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Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas)

Arapaima gigas, known as Pirarucu in Brazil, is one of the largest freshwater fish in the world. Some individuals could reach 3 m in length and weight up to 200 kg. Due to extinction risks and its economic value, the species has been a focus for preservation and reproduction studies. Thyrotropin (TS...

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Autores principales: Freire, Renan Passos, Hernandez-Gonzalez, Jorge Enrique, Lima, Eliana Rosa, Suzuki, Miriam Fussae, de Oliveira, João Ezequiel, Torai, Lucas Simon, Bartolini, Paolo, Soares, Carlos Roberto Jorge
Formato: Online Artículo Texto
Lenguaje:English
Publicado: SAGE Publications 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9926385/
https://www.ncbi.nlm.nih.gov/pubmed/36798082
http://dx.doi.org/10.1177/11779322231154148
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author Freire, Renan Passos
Hernandez-Gonzalez, Jorge Enrique
Lima, Eliana Rosa
Suzuki, Miriam Fussae
de Oliveira, João Ezequiel
Torai, Lucas Simon
Bartolini, Paolo
Soares, Carlos Roberto Jorge
author_facet Freire, Renan Passos
Hernandez-Gonzalez, Jorge Enrique
Lima, Eliana Rosa
Suzuki, Miriam Fussae
de Oliveira, João Ezequiel
Torai, Lucas Simon
Bartolini, Paolo
Soares, Carlos Roberto Jorge
author_sort Freire, Renan Passos
collection PubMed
description Arapaima gigas, known as Pirarucu in Brazil, is one of the largest freshwater fish in the world. Some individuals could reach 3 m in length and weight up to 200 kg. Due to extinction risks and its economic value, the species has been a focus for preservation and reproduction studies. Thyrotropin (TSH) is a glycoprotein hormone formed by 2 subunits α and β whose main activity is related to the synthesis of thyroid hormones (THs)—T3 and T4. In this work, we present a combination of bioinformatics tools to identify Arapaima gigas βTSH (ag-βTSH), modeling its molecular structure and express the recombinant heterodimer form in mammalian cells. Using the combination of computational biology, based on genome-related information, in silico molecular cloning and modeling led to confirm results of the ag-βTSH sequence by reverse transcriptase-polymerase chain reaction (RT-PCR) and transient expression in human embryonic kidney (HEK293F) cells. Molecular cloning of ag-βTSH retrieved 146 amino acids with a signal peptide of 21 amino acid residues and 6 disulfide bonds. The sequence has a similarity to 39 fish species, ranging between 43.1% and 81.6%, whose domains are extremely conserved, such as cystine knot motif and N-glycosylation site. The Arapaima gigas thyrotropin (ag-TSH) model, solved by AlphaFold, was used in molecular dynamics simulations with Scleropages formosus receptor, providing similar values of free energy ΔG(bind) and ΔG(PMF) in comparison with Homo sapiens model. The recombinant expression in HEK293F cells reached a yield of 25 mg/L, characterized via chromatographic and physical-chemical techniques. This work shows that other Arapaima gigas proteins could be studied in a similar way, using the combination of these techniques, recovering more information from its genome and improving the reproduction and preservation of this prehistoric fish.
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spelling pubmed-99263852023-02-15 Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas) Freire, Renan Passos Hernandez-Gonzalez, Jorge Enrique Lima, Eliana Rosa Suzuki, Miriam Fussae de Oliveira, João Ezequiel Torai, Lucas Simon Bartolini, Paolo Soares, Carlos Roberto Jorge Bioinform Biol Insights Original Research Article Arapaima gigas, known as Pirarucu in Brazil, is one of the largest freshwater fish in the world. Some individuals could reach 3 m in length and weight up to 200 kg. Due to extinction risks and its economic value, the species has been a focus for preservation and reproduction studies. Thyrotropin (TSH) is a glycoprotein hormone formed by 2 subunits α and β whose main activity is related to the synthesis of thyroid hormones (THs)—T3 and T4. In this work, we present a combination of bioinformatics tools to identify Arapaima gigas βTSH (ag-βTSH), modeling its molecular structure and express the recombinant heterodimer form in mammalian cells. Using the combination of computational biology, based on genome-related information, in silico molecular cloning and modeling led to confirm results of the ag-βTSH sequence by reverse transcriptase-polymerase chain reaction (RT-PCR) and transient expression in human embryonic kidney (HEK293F) cells. Molecular cloning of ag-βTSH retrieved 146 amino acids with a signal peptide of 21 amino acid residues and 6 disulfide bonds. The sequence has a similarity to 39 fish species, ranging between 43.1% and 81.6%, whose domains are extremely conserved, such as cystine knot motif and N-glycosylation site. The Arapaima gigas thyrotropin (ag-TSH) model, solved by AlphaFold, was used in molecular dynamics simulations with Scleropages formosus receptor, providing similar values of free energy ΔG(bind) and ΔG(PMF) in comparison with Homo sapiens model. The recombinant expression in HEK293F cells reached a yield of 25 mg/L, characterized via chromatographic and physical-chemical techniques. This work shows that other Arapaima gigas proteins could be studied in a similar way, using the combination of these techniques, recovering more information from its genome and improving the reproduction and preservation of this prehistoric fish. SAGE Publications 2023-02-12 /pmc/articles/PMC9926385/ /pubmed/36798082 http://dx.doi.org/10.1177/11779322231154148 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by-nc/4.0/This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage).
spellingShingle Original Research Article
Freire, Renan Passos
Hernandez-Gonzalez, Jorge Enrique
Lima, Eliana Rosa
Suzuki, Miriam Fussae
de Oliveira, João Ezequiel
Torai, Lucas Simon
Bartolini, Paolo
Soares, Carlos Roberto Jorge
Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas)
title Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas)
title_full Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas)
title_fullStr Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas)
title_full_unstemmed Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas)
title_short Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas)
title_sort molecular cloning and alphafold modeling of thyrotropin (ag-tsh) from the amazonian fish pirarucu (arapaima gigas)
topic Original Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9926385/
https://www.ncbi.nlm.nih.gov/pubmed/36798082
http://dx.doi.org/10.1177/11779322231154148
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