Estudos estruturais comparativos entre as importinas-α de Homo sapiens e de Mus musculus
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2022-01-31
Autores
Silva, Tainá Dorte da
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Universidade Estadual Paulista (Unesp)
Resumo
Uma das vias de transporte melhor caracterizada é mediada pela Importina-α (Impα), chamado via clássica de importação nuclear. Nesta via, a Importina-β (Impβ) se liga à Impα, afastando o domínio auto inibitório da Impα, permitindo o reconhecimento e a ligação das sequências de localização nuclear (NLS) presentes nas proteínas a serem importadas. Já foram identificadas diversas variantes da Impα em diferentes organismos, as quais foram classificadas em três subfamílias (α1, α2 e α3) baseado na similaridade sequencial entre elas. A Impα de Homo sapiens possui sete variantes, sendo que a sua variante α1 possui alta similaridade com a variante α2 de Mus musculus (MmImpα), primeira Impα de mamífero a ser estruturalmente elucidada e amplamente utilizada no estudo de processos de importação nuclear. Além disso, com o avanço nos estudos nesta área, as estruturas das Impα de humano, fungo e arroz foram elucidadas, revelando que estas estruturas são altamente conservadas, mas apresentam diferentes características, como a afinidade à diferentes NLSs, em razão das regiões adjacentes aos sítios de ligação da Impα. Ainda existem poucas informações acerca destas diferenças, apesar da grande importância acadêmica e farmacológica, devido a especificidade do transporte proteico. Sendo assim, este trabalho teve por objetivo a produção da variante α1 da HsImpα e sua comparação estrutural com a MmImpα, e o estudo da interação entre a HsImpα e os peptídeos NLS SV40, XPG1 e PAC-3. Para tal feito, foram realizadas a transformação, expressão e purificação da proteína HsImpα, bem como a análise estrutural com as técnicas de espalhamento de luz dinâmico (DLS), dicroísmo circular (CD) e espectroscopia de fluorescência (FS). A análise da afinidade dos complexos HsImpα/XPG1-NLS, HsImpα/SV40-NLS e HsImpα/PAC-3 foi realizada com a técnica de calorimetria por titulação isotérmica (ITC). O CD mostrou que a HsImpα possui estrutura similar à outras Impα, apresentando, majoritariamente, hélices-α em sua estrutura. O DLS e o FS da amostra da HsImpα isolada mostram que há uma possível oligomerização quando submetida ao aumento gradual da temperatura (5 ºC a 25 ºC), sendo que a 5 ºC ela se mostra majoritariamente monomérica. Já a análise dos resultados de DLS e FS do complexo HsImpα/SV40-NLS mostram que há uma melhor estabilização do complexo na presença da NLS. Quando analisada a amostra da HsImpα isolada pela técnica de FS, medindo diferentes amostras para cada temperatura (5, 10, 20 e 25 ºC, foi observado que não há a oligomerização. A FS do complexo HsImpα/SV40-NLS, também medida uma amostra para cada temperatura (5, 10, 20 e 25 ºC), sendo observado pequenas variações na exposição dos triptofanos, indicando que a afinidade do complexo é baixa, posteriormente confirmada com a técnica de ITC. A mesma análise de FS foi empregada para o complexo HsImpα/XPG1-NLS, sendo observado que há ligação em todas as temperaturas analisadas. Esta ligação se mostra forte e favorável, quando analisada pelo ITC, assim como já observado com a MmImpα em outro estudo realizado por nosso grupo. Foi realizado também a análise da HsImpα com peptídeo NLS de outro organismo (fungo), PAC-3-NLS com ITC, resultando que a afinidade do complexo não pode ser determinada, pois os termogramas obtidos não foram reprodutíveis, diferente do observado em outro estudo com a MmImpα, em que a afinidade do complexo é alta. Experimentos de co-cristalização estão sendo realizados visando a obtenção dos detalhes da interação dos complexos, sendo obtidos pequenos cristais que precisam ser otimizados. Os resultados apresentados indicaram uma possível oligomerização da Impα truncada quando isolada, porém, quando complexada com peptídeos NLSs de alta afinidade, como a XPG1, a proteína é majoritariamente monomérica. Possíveis diferenças na estequiometria e na afinidade do complexo HsImpα/XPG1-NLS em comparação com o MmImpα/XPG1-NLS podem indicar o modo de interação diferente entre eles. Estes resultados fortalecem a importância dos estudos entre as Impα e NLSs de diferentes organismos, extremamente importante para futuros estudos farmacológicos.
