Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana
Autor(a) principal: | |
---|---|
Data de Publicação: | 2024 |
Tipo de documento: | Trabalho de conclusão de curso |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/19430 |
Resumo: | The 70 kDa Heat shock protein (Hsp70 or HSPA in humans) is a highly conserved family of molecular chaperones that is expressed in various cellular compartments and is associated with several pathologies, including neurodegenerative diseases and cancer. Among HSPAs, the mitochondrial HSPA (HSPA9 or mortalin) plays a crucial role in the import of proteins synthesized in the cytosol into the mitochondria. This function requires interaction with a specific co-chaperone called human Hsp70-escort protein 1 (hHep1), whose L-shaped structure, containing a "zinc finger" domain, is fundamental for such interactions. hHep1 holds great importance in the maintenance of HSPA9 and HSPA1A (human Hsp701A) solubility, as well as the remodeling and stimulation of their ATPase activity. It is present in both the mitochondria and the cell nucleus, where it interacts with HSPAs such as HSPA1A. Due to its role in HSPA homeostasis, this study aimed to express, purify, and characterize hHep1 mutants with truncated N- and C-terminal regions to identify the primary regions responsible for its function. The hHep1-Core mutant, containing the "zinc finger" domain, was also studied to elucidate its structural and functional roles. The secondary and tertiary structures of the hHep1-Ndel, hHep1-Cdel, and hHep1-Core mutants were investigated using circular dichroism and fluorescence spectroscopy, respectively. The results revealed similarities in structure between the hHep1-Ndel and hHep1Δmts mutants, as well as between the hHep1-Cdel and hHep1-Core mutants. Moreover, the stability of the proteins was demonstrated for up to 72 hours, and their resistance to denaturing agents was demonstrated, with Cm values of 1.4 M, 1.9 M, and 1.5 M for hHep1-Ndel, hHep1-Cdel, and hHep1-Core, respectively. The size and shape of the mutants were also analyzed using analytical size exclusion chromatography, revealing that all proteins were monomeric, and that hHep1-Core had a more globular structure compared to the complete structure. The study concluded that the methods and conditions used were suitable for the expression, purification, and characterization of these recombinant proteins, providing a valuable contribution to the field of research. |
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Gomes, Maria Júlia MattosBorges, Júlio Césarhttp://lattes.cnpq.br/6033516785481779Teixeira, Felipe Robertihttp://lattes.cnpq.br/3568850159381693http://lattes.cnpq.br/78213104961813172024-02-23T12:54:58Z2024-02-23T12:54:58Z2024-02-15GOMES, Maria Júlia Mattos. Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana. 2024. Trabalho de Conclusão de Curso (Graduação em Biotecnologia) – Universidade Federal de São Carlos, São Carlos, 2024. Disponível em: https://repositorio.ufscar.br/handle/ufscar/19430.https://repositorio.ufscar.br/handle/ufscar/19430The 70 kDa Heat shock protein (Hsp70 or HSPA in humans) is a highly conserved family of molecular chaperones that is expressed in various cellular compartments and is associated with several pathologies, including neurodegenerative diseases and cancer. Among HSPAs, the mitochondrial HSPA (HSPA9 or mortalin) plays a crucial role in the import of proteins synthesized in the cytosol into the mitochondria. This function requires interaction with a specific co-chaperone called human Hsp70-escort protein 1 (hHep1), whose L-shaped structure, containing a "zinc finger" domain, is fundamental for such interactions. hHep1 holds great importance in the maintenance of HSPA9 and HSPA1A (human Hsp701A) solubility, as well as the remodeling and stimulation of their ATPase activity. It is present in both the mitochondria and the cell nucleus, where it interacts with HSPAs such as HSPA1A. Due to its role in HSPA homeostasis, this study aimed to express, purify, and characterize hHep1 mutants with truncated N- and C-terminal regions to identify the primary regions responsible for its function. The hHep1-Core mutant, containing the "zinc finger" domain, was also studied to elucidate its structural and functional roles. The secondary and tertiary structures of the hHep1-Ndel, hHep1-Cdel, and hHep1-Core mutants were investigated using circular dichroism and fluorescence spectroscopy, respectively. The results revealed similarities in structure between the hHep1-Ndel and hHep1Δmts mutants, as well as between the hHep1-Cdel and hHep1-Core mutants. Moreover, the stability of the proteins was demonstrated for up to 72 hours, and their resistance to denaturing agents was demonstrated, with Cm values of 1.4 M, 1.9 M, and 1.5 M for hHep1-Ndel, hHep1-Cdel, and hHep1-Core, respectively. The size and shape of the mutants were also analyzed using analytical size exclusion chromatography, revealing that all proteins were monomeric, and that hHep1-Core had a more globular structure compared to the complete structure. The study concluded that the methods and conditions