The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10773/30766 |
Resumo: | Parkinson’s disease (PD) is a progressive neurodegenerative disorder with no cure, characterized by the loss of dopaminergic neurons in the brain's substantia nigra pars compacta. Despite extensive research, the exac t causes and mechanisms leading to the development of PD are still unknown. Among the hereditary forms of PD, mutations in the DJ - 1 gene have been associated with an autosomal recessive early -onset disease type. The DJ -1 protein plays an important role in the defensive response to oxidative stress, in which the modulation of signaling pathways is pointed out as a central mechanism. While such cytoprotective function has been most described at the intracellular level, increasing evidence also confirm s the ability of the protein to mediate a protective effect from the extracellular space upon its secretion. However, the precise mode of action of the extracellular DJ -1 or the pathological implications of its mutations is still misunderstood. In this sense, the aim of this work was to elucidate the mechanisms of action of extracellular DJ -1 with particular consideration to its role in signaling pathways modulation. To accomplish this, an interatomic study was performed to identify the network of interactions esta blished by DJ - 1 in the extracellular space, followed by a phospho -motif profiling analysis to assess the pathological impact of DJ -1 mutants in kinase - related signaling. From the interactomic analysis of the secretome of SH -S5Y5 cells after the addition o f exogenous DJ - 1 in the presence or absence of oxidative stress , it was possible to identify a set of 28 potential extracellular DJ - 1 -interactors possibly involved in the neuroprotective action exerted by DJ - 1. Additionally, the modulation of such interactions was also assessed in oxidative stress conditions. The list of potential interactors identified in this work confirms an important involvement of DJ -1 in the modulation of signaling pathways from the extracellular environment. TSP1, THBR, MIF , and CAD1 1 , some of the binding partners of DJ - 1 identified in this work, are known to be involved in the regulation of some signaling pathways such as PI3K/Akt, TGF -β, ERK1/2, Wnt , and PLD - related signaling. In turn, from the “semi -targeted” phospho -motif profiling assay aimed to assess the differential modulated signaling events by the exogenous addition of DJ - 1 native and missense mutants M26I and E163K, it was possible to confirm that both mutations elicited alterations in the kinase - mediated substrate phosphorylation profile in comparison to the condition of WT -DJ -1 extracellular stimulated SH -S5Y5 cells. In fact, a generally decreased kinase activity was observed, with the E163K -DJ -1 isoform exhibiting a more prominent impact associated with a significant reduction i n the activity of Akt, ATM/ATR, AMPK, CK2, CDKs , and PKC. Nevertheless, the results also denote that both mutants of DJ -1 had a similar pattern of alteration in the phosphorylation of some of the substrates analyzed , which could be an indication of convergent pathological mechanisms. In conclusion, this work contributed with important knowledge into the biological role of extracellular DJ -1 in the modulation of signaling pathways and elucidated the impact of mutations in the activity of the protein. Following studies should be conducted to validate the obtained results and support the hypotheses raised in this work. Ultimately, the presen t findings also reflect valuable insights into the understanding of the molecular mechanisms implicated in PD. |
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The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s diseaseParkinson’s diseaseDJ -1NeuroprotectionOxidative stressSignaling pathwaysInteractomicsPhosphorylation patternsParkinson’s disease (PD) is a progressive neurodegenerative disorder with no cure, characterized by the loss of dopaminergic neurons in the brain's substantia nigra pars compacta. Despite extensive research, the exac t causes and mechanisms leading to the development of PD are still unknown. Among the hereditary forms of PD, mutations in the DJ - 1 gene have been associated with an autosomal recessive early -onset disease type. The DJ -1 protein plays an important role in the defensive response to oxidative stress, in which the modulation of signaling pathways is pointed out as a central mechanism. While such cytoprotective function has been most described at the intracellular level, increasing evidence also confirm s the ability of the protein to mediate a protective effect from the extracellular space upon its secretion. However, the precise mode of action of the extracellular DJ -1 or the pathological implications of its mutations is still misunderstood. In this sense, the aim of this work was to elucidate the mechanisms of action of extracellular DJ -1 with particular consideration to its role in signaling pathways modulation. To accomplish this, an interatomic study was performed to identify the network of interactions esta blished by DJ - 1 in the extracellular space, followed by a phospho -motif profiling analysis to assess the pathological impact of DJ -1 mutants in kinase - related signaling. From the interactomic analysis of the secretome of SH -S5Y5 cells after the addition o f exogenous DJ - 1 in the presence or absence of oxidative stress , it was possible to identify a set of 28 potential extracellular DJ - 1 -interactors