maquicystatins in inhibiting human cathepsin indicates a potential in controlling distinct pathologies.Cistatinas são inibidores competitivos, do tipo tight-binding, de cisteíno peptidases. Em plantas, elas atuam regulando diversos mecanismos fisiológicos, bem como protegendo a planta de estresses bióticos e abióticos, o que as torna de uso potencial para aplicações biotecnológicas na agricultura. Em humanos, as cistatinas estão associadas ao controle das cisteíno-catepsinas, que, quando desreguladas, podem desencadear patologias diversas. Desse modo, a intervenção com cistatinas exógenas, como as fitocistatinas, vêm sendo estudada como possível terapia contra diversos tipos de cânceres ou doenças inflamatórias, por exemplo. Diante da grande gama de aplicações das cistatinas, é de relevância a busca de novas variantes em espécies vegetais diversas. O maqui (Aristotelia chilensis) é uma planta nativa da América do Sul, intimamente associada à cultura da população chilena. Atualmente, ela vem ganhando destaque devido ao seu alto conteúdo de antocianinas, sendo considerada uma superfruta. Como é uma espécie pouco estudada, pouco se sabe sobre o potencial biotecnológico das suas proteínas, até então, pouco investigadas. Dessa forma, este trabalho consistiu na identificação, produção recombinante e caracterização das cistatinas de maqui. A partir de um transcriptoma da planta cultivada em biorreatores de imersão temporária, foram identificadas seis sequências, das quais cinco foram obtidas por amplificação do cDNA. Estas foram clonadas em vetor de expressão pET-28a para a produção recombinante em Escherichia coli Rosetta (DE3). As proteínas purificadas foram submetidas a ensaios fluorimétricos de inibição de cisteíno peptidases. Todas as cistatinas foram capazes de inibir a papaína, com Ki de 7,13, 1,42, 3,29, 2,99, 5,05 nM para as MaquiCPIs de 1 a 5 respectivamente, indicando que foram expressas em conformação correta. Elas também foram capazes de inibir a Catepsina L humana de maneira eficiente (Ki de 0,34, 0,33, 0,38, 0,57 e 1,25 nM), mas somente as MaquiCPIs 1, 2 e 3 foram capazes de inibir a Catepsina B humana na ordem de nanomolar (Ki de 35,74, 20,97 e 21,94 nM, respectivamente), as demais inibiram a proteína na ordem de micromolar. A capacidade das maquicistatinas em inibir as catepsinas humanas refletem em um potencial no controle de diversas patologias.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)131081/2020-9porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Genética Evolutiva e Biologia Molecular - PPGGEvUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessCatepsinaFitocistatinaInibição tight bindingMaquicistatinaCathepsinPhytocystatinTight binding inhibitionMaquicystatinAristotelia chilensisCIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARCistatinas de maqui: produção recombinante e inibição de cisteíno peptidasesMaquicystatins: recombinant production and cysteine peptidase inhibitioninfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis600600e2c04fa9-1e62-4316-915c-35a38d859aaereponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARCC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufscar.br/bitstream/ufscar/15798/3/license_rdfe39d27027a6cc9cb039ad269a5db8e34MD53ORIGINALEduardo Pereira de Souza - Dissertação.pdfEduardo Pereira de Souza - Dissertação.pdfDissertação de Mestradoapplication/pdf3858833https://repositorio.ufscar.br/bitstream/ufscar/15798/1/Eduardo%20Pereira%20de%20Souza%20-%20Disserta%c3%a7%c3%a3o.pdf6e064aab77ef6c50d1ea9a7b8963e0c8MD51Eduardo Pereira de Souza - Carta comprovante.pdfEduardo Pereira de Souza - Carta comprovante.pdfCarta de Comprovaçãoapplication/pdf154539https://repositorio.ufscar.br/bitstream/ufscar/15798/2/Eduardo%20Pereira%20de%20Souza%20-%20Carta%20comprovante.pdfb49067b4c622715c1a099250b2716926MD52TEXTEduardo Pereira de Souza - Dissertação.pdf.txtEduardo Pereira de Souza - Dissertação.pdf.txtExtracted texttext/plain154429https://repositorio.ufscar.br/bitstream/ufscar/15798/4/Eduardo%20Pereira%20de%20Souza%20-%20Disserta%c3%a7%c3%a3o.pdf.txteec83700c85cefea54ccc1954275f713MD54Eduardo Pereira de Souza - Carta comprovante.pdf.txtEduardo Pereira de Souza - Carta comprovante.pdf.txtExtracted texttext/plain1511https://repositorio.ufscar.br/bitstream/ufscar/15798/6/Eduardo%20Pereira%20de%20Souza%20-%20Carta%20comprovante.pdf.txt64296235a8d44a515534bf62c402ba89MD56THUMBNAILEduardo Pereira de Souza - Dissertação.pdf.jpgEduardo Pereira de Souza - Dissertação.pdf.jpgIM Thumbnailimage/jpeg6425https://repositorio.ufscar.br/bitstream/ufscar/15798/5/Eduardo%20Pereira%20de%20Souza%20-%20Disserta%c3%a7%c3%a3o.pdf.jpg208adc622633bfe529c751ad9d442735MD55Eduardo Pereira de Souza - Carta comprovante.pdf.jpgEduardo Pereira de Souza - Carta comprovante.pdf.jpgIM Thumbnailimage/jpeg14033https://repositorio.ufscar.br/bitstream/ufscar/15798/7/Eduardo%20Pereira%20de%20Souza%20-%20Carta%20comprovante.pdf.jpg66839abdfd03aa6cd5aeca0cb68b68efMD57ufscar/157982023-09-18 18:32:32.027oai:repositorio.ufscar.br:ufscar/15798Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:32:32Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases |
dc.title.alternative.eng.fl_str_mv |
Maquicystatins: recombinant production and cysteine peptidase inhibition |
title |
Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases |
spellingShingle |
Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases Souza, Eduardo Pereira de Catepsina Fitocistatina Inibição tight binding Maquicistatina Cathepsin Phytocystatin Tight binding inhibition Maquicystatin Aristotelia chilensis CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
title_short |
Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases |
title_full |
Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases |
title_fullStr |
Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases |
title_full_unstemmed |
Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases |
title_sort |
Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases |
author |
Souza, Eduardo Pereira de |
author_facet |
Souza, Eduardo Pereira de |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/0850771035889805 |
dc.contributor.author.fl_str_mv |
Souza, Eduardo Pereira de |
dc.contributor.advisor1.fl_str_mv |
Silva, Flávio Henrique da |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/1757309852446263 |
dc.contributor.authorID.fl_str_mv |
d1ffe123-a8c6-4525-af50-27555a380164 |
contributor_str_mv |
Silva, Flávio Henrique da |
dc.subject.por.fl_str_mv |
Catepsina Fitocistatina Inibição tight binding Maquicistatina |
topic |
Catepsina Fitocistatina Inibição tight binding Maquicistatina Cathepsin Phytocystatin Tight binding inhibition Maquicystatin Aristotelia chilensis CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
dc.subject.eng.fl_str_mv |
Cathepsin Phytocystatin Tight binding inhibition Maquicystatin Aristotelia chilensis |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
description |
Cystatins are tight binding competitive inhibitors of cysteine peptidases. In plants, they act regulating different physiological mechanisms, as well as defending from biotic and abiotic stresses, making them of potential use for biotechnological applications in agriculture. In humans, cystatins are associated with the control of cysteine-cathepsins, which, when deregulated, can trigger distinct pathologies. Then, the intervention with exogen cystatins, like the phytocystatins, have been studied as a therapy for cancer or inflamatory diseases, for example. In the face of the wide variety of cystatins applications, the search for new variants in different plant species is relevant. The maquiberry (Aristotelia chilensis) is a native plant from South America, closely related to the Chilean culture. Recently, it has been coming into light due to its high anthocyanin content, and now the species is considered a superfruit. As an understudied species, little is known about the biotechnological potential of its proteins, until then, underinvestigated. In light of this, this work consisted in the identification, recombinant production and characterization of maquicystatins. From a trascriptome of the plant cultivated in Temporary Immersion Bioreactor, six sequences were identified, from which five were obtained by cDNA amplification. These were cloned in pET-28a expression vector for production in Escherichia coli Rosetta (DE3). The purified proteins were submitted to fluorimetric assays of inhibition against cysteine peptidases. All cystatins were able to inhibit papain with Ki of 7.13, 1.42, 3.29, 2.99, 5.05 nM, for MaquiCPIs 1 to 5, respectively, proving that they were expressed in the right conformation. They were also able to inhibit human cathepsin L efficiently (Ki of 0.34, 0.33, 0.38, 0.57 and 1.25 nM), but only MaquiCPIs 1, 2 and 3 could inhibit human cathepsin B (Ki of 35.74, 20.97 e 21.94 nM, respectively) in nanomolar order, while the others inhibited the protein in micromolar order. The capacity of maquicystatins in inhibiting human cathepsin indicates a potential in controlling distinct pathologies. |
publishDate |
2022 |
dc.date.accessioned.fl_str_mv |
2022-04-04T16:08:54Z |
dc.date.available.fl_str_mv |
2022-04-04T16:08:54Z |
dc.date.issued.fl_str_mv |
2022-02-15 |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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dc.identifier.citation.fl_str_mv |
SOUZA, Eduardo Pereira de. Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases. 2022. Dissertação (Mestrado em Genética Evolutiva e Biologia Molecular) – Universidade Federal de São Carlos, São Carlos, 2022. Disponível em: https://repositorio.ufscar.br/handle/ufscar/15798. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/15798 |
identifier_str_mv |
SOUZA, Eduardo Pereira de. Cistatinas de maqui: produção recombinante e inibição de cisteíno peptidases. 2022. Dissertação (Mestrado em Genética Evolutiva e Biologia Molecular) – Universidade Federal de São Carlos, São Carlos, 2022. Disponível em: https://repositorio.ufscar.br/handle/ufscar/15798. |
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https://repositorio.ufscar.br/handle/ufscar/15798 |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ info:eu-repo/semantics/openAccess |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ |
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openAccess |
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Universidade Federal de São Carlos Câmpus São Carlos |
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Programa de Pós-Graduação em Genética Evolutiva e Biologia Molecular - PPGGEv |
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UFSCar |
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Universidade Federal de São Carlos Câmpus São Carlos |
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