used were suitable for the expression, purification, and characterization of these recombinant proteins, providing a valuable contribution to the field of research.A Heat shock protein de 70 kDa (Hsp70 ou HSPA em humanos) representa uma família de chaperonas moleculares altamente conservada, expressa em diversos compartimentos celulares, e associada a diversas patologias, como doenças neurodegenerativas e câncer. Entre as HSPAs, destaca-se a HSPA mitocondrial (HSPA9 ou mortalina), que desempenha um papel crucial na importação de proteínas sintetizadas no citosol para a mitocôndria. Essa função requer a interação com uma co-chaperona específica, denominada Hsp70-escort protein 1 humana (hHep1), cuja estrutura em forma de L contendo um domínio "dedo de zinco" é fundamental para tal interação. A hHep1 desempenha papéis significativos na manutenção da solubilidade da HSPA9 e HSPA1A (Hsp701A humana), remodelação de seus complexos supramoleculares e estímulo à atividade ATPásica dessas HSPAs. Além de sua localização mitocondrial, a hHep1 também é encontrada no núcleo celular, onde pode interagir com outras HSPAs, como a HSPA1A em situação de estresse. Diante da relevância da hHep1 para a estabilidade das HSPAs, este estudo propõe a expressão, purificação e caracterização estrutural de mutantes da hHep1, nos quais as regiões N- e C-terminais foram truncadas, visando identificar as principais regiões responsáveis por sua função. O mutante hHep1-Core, contendo o domínio "dedo de zinco", também foi caracterizado, visando elucidar seus papéis estruturais e funcionais. Foram conduzidos experimentos para análise da estrutura secundária e terciária por meio de dicroísmo circular e espectroscopia de fluorescência, respectivamente. Os resultados indicaram semelhanças estruturais entre hHep1-Ndel e hHep1Δmts, assim como entre hHep1-Cdel e hHep1-Core. Além disso, foi demonstrada a estabilidade temporal das proteínas por até 72 horas a 4°C, bem como sua estabilidade química frente ao agente desnaturante GndHCl, revelando valores de Cm de 1,4 M, 1,9 M e 1,5 M para hHep1-Ndel, hHep1-Cdel e hHep1-Core, respectivamente. As propriedades hidrodinâmicas dos mutantes foram determinadas por meio de cromatografia de exclusão por tamanho analítica, evidenciando que todas as proteínas são monoméricas, com destaque para a estrutura mais globular observada em hHep1-Core em comparação com a estrutura completa. Conclui-se que os protocolos e condições testadas são as ideais para a expressão, purificação e caracterização dessas proteínas recombinantes, o que proporciona um avanço científico nessa área de estudo.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)2021/04799-0porUniversidade Federal de São CarlosCâmpus São CarlosBiotecnologia - BiotecUFSCarAttribution-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nd/3.0/br/info:eu-repo/semantics/openAccessChaperonas molecularesHsp70Proteínas recombinantesCaracterização biofísicaMolecular chaperonesRecombinant proteinsBiophysical characterizationCIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULASExpressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humanaExpression, purification and structural characterization of truncated mutants of human Hsp70-escort protein 1info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/bachelorThesisreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINAL761228_MariaJúliaMattosGomes_TCC_VersãoFinal.pdf761228_MariaJúliaMattosGomes_TCC_VersãoFinal.pdfapplication/pdf2357401https://repositorio.ufscar.br/bitstream/ufscar/19430/1/761228_MariaJ%c3%baliaMattosGomes_TCC_Vers%c3%a3oFinal.pdfb1b091a0e2029b4604252f0d84396ff6MD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8804https://repositorio.ufscar.br/bitstream/ufscar/19430/2/license_rdf4774e414fb27824b0dfca5f33e4ff24fMD52TEXT761228_MariaJúliaMattosGomes_TCC_VersãoFinal.pdf.txt761228_MariaJúliaMattosGomes_TCC_VersãoFinal.pdf.txtExtracted texttext/plain111079https://repositorio.ufscar.br/bitstream/ufscar/19430/3/761228_MariaJ%c3%baliaMattosGomes_TCC_Vers%c3%a3oFinal.pdf.txtb54a0c49ca4febd2c05807c80ee21bd5MD53ufscar/194302024-05-14 17:35:03.87oai:repositorio.ufscar.br:ufscar/19430Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222024-05-14T17:35:03Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana |
dc.title.alternative.eng.fl_str_mv |
Expression, purification and structural characterization of truncated mutants of human Hsp70-escort protein 1 |
title |
Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana |
spellingShingle |
Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana Gomes, Maria Júlia Mattos Chaperonas moleculares Hsp70 Proteínas recombinantes Caracterização biofísica Molecular chaperones Recombinant proteins Biophysical characterization CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS |
title_short |
Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana |
title_full |
Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana |
title_fullStr |
Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana |
title_full_unstemmed |
Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana |
title_sort |
Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana |
author |
Gomes, Maria Júlia Mattos |
author_facet |
Gomes, Maria Júlia Mattos |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/7821310496181317 |
dc.contributor.author.fl_str_mv |
Gomes, Maria Júlia Mattos |
dc.contributor.advisor1.fl_str_mv |
Borges, Júlio César |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/6033516785481779 |
dc.contributor.advisor-co1.fl_str_mv |
Teixeira, Felipe Roberti |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/3568850159381693 |
contributor_str_mv |
Borges, Júlio César Teixeira, Felipe Roberti |
dc.subject.por.fl_str_mv |
Chaperonas moleculares Hsp70 Proteínas recombinantes Caracterização biofísica Molecular chaperones |
topic |
Chaperonas moleculares Hsp70 Proteínas recombinantes Caracterização biofísica Molecular chaperones Recombinant proteins Biophysical characterization CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS |
dc.subject.eng.fl_str_mv |
Recombinant proteins Biophysical characterization |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS |
description |
The 70 kDa Heat shock protein (Hsp70 or HSPA in humans) is a highly conserved family of molecular chaperones that is expressed in various cellular compartments and is associated with several pathologies, including neurodegenerative diseases and cancer. Among HSPAs, the mitochondrial HSPA (HSPA9 or mortalin) plays a crucial role in the import of proteins synthesized in the cytosol into the mitochondria. This function requires interaction with a specific co-chaperone called human Hsp70-escort protein 1 (hHep1), whose L-shaped structure, containing a "zinc finger" domain, is fundamental for such interactions. hHep1 holds great importance in the maintenance of HSPA9 and HSPA1A (human Hsp701A) solubility, as well as the remodeling and stimulation of their ATPase activity. It is present in both the mitochondria and the cell nucleus, where it interacts with HSPAs such as HSPA1A. Due to its role in HSPA homeostasis, this study aimed to express, purify, and characterize hHep1 mutants with truncated N- and C-terminal regions to identify the primary regions responsible for its function. The hHep1-Core mutant, containing the "zinc finger" domain, was also studied to elucidate its structural and functional roles. The secondary and tertiary structures of the hHep1-Ndel, hHep1-Cdel, and hHep1-Core mutants were investigated using circular dichroism and fluorescence spectroscopy, respectively. The results revealed similarities in structure between the hHep1-Ndel and hHep1Δmts mutants, as well as between the hHep1-Cdel and hHep1-Core mutants. Moreover, the stability of the proteins was demonstrated for up to 72 hours, and their resistance to denaturing agents was demonstrated, with Cm values of 1.4 M, 1.9 M, and 1.5 M for hHep1-Ndel, hHep1-Cdel, and hHep1-Core, respectively. The size and shape of the mutants were also analyzed using analytical size exclusion chromatography, revealing that all proteins were monomeric, and that hHep1-Core had a more globular structure compared to the complete structure. The study concluded that the methods and conditions used were suitable for the expression, purification, and characterization of these recombinant proteins, providing a valuable contribution to the field of research. |
publishDate |
2024 |
dc.date.accessioned.fl_str_mv |
2024-02-23T12:54:58Z |
dc.date.available.fl_str_mv |
2024-02-23T12:54:58Z |
dc.date.issued.fl_str_mv |
2024-02-15 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/bachelorThesis |
format |
bachelorThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
GOMES, Maria Júlia Mattos. Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana. 2024. Trabalho de Conclusão de Curso (Graduação em Biotecnologia) – Universidade Federal de São Carlos, São Carlos, 2024. Disponível em: https://repositorio.ufscar.br/handle/ufscar/19430. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/19430 |
identifier_str_mv |
GOMES, Maria Júlia Mattos. Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana. 2024. Trabalho de Conclusão de Curso (Graduação em Biotecnologia) – Universidade Federal de São Carlos, São Carlos, 2024. Disponível em: https://repositorio.ufscar.br/handle/ufscar/19430. |
url |
https://repositorio.ufscar.br/handle/ufscar/19430 |
dc.language.iso.fl_str_mv |
por |
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por |
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Attribution-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nd/3.0/br/ info:eu-repo/semantics/openAccess |
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Attribution-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nd/3.0/br/ |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Universidade Federal de São Carlos Câmpus São Carlos Biotecnologia - Biotec |
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UFSCar |
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Universidade Federal de São Carlos Câmpus São Carlos Biotecnologia - Biotec |
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UFSCAR |
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