possibly involved in the neuroprotective action exerted by DJ - 1. Additionally, the modulation of such interactions was also assessed in oxidative stress conditions. The list of potential interactors identified in this work confirms an important involvement of DJ -1 in the modulation of signaling pathways from the extracellular environment. TSP1, THBR, MIF , and CAD1 1 , some of the binding partners of DJ - 1 identified in this work, are known to be involved in the regulation of some signaling pathways such as PI3K/Akt, TGF -β, ERK1/2, Wnt , and PLD - related signaling. In turn, from the “semi -targeted” phospho -motif profiling assay aimed to assess the differential modulated signaling events by the exogenous addition of DJ - 1 native and missense mutants M26I and E163K, it was possible to confirm that both mutations elicited alterations in the kinase - mediated substrate phosphorylation profile in comparison to the condition of WT -DJ -1 extracellular stimulated SH -S5Y5 cells. In fact, a generally decreased kinase activity was observed, with the E163K -DJ -1 isoform exhibiting a more prominent impact associated with a significant reduction i n the activity of Akt, ATM/ATR, AMPK, CK2, CDKs , and PKC. Nevertheless, the results also denote that both mutants of DJ -1 had a similar pattern of alteration in the phosphorylation of some of the substrates analyzed , which could be an indication of convergent pathological mechanisms. In conclusion, this work contributed with important knowledge into the biological role of extracellular DJ -1 in the modulation of signaling pathways and elucidated the impact of mutations in the activity of the protein. Following studies should be conducted to validate the obtained results and support the hypotheses raised in this work. Ultimately, the presen t findings also reflect valuable insights into the understanding of the molecular mechanisms implicated in PD.A doença de Parkinson (DP) é uma doença neurodegenerativa progressiva sem cura, que é caracterizada pela perda de neurónios dopaminérgicos na substantia nigra pars compacta no cérebro. Apesar da ampla investigação, as causas e mecanismos exatos que despoletam o seu desenvolvimento permanecem desconhecidos. Entre as formas hereditárias da DP, mutações no gene DJ-1 foram associadas a um tipo autossómico recessivo da doença com início precoce. A proteína DJ-1 tem um papel fundamental na resposta defensora ao stress oxidativo, na qual a modulação de vias de sinalização revela ser um mecanismo central. Embora essa função citoprotetora tenha sido mais estudada a nível intracelular, crescentes evidências também comprovam a capacidade da proteína de desencadear um efeito protetor ao nível extracelular quando secretada. Porém, o modo de ação da DJ-1 no ambiente extracelular bem como as implicações patológicas de mutações da proteína nessa função permanecem por clarificar. Neste sentido, o objetivo deste trabalho foi elucidar os mecanismos de ação da DJ-1 extracelular, considerando em particular o seu papel de regulação de vias de sinalização. Para tal, foi realizado um ensaio de interactómica para identificar a rede de interações estabelecidas pela DJ-1 no espaço extracelular, seguido de uma análise semi-direccionada para avaliar o impacto patológico de formas mutantes da proteína na sinalização mediada por cinases. O ensaio de interatómica realizado em secretomas de células SH-SY5Y após a adição extracelular de DJ-1 na presença ou ausência de stress oxidativo permitiu identificar um grupo de 28 potenciais interatores extracelulares da DJ1, que poderão estar envolvidos na ação neuroprotetora mediada pela proteína. Além disso, foi também determinada a modulação destas interações em condições de stress oxidativo. Os potenciais interatores encontrados confirmam um importante envolvimento da DJ-1 na modulação de vias de sinalização a partir do espaço extracelular. TSP1, THBR, MIF e CAD11, alguns dos potenciais interatores da DJ-1 identificados neste trabalho, estão envolvidos na regulação de diversas vias de sinalização, tais como: PI3K/Akt, TGF-β, ERK1/2, Wnt e PLD. Por sua vez, da análise “semi-direccionada” de perfis de fosforilação focada na identificação dos eventos de sinalização mediados diferencialmente pela adição exógena de DJ-1 nas formas nativa ou associadas às mutações missense M26I e E163K, foi possível confirmar que ambas as mutações consideradas promoveram alterações no perfil de fosforilação mediada por cinases, em comparação com a adição extracelular da forma nativa da proteína DJ-1 em células SH-SY5Y. De facto, foi observada uma diminuição geral de atividade de cinases, e a isoforma E163K-DJ-1 exibiu um maior impacto levando a um significativo decréscimo de atividade das cinases Akt, ATM/ATR, AMPK, CK2, CDKs e PKC. Porém, os resultados também denotam que as duas formas mutantes da proteína exibem um padrão semelhante de alteração de fosforilação de alguns substratos, o que poderá ser uma indicação de potenciais mecanismos patológicos convergentes. Assim, o presente trabalho contribuiu para o aumento do conhecimento do papel extracelular da DJ-1 na regulação de vias de sinalização e permitiu verificar o impacto de mutações na atividade da proteína. Deverão ser conduzidos estudos posteriores de forma a validar os resultados obtidos e corroborar as hipóteses apresentadas. Por fim, as evidências adquiridas refletem também informações relevantes para a compreensão dos mecanismos moleculares implicados na DP.2023-02-22T00:00:00Z2021-02-12T00:00:00Z2021-02-12info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/30766engNeves, Margarida Seco Martins Marquesinfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-22T11:59:25Zoai:ria.ua.pt:10773/30766Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:02:46.377258Repositório 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 |
