RESUMO
A bioactive compound isolated from the stem extract of Aristolochia sprucei through High Performance Liquid Chromatography (HPLC) was identified via Nuclear Magnetic Resonance (NMR) as the aristolochic acid (AA). This compound showed an inhibitory effect over the myotoxic activity of Bothrops jararacussu and Bothrops asper venoms, being also effective against the indirect hemolytic activity of B. asper venom. Besides, AA also inhibited the myotoxic activity of BthTX-I and MTX-II with an efficiency greater than 60% against both myotoxins. Docking predictions revealed an interesting mechanism, through which the AA displays an interaction profile consistent with its inhibiting abilities, binding to both active and putative sites of svPLA2. Overall, the present findings indicate that AA may bind to critical regions of myotoxic Asp 49 and Lys49-PLA2s from snake venoms, highlighting the relevance of domains comprising the active and putative sites to inhibit these toxins.
RESUMO
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
The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP-NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Fatores de Transcrição/metabolismo , alfa Carioferinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Células HeLa , Humanos , Neurospora crassa/genética , Estrutura Secundária de Proteína , Esporos Fúngicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Difração de Raios X , alfa Carioferinas/química , alfa Carioferinas/genéticaRESUMO
Plant aquaporins are water channels implicated in various physiological processes, including growth, development and adaptation to stress. In this study, the Tonoplast Intrinsic Protein (TIP) gene subfamily of Eucalyptus, an economically important woody species, was investigated and characterized. A genome-wide survey of the Eucalyptus grandis genome revealed the presence of eleven putative TIP genes (referred as EgTIP), which were individually assigned by phylogeny to each of the classical TIP1-5 groups. Homology modeling confirmed the presence of the two highly conserved NPA (Asn-Pro-Ala) motifs in the identified EgTIPs. Residue variations in the corresponding selectivity filters, that might reflect differences in EgTIP substrate specificity, were observed. All EgTIP genes, except EgTIP5.1, were transcribed and the majority of them showed organ/tissue-enriched expression. Inspection of the EgTIP promoters revealed the presence of common cis-regulatory elements implicated in abiotic stress and hormone responses pointing to an involvement of the identified genes in abiotic stress responses. In line with these observations, additional gene expression profiling demonstrated increased expression under polyethylene glycol-imposed osmotic stress. Overall, the results obtained suggest that these novel EgTIPs might be functionally implicated in eucalyptus adaptation to stress.
RESUMO
Neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that N. crassa Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.
Assuntos
Neurospora crassa/metabolismo , Sinais de Localização Nuclear , alfa Carioferinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Neurospora crassa/genética , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Ligação Proteica , Conformação Proteica , alfa Carioferinas/química , alfa Carioferinas/genéticaRESUMO
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.
Assuntos
Cristalização/métodos , Cristalografia por Raios X/métodos , Neurospora crassa/metabolismo , Oligopeptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , alfa Carioferinas/química , alfa Carioferinas/metabolismo , Núcleo Celular/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , alfa Carioferinas/genéticaRESUMO
The influenza virus has been a challenge to science due to its ability to withstand new environmental conditions. Taking into account the development of virus sequence databases, computational approaches can be helpful to understand virus behavior over time. Furthermore, they can suggest new directions to deal with influenza. This work presents triplet entropy analysis as a potential phylodynamic tool to quantify nucleotide organization of viral sequences. The application of this measure to segments of hemagglutinin (HA) and neuraminidase (NA) of H1N1 and H3N2 virus subtypes has shown some variability effects along timeline, inferring about virus evolution. Sequences were divided by year and compared for virus subtype (H1N1 and H3N2). The nonparametric Mann-Whitney test was used for comparison between groups. Results show that differentiation in entropy precedes differentiation in GC content for both groups. Considering the HA fragment, both triplet entropy as well as GC concentration show intersection in 2009, year of the recent pandemic. Some conclusions about possible flu evolutionary lines were drawn.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Neuraminidase/genética , Composição de Bases , Evolução Molecular , Humanos , Modelos Genéticos , Filogenia , Análise de Sequência de DNA , Estatísticas não Paramétricas , TermodinâmicaRESUMO
Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins.
Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear/metabolismo , alfa Carioferinas/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Neurospora crassa/metabolismo , Estabilidade Proteica , Alinhamento de Sequência , alfa Carioferinas/química , alfa Carioferinas/genética , alfa Carioferinas/isolamento & purificação , beta Carioferinas/metabolismoRESUMO
Flap endonuclease 1 (FEN1) is a member of the nuclease family and is structurally conserved from bacteriophages to humans. This protein is involved in multiple DNA-processing pathways, including Okazaki fragment maturation, stalled replication-fork rescue, telomere maintenance, long-patch base-excision repair and apoptotic DNA fragmentation. FEN1 has three functional motifs that are responsible for its nuclease, PCNA-interaction and nuclear localization activities, respectively. It has been shown that the C-terminal nuclear localization sequence (NLS) facilitates nuclear localization of the enzyme during the S phase of the cell cycle and in response to DNA damage. To determine the structural basis of the recognition of FEN1 by the nuclear import receptor importin α, the crystal structure of the complex of importin α with a peptide corresponding to the FEN1 NLS was solved. Structural studies confirmed the binding of the FEN1 NLS as a classical bipartite NLS; however, in contrast to the previously proposed (354)KRKX(8)KKK(367) sequence, it is the (354)KRX(10)KKAK(369) sequence that binds to importin α. This result explains the incomplete inhibition of localization that was observed on mutating residues (365)KKK(367). Acidic and polar residues in the X(10) linker region close to the basic clusters play an important role in binding to importin α. These results suggest that the basic residues in the N-terminal basic cluster of bipartite NLSs may play roles that are more critical than those of the many basic residues in the C-terminal basic cluster.
Assuntos
Endonucleases Flap/metabolismo , Sinais de Localização Nuclear/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Endonucleases Flap/química , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Sinais de Localização Nuclear/química , Ligação Proteica , Conformação Proteica , alfa Carioferinas/químicaRESUMO
Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-ß-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.