Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method

Detalhes bibliográficos
Autor(a) principal: Ribeiro, Priscila Rafaela
Data de Publicação: 2018
Outros Autores: Chicaybam, Leonardo, Pretti, Marco Antônio, Bonamino, Martín Hernán
Tipo de documento: Artigo de conferência
Idioma: eng
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/27272
Resumo: Instituto Nacional de Câncer José Alencar Gomes da Silva. Rio de Janeiro, RJ, Brasil.
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spelling Ribeiro, Priscila RafaelaChicaybam, LeonardoPretti, Marco AntônioBonamino, Martín Hernán2018-07-04T12:37:24Z2018-07-04T12:37:24Z2018RIBEIRO, Priscila Rafaela et al. Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method. In: SEMINÁRIO ANUAL CIENTÍFICO E TECNOLÓGICO DE BIO-MANGUINHOS, 6., 2018, Rio de Janeiro. Anais... Rio de Janeiro: Bio-Manguinhos, 2018. p. 128-129.https://www.arca.fiocruz.br/handle/icict/2727210.35259/isi.sact.2018_27272engFiocruz/Instituto de Tecnologia em Imunobiológicoshttps://www.arca.fiocruz.br/handle/icict/27049LAG3Receptor de antigénio quiméricoBioIDSACTLAG3Chimeric antigen receptorBioIDScreening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID methodinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectInstituto Nacional de Câncer José Alencar Gomes da Silva. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Presidência. Vice-Presidência de Pesquisa e Laboratórios de Referência. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer José Alencar Gomes da Silva. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer José Alencar Gomes da Silva. Rio de Janeiro, RJ, Brasil.Introduction: Inhibitory receptors such as PD-1, LAG-3, TIM-3 and CTLA-4 have gained special attention as potential targets for immunotherapy, since manipulation of negative signals mediated by these receptors may provide new therapeutic forms for several diseases, as cancer. More recently, Lymphocyte activation gene-3 (LAG-3) was described as a cell surface molecule interacting with MHC class II molecules. Identifying how proteins transduce the signal from these receptors has been a challenge. Once identified, these molecules can also be targets for novel therapeutics. In 2012, in order to identify interactions between proteins in a proximity-dependent manner, Roux et al (2012) created a method called BioID based on the fusion of a protein of interest to a mutated biotin ligase (R118G) (CHOI-RHEE et al., 2004; CRONAN, 2005; ROUX et al., 2012) which has the ability to add biotin to molecules that are at 20 nm or less from the protein of interest. Once biotinylated, the proteins can be recovered by binding to beads conjugated to streptavidin and identified by mass spectrometry. Objective: To perform a screening of proteins interacting with LAG-3 through the BioID method as well as to identify the possible signaling pathways modulated by LAG-3 signaling Methodology: Chimeric antigen receptors (CARs) were constructed with the anti-CD20 scFv fused to the intracellular domains consisting of: Lag-3 WT, Lag-3 Kmut (K=> A mutation in the KIEELE domain), Lag3 EPdel (deleted EP domain), all fused to the BirA domain, with further induction of expression of these CARs in the HEK293T and Jurkat (CD4+ T lymphocyte) cell lines. Biotin at a final concentration of 50uM will be added directly to the culture medium and subsequently the cells will be lysed; the proteins recovered through beads conjugated to streptavidin and, after trypsin digestion, analyzed in mass spectrometer. In silico analysis of possible signaling pathways involved downstream to LAG-3 will also be performed based on the labeled proteins identified in mass spectrometry. Results: CAR based receptors were synthetized and cloned into pcDNA3.1 vector MCSBirA (R118G) -HA, using the AgeI and BamHI sites, in frame with the BirA domain. The CAR anti-CD20 / Lag3Wild Type-BirA was electroporated in the HEK293T cell lineage, presenting 80% of expression. CARs Ep del and Kmut were also electroporated into HEK 293T cell lineage showing 39% and 41% of CAR expression. Analysis of the biotinylation pattern by Western blotting was performed (incubation with Pierce ™ High Sensitivity Streptavidin-HRP) for CAR anti-CD20 / Lag3Wild Type-BirA, and the expected ladder pattern of biotinylation was observed. Conclusion: The constructed CARs were expressed in the target cell lines leading to the expected biotinilated patterns.