Estudos estruturais com a Importina-α de Leishmania major e sequência de localização nuclear de proteína do metabolismo de Leishmania
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Data
2021-05-26
Autores
Fukuda, Cintia Akemi
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Universidade Estadual Paulista (Unesp)
Resumo
A comunicação entre o núcleo celular e o citoplasma acontece por mecanismos de transporte que permitem a passagem de moléculas por poros presentes no envoltório nuclear. Dentre as vias de transporte entre o núcleo e o citoplasma celular conhecidas, a chamada Via Clássica de Importação Nuclear é a mais bem caracterizada. Nessa via, a proteína Importina-α (Impα) se liga às proteínas que serão transportadas ao núcleo a partir do reconhecimento de sequências de localização nuclear (NLS). A estrutura da proteína Impα já foi elucidada e caracterizada em alguns organismos. Atualmente estas proteínas são classificadas em três subfamílias: α1, α2 e α3. As diferenças entre as proteínas Impα de cada família evidenciam as especificidades destas no reconhecimento de NLSs, dependendo do organismo e função que exercem. Ou seja, uma mesma região da proteína pode apresentar variações na afinidade e no modo de ligação quando interage com Impα de famílias distintas. A leishmaniose é uma doença presente em diversas regiões do Brasil e no mundo. Novos casos da doença vêm aumentando progressivamente e por isso a Organização Mundial da Saúde (OMS) tem incentivado a busca de novos alvos para desenvolver drogas que eliminem eficazmente o parasita. Estudos prévios realizados por nosso grupo e outros mostraram que algumas proteínas são encontradas exclusivamente em parasitas da família Trypanosomatidae, como por exemplo a proteína Rbp38, que está envolvida em diversas atividades no núcleo celular. Desta maneira, o estudo do mecanismo de importação nuclear desta família de parasitas pode representar um importante alvo na busca por novos alvos moleculares no combate à leishmaniose. Assim, este trabalho teve como objetivo avançar no entendimento do transporte de proteínas de leishmania para o interior do núcleo celular. Para tanto, produzimos Impα recombinantes de Leishmania major (LmImpα), Mus musculus (MmImpα) e Homo sapiens (HsImpα) para obter informações comparativas através de técnicas biofísicas. Utilizamos também técnicas bioinformáticas para obter informações sobre um possível modelo do complexo formado da LmImpα com a NLS da proteína Rbp38.
Among the transport pathways, that enable the transport of macromolecules in or out the nucleus through the recognition of specific signaling sequences, the Classical Import Nuclear Pathway is the best characterized. In this way, the Importin-α (Impα) protein acts on the identification of proteins to be transported to the nucleus by the recognition of nuclear localization sequences (NLS). The structure of the Impα protein has already been elucidated and characterized in some organisms and is classified into three subfamilies: α1, α2 and α3. The differences between the Impα proteins of each family show the specificities of these proteins in the recognition of NLSs, depending on the organism and function they carry out. The same peptide may exhibit variations in affinity and binding when interacting with Impα from different families. Leishmaniasis is a disease present in several regions of Brazil and the world. New cases of the disease have been increasing, so the World Health Organization (WHO) has encouraged researches in order to promote new mechanisms for the development of drugs and methodologies against the parasite. Previous studies performed by our group and others showed that some proteins are found exclusively in Trypanosomatidae family, for instance, the Rpb38, who is involved in many cellular nucleus activities. This way, the study of nuclear importation mechanism of this parasite family can represent a major target to find new molecular targets in the combat to the leishmaniasis. This work had the purpose to advance the understanding of the protein transportation of leishmania to the nucleus. For this purpose, we produced Impα from Leishmania major (LmImpα), Mus musculus (MmImpα) and Homo sapiens (HsImpα), all recombinant to obtain comparative information through biophysical techniques and through bioinformatics techniques, information about a possible model of the complex formed from LmImpα with the Rbp38 protein NLS
Among the transport pathways, that enable the transport of macromolecules in or out the nucleus through the recognition of specific signaling sequences, the Classical Import Nuclear Pathway is the best characterized. In this way, the Importin-α (Impα) protein acts on the identification of proteins to be transported to the nucleus by the recognition of nuclear localization sequences (NLS). The structure of the Impα protein has already been elucidated and characterized in some organisms and is classified into three subfamilies: α1, α2 and α3. The differences between the Impα proteins of each family show the specificities of these proteins in the recognition of NLSs, depending on the organism and function they carry out. The same peptide may exhibit variations in affinity and binding when interacting with Impα from different families. Leishmaniasis is a disease present in several regions of Brazil and the world. New cases of the disease have been increasing, so the World Health Organization (WHO) has encouraged researches in order to promote new mechanisms for the development of drugs and methodologies against the parasite. Previous studies performed by our group and others showed that some proteins are found exclusively in Trypanosomatidae family, for instance, the Rpb38, who is involved in many cellular nucleus activities. This way, the study of nuclear importation mechanism of this parasite family can represent a major target to find new molecular targets in the combat to the leishmaniasis. This work had the purpose to advance the understanding of the protein transportation of leishmania to the nucleus. For this purpose, we produced Impα from Leishmania major (LmImpα), Mus musculus (MmImpα) and Homo sapiens (HsImpα), all recombinant to obtain comparative information through biophysical techniques and through bioinformatics techniques, information about a possible model of the complex formed from LmImpα with the Rbp38 protein NLS
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Palavras-chave
Biologia molecular, Cromatografia líquida de alta eficiência, Proteínas recombinantes, Importin-alpha, Protein chromatography, Recombinant protein, Leishmania major, NLS peptide