The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease |
title |
The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease |
spellingShingle |
The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease Neves, Margarida Seco Martins Marques Parkinson’s disease DJ -1 Neuroprotection Oxidative stress Signaling pathways Interactomics Phosphorylation patterns |
title_short |
The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease |
title_full |
The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease |
title_fullStr |
The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease |
title_full_unstemmed |
The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease |
title_sort |
The extracellular role of DJ-1 in the regulation of signaling pathways: implications for Parkinson’s disease |
author |
Neves, Margarida Seco Martins Marques |
author_facet |
Neves, Margarida Seco Martins Marques |
author_role |
author |
dc.contributor.author.fl_str_mv |
Neves, Margarida Seco Martins Marques |
dc.subject.por.fl_str_mv |
Parkinson’s disease DJ -1 Neuroprotection Oxidative stress Signaling pathways Interactomics Phosphorylation patterns |
topic |
Parkinson’s disease DJ -1 Neuroprotection Oxidative stress Signaling pathways Interactomics Phosphorylation patterns |
description |
Parkinson’s disease (PD) is a progressive neurodegenerative disorder with no cure, characterized by the loss of dopaminergic neurons in the brain's substantia nigra pars compacta. Despite extensive research, the exac t causes and mechanisms leading to the development of PD are still unknown. Among the hereditary forms of PD, mutations in the DJ - 1 gene have been associated with an autosomal recessive early -onset disease type. The DJ -1 protein plays an important role in the defensive response to oxidative stress, in which the modulation of signaling pathways is pointed out as a central mechanism. While such cytoprotective function has been most described at the intracellular level, increasing evidence also confirm s the ability of the protein to mediate a protective effect from the extracellular space upon its secretion. However, the precise mode of action of the extracellular DJ -1 or the pathological implications of its mutations is still misunderstood. In this sense, the aim of this work was to elucidate the mechanisms of action of extracellular DJ -1 with particular consideration to its role in signaling pathways modulation. To accomplish this, an interatomic study was performed to identify the network of interactions esta blished by DJ - 1 in the extracellular space, followed by a phospho -motif profiling analysis to assess the pathological impact of DJ -1 mutants in kinase - related signaling. From the interactomic analysis of the secretome of SH -S5Y5 cells after the addition o f exogenous DJ - 1 in the presence or absence of oxidative stress , it was possible to identify a set of 28 potential extracellular DJ - 1 -interactors possibly involved in the neuroprotective action exerted by DJ - 1. Additionally, the modulation of such interactions was also assessed in oxidative stress conditions. The list of potential interactors identified in this work confirms an important involvement of DJ -1 in the modulation of signaling pathways from the extracellular environment. TSP1, THBR, MIF , and CAD1 1 , some of the binding partners of DJ - 1 identified in this work, are known to be involved in the regulation of some signaling pathways such as PI3K/Akt, TGF -β, ERK1/2, Wnt , and PLD - related signaling. In turn, from the “semi -targeted” phospho -motif profiling assay aimed to assess the differential modulated signaling events by the exogenous addition of DJ - 1 native and missense mutants M26I and E163K, it was possible to confirm that both mutations elicited alterations in the kinase - mediated substrate phosphorylation profile in comparison to the condition of WT -DJ -1 extracellular stimulated SH -S5Y5 cells. In fact, a generally decreased kinase activity was observed, with the E163K -DJ -1 isoform exhibiting a more prominent impact associated with a significant reduction i n the activity of Akt, ATM/ATR, AMPK, CK2, CDKs , and PKC. Nevertheless, the results also denote that both mutants of DJ -1 had a similar pattern of alteration in the phosphorylation of some of the substrates analyzed , which could be an indication of convergent pathological mechanisms. In conclusion, this work contributed with important knowledge into the biological role of extracellular DJ -1 in the modulation of signaling pathways and elucidated the impact of mutations in the activity of the protein. Following studies should be conducted to validate the obtained results and support the hypotheses raised in this work. Ultimately, the presen t findings also reflect valuable insights into the understanding of the molecular mechanisms implicated in PD. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-02-12T00:00:00Z 2021-02-12 2023-02-22T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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http://hdl.handle.net/10773/30766 |
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eng |
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