Estudos estruturais com a importina-α do fungo Neurospora crassa e sequências de localização nuclear

The transport of macromolecules from the cytoplasm to the nucleus occurs by passage through the nuclear pore complex, present in the nuclear envelope. One of that nuclear transport pathway depends on carrier proteins called Importins. In this process, known as classical nuclear import pathway, the Importin-α protein (Impα) recognizes nuclear localization sequences (NLSs) in the protein to be transported to the formation of a complex with Importin-β (Impβ) allowing the transport of macromolecules. The aim of this work is the structural study of the protein Importin-α, from the filamentous fungus Neurospora crassa (ImpαNc) in order to recognize the regions that determine the specificity of the protein and to compare their behavior in its native state and in presence of a NLS peptide. The first experiments with ImpαNc allowed an initial characterization of the protein and its behavior in solution. Experiments of analytical size exclusion chromatography comparing two samples of Impα: (1) in the presence of the NLS peptide of the protein FEN1 ( FEN1 NLS) and, (2) without NLS peptide; indicated the formation of agglomerates in the sample 2 and the conformation predominantly monomodal, in the sample 1, suggesting a greater stability of the protein in the presence of NLS peptides. For further information about the ImpαNc, crystallization experiments were carried out with the peptide complexed to the classic NLS SV40 (SV40 NLS) protein as previous experiments have suggested the increased stability of the protein in the presence of NLSs. A first crystal obtained in the condition 0.2mM dibasic sodium phosphate dihydrate and 20% (w / v) polyethylene glycol 3350 were subjected to xray diffraction and showed satisfactory diffraction pattern for the elucidation of the structure of the complex at a resolution of 2.05 Å. A second crystal (ImpαNc-2) obtained under the condition of 0.2 mM bicine pH 8.5 and 20% PEG 6000, was subjected to x-ray ...Um dos mecanismos de transporte de macromoléculas do citoplasma para o núcleo celular ocorre através da passagem da macromolécula pelo complexo poro nuclear (CPN), presente no envoltório nuclear, e depende de proteínas transportadoras denominadas Importinas. Neste processo, conhecido como via clássica de importação nuclear, a proteína Importina-α (Impα) reconhece sequências de localização nuclear (NLSs) na proteína a ser transportada para a formação de um complexo junto a Importina-β (Impβ) permitindo o transporte da macromolécula. O objetivo do presente trabalho é o estudo estrutural da Impα proveniente do fungo filamentoso Neurospora crassa (ImpαNc), a fim de reconhecer as regiões que determinam especificidades da proteína, além de comparar seu comportamento nativo e na presença de um peptídeo NLS. Os primeiros experimentos com a ImpαNc permitiram uma caracterização inicial da proteína e seu comportamento em solução. Experimentos de cromatografia analítica de exclusão molecular, comparando duas amostras de ImpαNc: (1) na presença do peptídeo NLS da proteína FEN1 (FEN1 NLS) e (2) sem peptídeo NLS, indicaram a formação de aglomerados na amostra 2 e a conformação, predominantemente, monomodal na amostra 1, sugerindo uma maior estabilidade da proteína na presença de peptídeos NLS. Para aprofundar as informações sobre a ImpαNc, experimentos de cristalização foram conduzidos com a proteína complexada ao peptídeo de NLS clássica e monopartida do SV40 (SV40 NLS), conforme experimentos anteriores sugeriram a maior estabilidade da proteína na presença de NLSs. Um primeiro cristal (ImpαNc-1), obtido na condição 0,2mM fosfato de sódio dibásico dihidratado e 20% (w/v) de polietilenoglicol 3350, foi submetido á difração de raios X e apresentou padrão de difração satisfatório para elucidação da estrutura do complexo a uma resolução de 2,05 Å. Um segundo cristal (ImpαNc-2), obtido na ...Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Molecular Components of the <i>Neurospora crassa</i> pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor

<div><p>Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. <i>Neurospora crassa</i> shares all six components of the <i>Aspergillus nidulans</i> pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the <i>N</i>. <i>crassa pal</i> mutant strains, with the exception of Δ<i>pal-9</i>, which is the <i>A</i>. <i>nidulans palI</i> homolog, exhibit low conidiation and are unable to grow at alkaline pH. Moreover, they accumulate the pigment melanin, most likely via regulation of the tyrosinase gene by the pH signaling components. The PAC-3 transcription factor binds to the tyrosinase promoter and negatively regulates its gene expression. PAC-3 also binds to all <i>pal</i> gene promoters, regulating their expression at normal growth pH and/or alkaline pH, which indicates a feedback regulation of PAC-3 in the <i>pal</i> gene expression. In addition, PAC-3 binds to the <i>pac-3</i> promoter only at alkaline pH, most likely influencing the <i>pac-3</i> expression at this pH suggesting that the activation of PAC-3 in <i>N</i>. <i>crassa</i> results from proteolytic processing and gene expression regulation by the pH signaling components. In <i>N</i>. <i>crassa</i>, PAC-3 is proteolytically processed in a single cleavage step predominately at alkaline pH; however, low levels of the processed protein can be observed at normal growth pH. We also demonstrate that PAC-3 preferentially localizes in the nucleus at alkaline pH stress and that the translocation may require the <i>N</i>. <i>crassa</i> importin-α since the PAC-3 nuclear localization signal (NLS) has a strong <i>in vitro</i> affinity with importin-α. The data presented here show that the pH signaling pathway in <i>N</i>. <i>crassa</i> shares all the components with the <i>A</i>. <i>nidulans</i> and <i>S</i>. <i>cerevisiae</i> pathways; however, it exhibits some properties not previously described in either organism.</p></div

The expression of the <i>pac-3</i> gene at normal and alkaline pH.

