Caracterização funcional da proteína TMP-1 do fungo Neurospora crassa
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Data
2022-05-20
Autores
Paula, Mariana Sousa de
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Neurospora crassa é um organismo pertencente ao Reino dos Fungos e tem
desempenhado um papel muito importante no conhecimento de mecanismos
celulares em eucariotos, devido ao seu genoma já sequenciado e sua fácil
manipulação. Entretanto, cerca de 60% das proteínas de N. crassa não foram
caracterizadas funcionalmente. Dentre tais proteínas, destaca-se o objeto deste
estudo, a proteína transmembrana TMP-1, a qual foi revelada ser regulada pelo fator
de transcrição FLB-3. Em Aspergillus nidulans, a proteína TmpA, ortóloga a TMP-1 de
N. crassa, foi descrita como participante na regulação do desenvolvimento assexual.
O objetivo deste trabalho foi caracterizar funcionalmente a proteína TMP-1. Para tanto,
foi realizada a avaliação fenotípica da linhagem Δtmp-1 em diferentes condições de
estresse e em comparação à linhagem selvagem pela avaliação do crescimento em
placa e análises macro e microscópica. Para os estudos de localização celular foi
construída uma linhagem produzindo a proteína TMP-1 fusionada ao tag GFP (TMP-1-GFP). Os resultados obtidos sugerem que não houve diferença fenotípica entre os
micélios das linhagens WT e Δtmp-1, porém houve uma diminuição na produção de
microconídeos, em comparação à linhagem selvagem. Nas condições de estresse,
houve diferença relevante quando a linhagem foi exposta ao Congo Red, agente que
altera a integridade da membrana e ao meio sem carbono, sugerindo que esta
proteína possa estar envolvida com estresse de parede celular e metabolismo de
carboidratos. Quanto à sua localização, os resultados mostraram que a proteína TMP-1 está localizada na membrana e intracelularmente de forma pontual, mas parece não
estar relacionada ao processo de germinação de N. crassa. Novos estudos são
necessários para melhor compreensão do papel funcional desta proteína no fungo N.
crassa, bem como quais vias metabólicas a proteína está envolvida.
Neurospora crassa is an organism belonging to the Kingdom of Fungi and has played a very important role in the knowledge of the cellular mechanisms in eukaryotes due to its sequenced genome and its easy manipulation. However, about 60% of the N. crassa proteins have not been functionally characterized. Among such proteins, we highlight the object of this study, the transmembrane protein TMP-1, previously described as regulated by the transcription factor FLB-3. In Aspergillus nidulans, the TmpA protein, ortholog of the N. crassa TMP-1, was described to participate in the regulation of the asexual development. The objective of this work is to functionally characterize the TMP-1 protein and to evaluate its potential influence on the asexual development of the fungus and its localization. For this, we performed a phenotypic evaluation of the Δtmp-1 strain and compared to the wild-type (WT) strain by plaque growth assay and macro and microscopic analysis. For cellular localization studies, a strain producing the TMP-1 protein fused to the GFP tag (TMP-1-GFP) was constructed. The results showed that there was no phenotypic difference between mycelia of the WT and Δtmp-1 strains; however, there was a decrease in the production of microconidia compared to the WT strain. Under stress conditions, we observed a relevant difference when the mutant strain was exposed to Congo Red, an agent that affects the membrane integrity, and to a medium without carbon, suggesting that this protein may be involved in cell wall stress and carbohydrate metabolism. Regarding cellular localization, the results showed that the TMP-1 protein is localized in the membrane and intracellularly in a punctual way, and it is not related to the germination process of N. crassa. Further studies are needed to better understand the functional role of the protein, as well as which metabolic pathways the protein is involved.
Neurospora crassa is an organism belonging to the Kingdom of Fungi and has played a very important role in the knowledge of the cellular mechanisms in eukaryotes due to its sequenced genome and its easy manipulation. However, about 60% of the N. crassa proteins have not been functionally characterized. Among such proteins, we highlight the object of this study, the transmembrane protein TMP-1, previously described as regulated by the transcription factor FLB-3. In Aspergillus nidulans, the TmpA protein, ortholog of the N. crassa TMP-1, was described to participate in the regulation of the asexual development. The objective of this work is to functionally characterize the TMP-1 protein and to evaluate its potential influence on the asexual development of the fungus and its localization. For this, we performed a phenotypic evaluation of the Δtmp-1 strain and compared to the wild-type (WT) strain by plaque growth assay and macro and microscopic analysis. For cellular localization studies, a strain producing the TMP-1 protein fused to the GFP tag (TMP-1-GFP) was constructed. The results showed that there was no phenotypic difference between mycelia of the WT and Δtmp-1 strains; however, there was a decrease in the production of microconidia compared to the WT strain. Under stress conditions, we observed a relevant difference when the mutant strain was exposed to Congo Red, an agent that affects the membrane integrity, and to a medium without carbon, suggesting that this protein may be involved in cell wall stress and carbohydrate metabolism. Regarding cellular localization, the results showed that the TMP-1 protein is localized in the membrane and intracellularly in a punctual way, and it is not related to the germination process of N. crassa. Further studies are needed to better understand the functional role of the protein, as well as which metabolic pathways the protein is involved.
Descrição
Palavras-chave
Neurospora crassa, proteína TMP-1, localização celular, desenvolvimento assexual, TMP-1 protein, cellular location, asexual development