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txttext/plain1748https://www.arca.fiocruz.br/bitstream/icict/27272/1/license.txt8a4605be74aa9ea9d79846c1fba20a33MD51TEXTOTR.03.pdf.txtOTR.03.pdf.txtExtracted texttext/plain1https://www.arca.fiocruz.br/bitstream/icict/27272/3/OTR.03.pdf.txt68b329da9893e34099c7d8ad5cb9c940MD53ORIGINALOTR.03.pdfOTR.03.pdfapplication/pdf160894https://www.arca.fiocruz.br/bitstream/icict/27272/4/OTR.03.pdfa1c54c29747635ceccb78f7f9e801c87MD54icict/272722023-04-26 17:06:14.12oai:www.arca.fiocruz.br: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Repositório InstitucionalPUBhttps://www.arca.fiocruz.br/oai/requestrepositorio.arca@fiocruz.bropendoar:21352023-04-26T20:06:14Repositório Institucional da FIOCRUZ (ARCA) - Fundação Oswaldo Cruz (FIOCRUZ)false
dc.title.pt_BR.fl_str_mv Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method
title Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method
spellingShingle Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method
Ribeiro, Priscila Rafaela
LAG3
Receptor de antigénio quimérico
BioID
SACT
LAG3
Chimeric antigen receptor
BioID
title_short Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method
title_full Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method
title_fullStr Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method
title_full_unstemmed Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method
title_sort Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method
author Ribeiro, Priscila Rafaela
author_facet Ribeiro, Priscila Rafaela
Chicaybam, Leonardo
Pretti, Marco Antônio
Bonamino, Martín Hernán
author_role author
author2 Chicaybam, Leonardo
Pretti, Marco Antônio
Bonamino, Martín Hernán
author2_role author
author
author
dc.contributor.author.fl_str_mv Ribeiro, Priscila Rafaela
Chicaybam, Leonardo
Pretti, Marco Antônio
Bonamino, Martín Hernán
dc.subject.other.pt_BR.fl_str_mv LAG3
Receptor de antigénio quimérico
BioID
SACT
topic LAG3
Receptor de antigénio quimérico
BioID
SACT
LAG3
Chimeric antigen receptor
BioID
dc.subject.en.pt_BR.fl_str_mv LAG3
Chimeric antigen receptor
BioID
description Instituto Nacional de Câncer José Alencar Gomes da Silva. Rio de Janeiro, RJ, Brasil.
publishDate 2018
dc.date.accessioned.fl_str_mv 2018-07-04T12:37:24Z
dc.date.available.fl_str_mv 2018-07-04T12:37:24Z
dc.date.issued.fl_str_mv 2018
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/conferenceObject
format conferenceObject
status_str publishedVersion
dc.identifier.citation.fl_str_mv RIBEIRO, Priscila Rafaela et al. Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method. In: SEMINÁRIO ANUAL CIENTÍFICO E TECNOLÓGICO DE BIO-MANGUINHOS, 6., 2018, Rio de Janeiro. Anais... Rio de Janeiro: Bio-Manguinhos, 2018. p. 128-129.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/27272
dc.identifier.doi.none.fl_str_mv 10.35259/isi.sact.2018_27272
identifier_str_mv RIBEIRO, Priscila Rafaela et al. Screening of proteins related to the immunological checkpoint Lymphocyte activation gene-3 (LAG-3) through the BioID method. In: SEMINÁRIO ANUAL CIENTÍFICO E TECNOLÓGICO DE BIO-MANGUINHOS, 6., 2018, Rio de Janeiro. Anais... Rio de Janeiro: Bio-Manguinhos, 2018. p. 128-129.
10.35259/isi.sact.2018_27272
url https://www.arca.fiocruz.br/handle/icict/27272
dc.language.iso.fl_str_mv eng
language eng
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dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
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dc.publisher.none.fl_str_mv Fiocruz/Instituto de Tecnologia em Imunobiológicos
publisher.none.fl_str_mv Fiocruz/Instituto de Tecnologia em Imunobiológicos
dc.source.none.fl_str_mv reponame:Repositório Institucional da FIOCRUZ (ARCA)
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