Caracterização funcional, bioquímica e na resposta a estresse das ATPases RVB-1 e RVB-2 e da ciclina PCL-1 no fungo filamentoso Neurospora crassa
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Data
2022-08-25
Autores
Campanella, Jonatas Erick Maimoni
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Universidade Estadual Paulista (Unesp)
Resumo
O fungo Neurospora crassa está entre os organismos mais bem compreendidos, sendo um fungo filamentoso amplamente utilizado como organismo modelo. Em nosso laboratório, diferentes proteínas foram previamente identificadas como reguladoras do metabolismo de glicogênio em diferentes condições de crescimento. Neste trabalho, algumas destas proteínas foram mais detalhadamente caracterizadas do ponto de vista bioquímico e funcional. A função da ciclina PCL-1, anteriormente identificada como uma proteína parceira da quinase PHO85-1, foi mais amplamente investigada mostrando funções adicionais à regulação do ciclo celular e metabolismo de glicogênio. Foi demonstrado que esta ciclina atua também na resposta a estresse, principalmente aquele induzido por altas concentrações de cálcio extracelular, possivelmente através da fosforilação de CRZ-1, um fator de transcrição responsivo a cálcio. O conjunto de resultados mostra o papel multifuncional desta ciclina no fungo N. crassa, diferente da levedura Saccharomyces cerevisiae, na qual ciclinas de uma família são direcionadas às mesmas funções celulares. A proteína RVB-1, anteriormente identificada como capaz de se ligar ao promotor gsn, foi também amplamente caracterizada neste trabalho. RVB-1, juntamente com sua paráloga RVB-2, pertence à família AAA+ ATPases (ATPases Associated with Diverse Celular Activities) conhecidas por interagir com múltiplos complexos proteicos incluindo remodeladores da cromatina e amplamente associadas ao desenvolvimento celular e câncer. Ambas as proteínas são essenciais ao fungo, tem localização nuclear e influenciam no seu desenvolvimento e produção de metabolitos secundários. O complexo RVB-1/RVB-2 foi produzido na forma recombinante em E. coli e caracterizado quanto aos aspectos estruturais e bioquímicos. O complexo mostrou atividade ATPase, capacidade de se ligar a fragmentos de DNA e apresentou diferentes formas oligoméricas em solução, sendo que a presença dos nucleotídeos ATP/ADP influencia no estado de oligomerização. A expressão de ambas as proteínas é modulada por diferentes estresses e ensaios de imunoprecipitação da proteína RVB-1 levaram à identificação de numerosas proteínas parceiras. Algumas interações foram caracterizadas neste trabalho. A interação in vivo com a proteína glicogenina (GNN), juntamente com a redução no conteúdo de glicogênio nas linhagens mutantes rvb-1het e rvb-2het indicou a participação do complexo na regulação do metabolismo de glicogênio. A interação in vitro com a parceira shugoshin (SGO-1), descrita como guardiã da resposta ao estresse térmico em mamífero, foi demonstrada comprovando o papel na resposta ao estresse. Finalmente, linhagens do fungo mutantes em componentes de complexos de remodelagem de cromatina, identificados como parceiros, são severamente sensíveis a estresse, evidenciando um papel funcional ainda não descrito para estas proteínas: a participação na resposta a estresse.
