Restoring NKX6-2 function by protein complementation: a proof-of-concept
Autor(a) principal: | |
---|---|
Data de Publicação: | 2022 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/138619 |
Resumo: | NKX6-2 is a transcriptional repressor involved in the regulation of various genes associated with cell fate determination of neurons, oligodendrocytes, or pancreatic cells, as well as glucagon production, among other phenomena. Loss-of-function mutations in NKX6-2 are the cause of Spastic Ataxia 8 (SPAX8), a rare recessive hereditary disease that leads to an early-onset hypomyelinating leukodystrophy due to the lack of oligodendrocyte maturation. The most common disease-associated NKX6-2 mutations involve the formation of premature termination codons (PTCs). These PTCs will render the nascent mRNA a potential target for degradation via nonsense-mediated mRNA decay (NMD). The small fraction of mRNA that survives NMD and is eventually translated to protein will produce truncated, non-functional NKX6-2 proteins. We aimed to overcome these two issues to lay the groundwork for the development of new therapies for SPAX8. Using site-directed mutagenesis, we were able to recreate SPAX8-related mutations on a cDNA-based NKX6-2-Venus fusion protein, but not in a NKX6-2 minigene including its introns, exons, and 5 ́and 3 ́UTR regions. The effects of a SPAX8-related mutation, in the amino acid 41, was verified by microscopy, western blot, and a home-made luciferase activity assay where the NKX6-2 DNA response element was inserted in a pRL miniTK-luciferase system. We tried unsuccessfully to restore the normal phenotypes by means of a complementary NKX6- 2-Flagtag-P2A-T2A-mCherry fusion protein, that contains the amino acids 35 to 277. Normal, full-length NKX6-2 had an exclusively nuclear localization, and its overexpression induced a change in the morphology of cell nuclei. SPAX8-causing mutations in NKX6-2 led to an even distribution in both nuclei and cytoplasm, indicating a loss of the nuclear localization signal, and in one case protein aggregation. Transfection of cells with SPAX8-mutant NKX6-2 and its complementary fragment showed a partial increase in truncated NKX6-2 in the nucleus. Our NKX6-2 luciferase assay was able to detect NKX6-2 transcriptional repression, which was lost in a SPAX8 mutant and not recovered by the complementary fragment. We have created new cellular models for the study of SPAX8 cellular and molecular alterations. Our attempt to restore mutant NKX6-2 function by means of a complementary fragment was functionally ineffective, but the fact that we could increase the nuclear localization of the mutant protein suggests that other complementary fragments could be more effective. Furthermore, we obtained basic information about the domains involved in the nuclear localization and transcriptional repressor activity of NKX6-2. Although our attempts to study NMD in this context were unsuccessful, we are still trying to sort out its potential role in SPAX8, and the minigene we designed could be a good starting point. If we succeed, our efforts could be applied to other genetic disorders involving PTCs, which are associated to one-third of inherited human diseases. |
id |
RCAP_74f172ae06fe76369177726475088769 |
---|---|
oai_identifier_str |
oai:run.unl.pt:10362/138619 |
network_acronym_str |
RCAP |
network_name_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository_id_str |
7160 |
spelling |
Restoring NKX6-2 function by protein complementation: a proof-of-conceptNKX6-2Spastic Ataxia 8rotein complementationtranscriptional factorpRL miniTK-luciferase systemDomínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e TecnologiasNKX6-2 is a transcriptional repressor involved in the regulation of various genes associated with cell fate determination of neurons, oligodendrocytes, or pancreatic cells, as well as glucagon production, among other phenomena. Loss-of-function mutations in NKX6-2 are the cause of Spastic Ataxia 8 (SPAX8), a rare recessive hereditary disease that leads to an early-onset hypomyelinating leukodystrophy due to the lack of oligodendrocyte maturation. The most common disease-associated NKX6-2 mutations involve the formation of premature termination codons (PTCs). These PTCs will render the nascent mRNA a potential target for degradation via nonsense-mediated mRNA decay (NMD). The small fraction of mRNA that survives NMD and is eventually translated to protein will produce truncated, non-functional NKX6-2 proteins. We aimed to overcome these two issues to lay the groundwork for the development of new therapies for SPAX8. Using site-directed mutagenesis, we were able to recreate SPAX8-related mutations on a cDNA-based NKX6-2-Venus fusion protein, but not in a NKX6-2 minigene including its introns, exons, and 5 ́and 3 ́UTR regions. The effects of a SPAX8-related mutation, in the amino acid 41, was verified by microscopy, western blot, and