One of the best characterized transport routes is mediated by Importin-α (Impα), called the classical nuclear import pathway. In this pathway, Importin-β (Impβ) binds to Impα, pushing away the self-inhibitory domain of Impα, allowing the recognition and binding of nuclear localization sequences (NLS) present in the proteins to be imported. Several variants of Impα have been identified in different organisms, which have been classified into three subfamilies (α1, α2 and α3) based on the sequential similarity between them. The Homo sapiens Impα has seven variants, and its α1 variant has high similarity with the α2 variant of Mus musculus (MmImpα), the first mammalian Impα to be structurally elucidated and widely used in the study of nuclear import processes. Furthermore, with advances in studies in this area, the structures of Impα from human, fungus and rice were elucidated, revealing that these structures are highly conserved, but have different characteristics, such as affinity to different NLSs, due to the regions adjacent to the Impα binding sites. There is still little information about these differences, despite the great academic and pharmacological importance, due to the specificity of protein transport. Therefore, this work aimed to produce the α1 variant of HsImpα and its structural comparison with MmImpα, and to study the interaction between HsImpα and NLS SV40, XPG1 and PAC-3 peptides. For this purpose, transformation, expression and purification of the HsImpα protein were performed, as well as structural analysis with dynamic light scattering (DLS), circular dichroism (CD) and fluorescence spectroscopy (FS) techniques. The affinity analysis of the complexes HsImpα/XPG1-NLS, HsImpα/SV40-NLS and HsImpα/PAC-3 were performed using isothermal titration calorimetry (ITC). The CD showed that HsImpα has a structure similar to other Impα, mostly presenting α-helices in its structure. The DLS and FS of the isolated HsImpα sample show that there is a possible oligomerization when subjected to a gradual increase in temperature (5 ºC to 25 ºC), but at 5 ºC it is mostly monomeric. The analysis of the DLS and FS results of the HsImpα/SV40-NLS complex showed that there is better stabilization of the sample in the presence of the NLS. When the isolated HsImpα sample was analyzed by the FS technique, measuring different samples for each temperature (5, 10, 20 and 25 ºC, it was observed that there is not oligomerization. The FS of the complex HsImpα/SV40-NLS, with also one sample measured at each temperature (5, 10, 20 and 25 ºC), indicated small variations in the exposure of tryptophans, showing that the affinity of the complex is low, which was later confirmed by the ITC technique. The same FS analysis was performed for the HsImpα/XPG1-NLS complex, being observed that there was binding at all temperatures analyzed. This binding was strong and favorable when analyzed by the ITC, as has already been observed with MmImpα in another study carried out by our group. A analysis of HsImpα with NLS peptide from other organism (fungus) was also performed (HsImpα/PAC-3-NLS complex) by ITC, but its affinity could not be determined, as the thermograms obtained were not reproducible, unlike that observed in another study with MmImpα, which the affinity was high. Co-crystallization experiments are being carried out in order to obtain details of the interaction of the complexes, obtaining small crystals that need to be optimized. The results presented here indicated the possible oligomerization of truncated Impα when isolated, however, when complexed with high-affinity NLSs peptides, such as XPG1, the protein is mostly monomeric. Possible differences in the stoichiometry and affinity of the HsImpα/XPG1-NLS complex compared to MmImpα/XPG1-NLS may indicate different mode of interaction between them. These results strengthen the importance of studies between Impα and NLSs from different organisms, extremely important for future pharmacological studies.
One of the best characterized transport routes is mediated by Importin-α (Impα), called the classical nuclear import pathway. In this pathway, Importin-β (Impβ) binds to Impα, pushing away the self-inhibitory domain of Impα, allowing the recognition and binding of nuclear localization sequences (NLS) present in the proteins to be imported. Several variants of Impα have been identified in different organisms, which have been classified into three subfamilies (α1, α2 and α3) based on the sequential similarity between them. The Homo sapiens Impα has seven variants, and its α1 variant has high similarity with the α2 variant of Mus musculus (MmImpα), the first mammalian Impα to be structurally elucidated and widely used in the study of nuclear import processes. Furthermore, with advances in studies in this area, the structures of Impα from human, fungus and rice were elucidated, revealing that these structures are highly conserved, but have different characteristics, such as affinity to different NLSs, due to the regions adjacent to the Impα binding sites. There is still little information about these differences, despite the great academic and pharmacological importance, due to the specificity of protein transport. Therefore, this work aimed to produce the α1 variant of HsImpα and its structural comparison with MmImpα, and to study the interaction between HsImpα and NLS SV40, XPG1 and PAC-3 peptides. For this purpose, transformation, expression and purification of the HsImpα protein were performed, as well as structural analysis with dynamic light scattering (DLS), circular dichroism (CD) and fluorescence spectroscopy (FS) techniques. The affinity analysis of the complexes HsImpα/XPG1-NLS, HsImpα/SV40-NLS and HsImpα/PAC-3 were performed using isothermal titration calorimetry (ITC). The CD showed that HsImpα has a structure similar to other Impα, mostly presenting α-helices in its structure. The DLS and FS of the isolated HsImpα sample show that there is a possible oligomerization when subjected to a gradual increase in temperature (5 ºC to 25 ºC), but at 5 ºC it is mostly monomeric. The analysis of the DLS and FS results of the HsImpα/SV40-NLS complex showed that there is better stabilization of the sample in the presence of the NLS. When the isolated HsImpα sample was analyzed by the FS technique, measuring different samples for each temperature (5, 10, 20 and 25 ºC, it was observed that there is not oligomerization. The FS of the complex HsImpα/SV40-NLS, with also one sample measured at each temperature (5, 10, 20 and 25 ºC), indicated small variations in the exposure of tryptophans, showing that the affinity of the complex is low, which was later confirmed by the ITC technique. The same FS analysis was performed for the HsImpα/XPG1-NLS complex, being observed that there was binding at all temperatures analyzed. This binding was strong and favorable when analyzed by the ITC, as has already been observed with MmImpα in another study carried out by our group. A analysis of HsImpα with NLS peptide from other organism (fungus) was also performed (HsImpα/PAC-3-NLS complex) by ITC, but its affinity could not be determined, as the thermograms obtained were not reproducible, unlike that observed in another study with MmImpα, which the affinity was high. Co-crystallization experiments are being carried out in order to obtain details of the interaction of the complexes, obtaining small crystals that need to be optimized. The results presented here indicated the possible oligomerization of truncated Impα when isolated, however, when complexed with high-affinity NLSs peptides, such as XPG1, the protein is mostly monomeric. Possible differences in the stoichiometry and affinity of the HsImpα/XPG1-NLS complex compared to MmImpα/XPG1-NLS may indicate different mode of interaction between them. These results strengthen the importance of studies between Impα and NLSs from different organisms, extremely important for future pharmacological studies.
Descrição
Palavras-chave
Importinas, Carioferinas, Sequência de Localização Nuclear, NLS, Homo sapiens, Mus musculus, Complexo Poro Nuclear