Neurospora crassa is one of the most studied organisms being a filamentous fungus that is often used as a model organism. Several proteins have already been identified in our laboratory as regulators of glycogen metabolism under different growth conditions. In this work, some of these proteins were biochemically and functionally charactrized. The function of the PCL-1 cyclin, previously identified as a partner protein of the PHO85-1 kinase, was investigated and the results revealed additional functions rather than those in cell cycle regulation and glycogen metabolism. This cyclin has shown to act on the stress response mainly the one triggered by high concentrations of extracellular calcium possibly through phosphorylation of CRZ-1, a calcium-responsive transcription factor. The results demonstrate the multifunctional role of this cyclin in N. crassa, unlike the yeast Saccharomyces cerevisiae, in which different cyclins from one family target to the same cellular functions. The protein RVB-1, previously identified as capable of binding to the gsn promoter, was also extensively characterized in this work. RVB-1, together with its RVB-2 paralog, belongs to the family of AAA + ATPases (ATPases Associated with Diverse Cell Activities), which are known to interact with several protein complexes, including chromatin remodelers, and are widely associated with cell development and cancer. Both proteins are essential for the fungus, have nuclear localization, and influence the fungus development and production of secondary metabolites. The recombinant RVB-1/RVB-2 complex was produced in E. coli and characterized under structural and biochemical aspects. The complex showed ATPase activity, ability to bind to DNA fragments, and presented different oligomeric states in solution depending on the presence or not of the ATP/ADP nucleotides. The in vivo expression of both proteins is modulated by various stress agents, and RVB-1 immunoprecipitation assays led to the identification of numerous proteins partners. Some interactions were characterized in this work. The in vivo interaction with the glycogenin protein (GNN) and the reduction of the glycogen levels in the mutant strains Δrvb-1het and Δrvb-2het suggest the involvement of the complex in the regulation of glycogen metabolism. The in vitro interaction with the Shugoshin partner (SGO-1), described as a guardian of the mammalian heat stress response, was detected demonstrating its role in the stress response. Finally, mutant strains in components of chromatin remodeling complexes, identified as partners, are highly sensitive to stress, indicating a functional role not yet described for these proteins: their involvement in stress response.
Neurospora crassa is one of the most studied organisms being a filamentous fungus that is often used as a model organism. Several proteins have already been identified in our laboratory as regulators of glycogen metabolism under different growth conditions. In this work, some of these proteins were biochemically and functionally charactrized. The function of the PCL-1 cyclin, previously identified as a partner protein of the PHO85-1 kinase, was investigated and the results revealed additional functions rather than those in cell cycle regulation and glycogen metabolism. This cyclin has shown to act on the stress response mainly the one triggered by high concentrations of extracellular calcium possibly through phosphorylation of CRZ-1, a calcium-responsive transcription factor. The results demonstrate the multifunctional role of this cyclin in N. crassa, unlike the yeast Saccharomyces cerevisiae, in which different cyclins from one family target to the same cellular functions. The protein RVB-1, previously identified as capable of binding to the gsn promoter, was also extensively characterized in this work. RVB-1, together with its RVB-2 paralog, belongs to the family of AAA + ATPases (ATPases Associated with Diverse Cell Activities), which are known to interact with several protein complexes, including chromatin remodelers, and are widely associated with cell development and cancer. Both proteins are essential for the fungus, have nuclear localization, and influence the fungus development and production of secondary metabolites. The recombinant RVB-1/RVB-2 complex was produced in E. coli and characterized under structural and biochemical aspects. The complex showed ATPase activity, ability to bind to DNA fragments, and presented different oligomeric states in solution depending on the presence or not of the ATP/ADP nucleotides. The in vivo expression of both proteins is modulated by various stress agents, and RVB-1 immunoprecipitation assays led to the identification of numerous proteins partners. Some interactions were characterized in this work. The in vivo interaction with the glycogenin protein (GNN) and the reduction of the glycogen levels in the mutant strains Δrvb-1het and Δrvb-2het suggest the involvement of the complex in the regulation of glycogen metabolism. The in vitro interaction with the Shugoshin partner (SGO-1), described as a guardian of the mammalian heat stress response, was detected demonstrating its role in the stress response. Finally, mutant strains in components of chromatin remodeling complexes, identified as partners, are highly sensitive to stress, indicating a functional role not yet described for these proteins: their involvement in stress response.
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Palavras-chave
Bioquímica, Espectrometria de massas, Proteínas de choque térmico, Western blotting, Neurospora crassa