a home-made luciferase activity assay where the NKX6-2 DNA response element was inserted in a pRL miniTK-luciferase system. We tried unsuccessfully to restore the normal phenotypes by means of a complementary NKX6- 2-Flagtag-P2A-T2A-mCherry fusion protein, that contains the amino acids 35 to 277. Normal, full-length NKX6-2 had an exclusively nuclear localization, and its overexpression induced a change in the morphology of cell nuclei. SPAX8-causing mutations in NKX6-2 led to an even distribution in both nuclei and cytoplasm, indicating a loss of the nuclear localization signal, and in one case protein aggregation. Transfection of cells with SPAX8-mutant NKX6-2 and its complementary fragment showed a partial increase in truncated NKX6-2 in the nucleus. Our NKX6-2 luciferase assay was able to detect NKX6-2 transcriptional repression, which was lost in a SPAX8 mutant and not recovered by the complementary fragment. We have created new cellular models for the study of SPAX8 cellular and molecular alterations. Our attempt to restore mutant NKX6-2 function by means of a complementary fragment was functionally ineffective, but the fact that we could increase the nuclear localization of the mutant protein suggests that other complementary fragments could be more effective. Furthermore, we obtained basic information about the domains involved in the nuclear localization and transcriptional repressor activity of NKX6-2. Although our attempts to study NMD in this context were unsuccessful, we are still trying to sort out its potential role in SPAX8, and the minigene we designed could be a good starting point. If we succeed, our efforts could be applied to other genetic disorders involving PTCs, which are associated to one-third of inherited human diseases.NKX6-2 é um repressor de transcrição envolvido na regulação de vários genes associados na determinação do destino celular dos neurónios, oligodendrócito e células pancreáticas, estando também associado com a produção de glucagon. Mutações que levam a perda de função no NKX6-2 causam Spastic Ataxia 8 (SPAX8), uma patologia genética rara de transmissão recessiva, que leva a uma leucodistrofia hipomielinizante de início precoce devido à falta de maturação dos oligodendrócitos. As mutações mais associadas a SPAX8 são as que levam a formação de codões STOP prematuros (PTCs). Estes tornam o mRNA nascente num potencial alvo de degradação pelo sistema non-mediated mRNA decay (NMD). A pequena fração de mRNAs que escapam o sistema eventualmente são traduzidas numa proteína truncada não funcional. Neste projeto queríamos superar estes dois problemas de forma a desenvolver ferramentas que possam levar a novas terapias para SPAX8. Recriámos as várias mutações associadas a SPAX8 na proteína de fusão NKX6-2-Venus através de site-directed mutagenesis, mas não no plasmideo com a proteína NKX6-2 minigene, que inclui os intrões, exões e as regiões 5’ e 3’-UTR. Verificamos os efeitos nas mutações relacionadas com o SPAX8, no aminoácido 41, através de microscopia, western blot e nova ferramenta estabelecida, um ensaio de atividade a partir do sistema pRL miniTK-luciferase onde inserimos o elemento de resposta do NKX6-2. Tentamos, sem sucesso, restaurar o fenótipo normal por meio de complementação com a proteína de fusão NKX6-2-Flagtag-P2A-T2A-mCherry, que inclui os aminoácidos 35 a 277. A proteína NKX6-2 possui normalmente uma localização exclusivamente nuclear, porém quando expressa constitutivamente induz uma mudança morfológica no núcleo. As mutações associadas a SPAX8 levaram a uma distribuição uniforme no citoplasma e no núcleo. Células transfectadas com o mutante NKX6-2 e o seu fragmento complementar mostrou um aumento parcial do NKX6-2 truncado no núcleo. O nosso ensaio de atividade consegue detetar a atividade repressora do NKX6-2, porém não deteta recuperação no mutante SPAX8 após complementação. Criamos novos modelos celulares capazes de estudar as alterações celulares e moleculares causadas por SPAX8. A nossa tentativa de recuperar a função do mutante NKX6-2 através do complementação proteica foi ineficaz, contudo o facto que houve um aumento da sua presença no núcleo sugere que a modificação do fragmento complementar poderá provar-se mais eficazes. Obtemos, também, informação básica sobre os domínios envolvidos na localização nuclear e atividade repressora do NKX6-2. Embora as tentativas de estudar o NMD neste contexto não tenham resultado, ainda estamos a tentar descobrir o seu papel na doença SPAX8, e o plasmídeo contento o minigene será um bom ponto de partida. Se tivermos sucesso, o método poderá ser aplicado em outras doenças genéticas que envolvem PTCs, que estão associados a um terço das doenças hereditárias.Herrera, FedericoCaldas, MargaridaRUNPeralta, Pedro Emanuel Ferreira2022-05-25T14:55:18Z2022-012022-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/138619enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:15:56Zoai:run.unl.pt:10362/138619Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:49:09.483246Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Restoring NKX6-2 function by protein complementation: a proof-of-concept |
