RESUMO
NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/ß and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.
Assuntos
DNA Glicosilases , alfa Carioferinas , Animais , Camundongos , Humanos , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Sinais de Localização Nuclear/genética , DNA Glicosilases/metabolismoRESUMO
The establishment of persistent dengue virus infection within the cells of the mosquito vector is an essential requirement for viral transmission to a new human host. The mechanisms involved in the establishment and maintenance of persistent infection are not well understood, but it has been suggested that both viral and cellular factors might play an important role. In the present work, we evaluated differential gene expression in Aedes albopictus cells acutely (C6/36-HT) and persistently infected (C6-L) with Dengue virus 2 by cDNA-AFLP. We observed that importin ß3 was upregulated in noninfected cells compared with C6-L cells. Using RT-qPCR and plaque assays, we observed that Dengue virus levels in C6-L cells essentially do not vary over time, and peak viral titers in acutely infected cells are observed at 72 and 120 h postinfection. The expression level of importin ß3 was higher in acutely infected cells than in persistently infected cells; this correlates with higher levels of NS5 in the nucleus of the cell. The differential pattern of importin ß3 expression between acute and persistent infection with Dengue virus 2 could be a mechanism to maintain viral infection over time, reducing the antiviral response of the cell and the viral replicative rate.
RESUMO
The classical nuclear import pathway is mediated by importin (Impα and Impß), which recognizes the cargo protein by its nuclear localization sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high-affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3, the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.
Assuntos
Calorimetria/métodos , Simulação de Acoplamento Molecular , Sinais de Localização Nuclear/genética , alfa Carioferinas/genética , Transporte Ativo do Núcleo Celular/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Ligação Competitiva , Núcleo Celular/metabolismo , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Camundongos , Mutação , Ligação Proteica , Domínios Proteicos , Eletricidade Estática , alfa Carioferinas/química , alfa Carioferinas/metabolismoRESUMO
Nuclear-cytoplasmic trafficking of proteins is a highly regulated process that modulates multiple biological processes in eukaryotic cells. In Giardia lamblia, shuttling has been described from the cytoplasm to nuclei of proteins during the biological cell cycle of the parasite. This suggests that a mechanism of nucleocytoplasmic transport is present and functional in G. lamblia. By means of computational biology analyses, we found that there are only two genes for nuclear transport in this parasite, named Importin α and Importin ß. When these transporters were overexpressed, both localized close to the nuclear envelope, and no change was observed in trophozoite growth rate. However, during the encystation process, both transporters induced an increase in the number of cysts produced. Importazole and Ivermectin, two known specific inhibitors of importins, separately influenced the encysting process by inducing an arrest in the trophozoite stage that prevents the production of cysts. This effect was more noticeable when Ivermectin, an anti-parasitic drug, was used. Finally, we tested whether the enzyme arginine deiminase, which shuttles from the cytoplasm to the nuclei during encystation, was influenced by these transporters. We found that treatment with each of the inhibitors abrogates arginine deiminase nuclear translocation and favors perinuclear localization. This suggests that Importin α and Importin ß are key transporters during the encystation process and are involved, at least, in the transport of arginine deiminase into the nuclei. Considering the effect produced by Ivermectin during growth and encystation, we postulate that this drug could be used to treat giardiasis.
Assuntos
Núcleo Celular/metabolismo , Giardia lamblia/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Antiparasitários/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Biologia Computacional , Giardia lamblia/efeitos dos fármacos , Giardia lamblia/genética , Giardia lamblia/crescimento & desenvolvimento , Hidrolases/metabolismo , Ivermectina/farmacologia , Encistamento de Parasitas/efeitos dos fármacos , Encistamento de Parasitas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Proteínas de Protozoários/genética , Quinazolinas/farmacologia , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
NUAK1 is a serine/threonine kinase member of the AMPK-α family. NUAK1 regulates several processes in tumorigenesis; however, its regulation and molecular targets are still poorly understood. Bioinformatics analysis predicted that the majority of NUAK1 localizes in the nucleus. However, there are no studies about the regulation of NUAK1 subcellular distribution. Here, we analyzed NUAK1 localization in several human cell lines, mouse embryo fibroblasts, and normal mouse tissues. We found that NUAK1 is located in the nucleus and also in the cytoplasm. Through bioinformatics analysis and studies comparing subcellular localization of wild type and NUAK1 mutants, we identified a conserved bipartite nuclear localization signal at the N-terminal domain of NUAK1. Based on mass spectrometry analysis, we found that NUAK1 interacts with importin-ß members including importin-ß1 (KPNB1), importin-7 (IPO7), and importin-9 (IPO9). We confirmed that importin-ß members are responsible for NUAK1 nuclear import through the inhibition of importin-ß by Importazole and the knockdown of either IPO7 or IPO9. In addition, we found that oxidative stress induces NUAK1 cytoplasmic accumulation, indicating that oxidative stress affects NUAK1 nuclear transport. Thus, our study is the first evidence of an active nuclear transport mechanism regulating NUAK1 subcellular localization. These data will lead to investigations of the molecular targets of NUAK1 according to its subcellular distribution, which could be new biomarkers or targets for cancer therapies.