title |
Restoring NKX6-2 function by protein complementation: a proof-of-concept |
spellingShingle |
Restoring NKX6-2 function by protein complementation: a proof-of-concept Peralta, Pedro Emanuel Ferreira NKX6-2 Spastic Ataxia 8 rotein complementation transcriptional factor pRL miniTK-luciferase system Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
title_short |
Restoring NKX6-2 function by protein complementation: a proof-of-concept |
title_full |
Restoring NKX6-2 function by protein complementation: a proof-of-concept |
title_fullStr |
Restoring NKX6-2 function by protein complementation: a proof-of-concept |
title_full_unstemmed |
Restoring NKX6-2 function by protein complementation: a proof-of-concept |
title_sort |
Restoring NKX6-2 function by protein complementation: a proof-of-concept |
author |
Peralta, Pedro Emanuel Ferreira |
author_facet |
Peralta, Pedro Emanuel Ferreira |
author_role |
author |
dc.contributor.none.fl_str_mv |
Herrera, Federico Caldas, Margarida RUN |
dc.contributor.author.fl_str_mv |
Peralta, Pedro Emanuel Ferreira |
dc.subject.por.fl_str_mv |
NKX6-2 Spastic Ataxia 8 rotein complementation transcriptional factor pRL miniTK-luciferase system Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
topic |
NKX6-2 Spastic Ataxia 8 rotein complementation transcriptional factor pRL miniTK-luciferase system Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
description |
NKX6-2 is a transcriptional repressor involved in the regulation of various genes associated with cell fate determination of neurons, oligodendrocytes, or pancreatic cells, as well as glucagon production, among other phenomena. Loss-of-function mutations in NKX6-2 are the cause of Spastic Ataxia 8 (SPAX8), a rare recessive hereditary disease that leads to an early-onset hypomyelinating leukodystrophy due to the lack of oligodendrocyte maturation. The most common disease-associated NKX6-2 mutations involve the formation of premature termination codons (PTCs). These PTCs will render the nascent mRNA a potential target for degradation via nonsense-mediated mRNA decay (NMD). The small fraction of mRNA that survives NMD and is eventually translated to protein will produce truncated, non-functional NKX6-2 proteins. We aimed to overcome these two issues to lay the groundwork for the development of new therapies for SPAX8. Using site-directed mutagenesis, we were able to recreate SPAX8-related mutations on a cDNA-based NKX6-2-Venus fusion protein, but not in a NKX6-2 minigene including its introns, exons, and 5 ́and 3 ́UTR regions. The effects of a SPAX8-related mutation, in the amino acid 41, was verified by microscopy, western blot, and a home-made luciferase activity assay where the NKX6-2 DNA response element was inserted in a pRL miniTK-luciferase system. We tried unsuccessfully to restore the normal phenotypes by means of a complementary NKX6- 2-Flagtag-P2A-T2A-mCherry fusion protein, that contains the amino acids 35 to 277. Normal, full-length NKX6-2 had an exclusively nuclear localization, and its overexpression induced a change in the morphology of cell nuclei. SPAX8-causing mutations in NKX6-2 led to an even distribution in both nuclei and cytoplasm, indicating a loss of the nuclear localization signal, and in one case protein aggregation. Transfection of cells with SPAX8-mutant NKX6-2 and its complementary fragment showed a partial increase in truncated NKX6-2 in the nucleus. Our NKX6-2 luciferase assay was able to detect NKX6-2 transcriptional repression, which was lost in a SPAX8 mutant and not recovered by the complementary fragment. We have created new cellular models for the study of SPAX8 cellular and molecular alterations. Our attempt to restore mutant NKX6-2 function by means of a complementary fragment was functionally ineffective, but the fact that we could increase the nuclear localization of the mutant protein suggests that other complementary fragments could be more effective. Furthermore, we obtained basic information about the domains involved in the nuclear localization and transcriptional repressor activity of NKX6-2. Although our attempts to study NMD in this context were unsuccessful, we are still trying to sort out its potential role in SPAX8, and the minigene we designed could be a good starting point. If we succeed, our efforts could be applied to other genetic disorders involving PTCs, which are associated to one-third of inherited human diseases. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-05-25T14:55:18Z 2022-01 2022-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10362/138619 |
url |
http://hdl.handle.net/10362/138619 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
_version_ |
1799138091021107200 |