Assuntos
Sinais de Localização Nuclear/metabolismo , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Citoplasma/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Camundongos , Estresse Oxidativo , Proteínas Quinases/genética , Proteínas Repressoras/genéticaRESUMO
The cauda epididymidis is the major sperm storage region whose androgenic supply, essential for the sperm viability, is provided by the vasculature and is dependent upon testosterone diffusion through the stromal tissue to reach the epithelial cells. We have focused our efforts on examining the regulation of this important epididymal region by evaluating the impact of the androgen disrupter cimetidine on the epithelial-stromal androgenic microenvironment. Male rats received 100 mg/kg cimetidine (CMTG) or saline (CG) for 50 days, serum testosterone levels were measured and the epididymal cauda region was processed for light and transmission electron microscopy. In the proximal cauda region, the duct diameter was measured and birefringent collagen in the stroma was quantified. TUNEL-labeled epithelial cells were quantified, and androgen receptor (AR), karyopherin alpha (KPNA) and sex hormone-binding globulin (SHBG) levels were analyzed by immunofluorescence and Western blot. CMTG showed reduced duct diameter and high number of apoptotic epithelial cells. In the epithelium, the total AR concentration and the KPNA immunoreactivity were reduced, and a weak/absent AR nuclear immunofluorescence was observed in contrast to the enhanced AR immunolabeling observed in the cytoplasm of the epithelial cells. A significant reduction of collagen and SHBG levels in the stroma was also observed. Cimetidine treatment impairs AR nuclear import in the epithelium, causing androgenic dysfunction and subsequent epithelial cell apoptosis and duct atrophy. The connective tissue atrophy and reduction of SHBG stromal levels associated with epithelial androgenic dysfunction indicate a possible role of stromal SHBG in the androgenic supply of the sperm storage region of the epididymis.
Assuntos
Epididimo/metabolismo , Epididimo/patologia , Células Epiteliais/metabolismo , Receptores Androgênicos/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Espermatozoides/fisiologia , Células Estromais/metabolismo , Animais , Células Cultivadas , Células Epiteliais/patologia , Masculino , Ratos , Espermatozoides/citologia , Células Estromais/patologia , Testosterona/metabolismoRESUMO
MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed.
Assuntos
Núcleo Celular/metabolismo , Reparo de Erro de Pareamento de DNA , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Humanos , Carioferinas/metabolismo , Camundongos , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Modelos Moleculares , Proteína 1 Homóloga a MutL/química , Conformação ProteicaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that is vectored by mosquitos and cycled in rodents. It can cause disease in equines and humans characterized by a febrile illness that may progress into encephalitis. Like the capsid protein of other viruses, VEEV capsid is an abundant structural protein that binds to the viral RNA and interacts with the membrane-bound glycoproteins. It also has protease activity, allowing cleavage of itself from the growing structural polypeptide during translation. However, VEEV capsid protein has additional nonstructural roles within the host cell functioning as the primary virulence factor for VEEV. VEEV capsid inhibits host transcription and blocks nuclear import in mammalian cells, at least partially due to its complexing with the host CRM1 and importin α/ß1 nuclear transport proteins. VEEV capsid also shuttles between the nucleus and cytoplasm and is susceptible to inhibitors of nuclear trafficking, making it a promising antiviral target. Herein, the role of VEEV capsid in viral replication and pathogenesis will be discussed including a comparison to proteins of other alphaviruses.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/patogenicidade , RNA Viral/metabolismo , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Vírus da Encefalite Equina do Leste , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/metabolismo , Vírus da Encefalite Equina do Oeste , Cavalos , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Virulência , Replicação Viral/genética , Proteína Exportina 1RESUMO
Importin-α recognizes cargo proteins that contain classical nuclear localization sequences (NLS) and, in complex with importin-ß, is able to translocate nuclear proteins through the nuclear pore complex. The filamentous fungus Neurospora crassa is a well studied organism that has been widely used as a model organism for fundamental aspects of eukaryotic biology, and is important for understanding the specific mechanisms of protein transport to the cell nucleus. In this work, the crystallization and preliminary X-ray diffraction analysis of importin-α from N. crassa (IMPα-Nc) complexed with a classical NLS peptide (SV40 NLS) are reported. IMPα-Nc-SV40 NLS crystals diffracted X-rays to 2.0â Å resolution and the structure was solved by molecular-replacement techniques, leading to a monomeric structure. The observation of the electron-density map indicated the presence of SV40 NLSs interacting at both the minor and major NLS-binding sites of the protein.