<p>Samples from the wild-type and from all <i>pal</i> mutant strains cultured at pH 5.8 for 24 h and then shifted to pH 7.8 for 1 h were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus^™ Real-Time PCR system (Applied Biosystems) using the Power SYBR^® Green and specific primers. At least three biological replicates were carried out, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. The <i>tub-2</i> gene was used as the reference gene, and the pH 5.8 wild-type was used as the reference sample. *Asterisks indicate significant differences compared to the wild-type at the same pH (Student’s <i>t</i>-test, <i>P</i> < 0.01).</p

Binding of PAC-3 to the <i>pal</i> gene promoters at normal and alkaline pH.

<p><b>(A)</b> Representation of the PAC-3 motifs in the <i>pal</i> gene promoters. The black dots indicate the position of the PAC-3 motifs, and the dashed boxes indicate regions that were analyzed by ChIP-PCR. <b>(B)</b> Genomic DNA samples from the Δ<i>pac-3</i> complemented strain both subjected to alkaline pH stress or not were immunoprecipitated with the anti-mCherry antibody and subjected to PCR to amplify DNA fragments containing the PAC-3 motif. A DNA fragment from the ubiquitin gene, which does not have a PAC-3 motif, was used as a negative control of binding. The input DNA (I) was used as a positive control and the non-immunoprecipitated reaction (no Ab) as the negative control. L, 1 kb DNA ladder. <b>(C)</b> The DNA bands after ChIP-PCR were quantified by ImageJ and the results are shown in the graphs. *Asterisks indicate significant differences between no Ab and IP at the same pH (Student’s <i>t</i>-test, <i>P</i> < 0.01). All results represent the average of at least two independent experiments. Bars indicate the standard deviation from the biological experiments.</p

Morphological analyses of the <i>pal</i> mutant strains.

<p><b>(A)</b> The strains (10⁷ conidia/mL) were cultured in Erlenmeyer flasks containing solid VM medium plus 2% sucrose at pH 5.8 for 8–10 days. Basal hyphae growth was examined after cultivating the strains on Petri dishes containing solid VM medium plus 2% sucrose at pH 5.8 and 7.8 for 24 h at 30°C. <b>(B)</b> Radial growth of the colonies measured in cm. The results represent at least two independent experiments. *Indicates significant difference between wild-type and mutant strains at the same pH (Student’s <i>t</i>-test, <i>P</i> < 0.01).</p

Expression of the tyrosinase gene in the wild-type and the Δ<i>pal</i> mutant strains at normal (5.8) and alkaline (7.8) pH.

<p><b>(A)</b> Mycelial samples from the wild-type and Δ<i>pal</i> mutant strains cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h were used to extract total RNA. Gene expression analysis was performed by RT-qPCR on the StepOnePlus^™ Real-Time PCR system (Applied Biosystems) using Power SYBR^® Green and specific primers. The <i>tub-2</i> gene was used as the reference gene. The asterisks indicate significant differences compared to the wild-type strain at the same pH, and circles indicate significant differences between the same mutant strain cultured at a different pH (Student’s <i>t</i>-test, <i>P</i> < 0.01). <b>(B)</b> Representation of the PAC-3 motifs (black circles) in the tyrosinase gene promoter. Dashed boxes indicate the region analyzed by ChIP-PCR. Genomic DNA samples from the Δ<i>pac-3</i> complemented strain subjected to pH stress or not were immunoprecipitated with an anti-mCherry antibody and subjected to PCR using specific primers. The input DNA (I) was used as the positive control, and the reactions without any antibody (no Ab) were used as the negative control. The intensity of the DNA bands in the gel (left side, arrow) was quantified by ImageJ and the results are shown in the right figure. L, 1 kb DNA ladder. *Asterisks indicate significant differences between the no Ab and IP samples at the same pH (Student’s <i>t</i>-test, <i>P</i> < 0.01). All results represent the average of at least three independent experiments. Bars indicate the standard deviation from the biological experiments.</p

Expression of the <i>pal</i> genes in the wild-type and Δ<i>pac-3</i> strains at normal and alkaline pH.

<p>Cells from the wild-type and Δ<i>pac-3</i> strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus^™ Real-Time PCR system (Applied Biosystems) using the Power SYBR^® Green and specific primers. The <i>tub-2</i> gene was used as the reference gene, and the pH 5.8 wild-type was used as the reference sample. At least three biological replicates were performed, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. <b>a, b, c:</b> Letters above the bars indicate statistical significance; different letters indicate significant differences between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s <i>t</i>-test, <i>P</i> < 0.01).</p

Proposed model for the alkaline pH signaling in <i>N</i>. <i>crassa</i>.

<p><b>(A)</b> The PAC-3 protein contains 621 amino acid residues and has three C₂H₂ zinc-finger domains encompassing the amino acid from 95 to 183. The NLS sequence is shown between amino acid residues 281 and 304. The black arrow indicates the putative protease-processing site at amino acid 490. <b>(B)</b> <i>N</i>. <i>crassa</i> has the six <i>A</i>. <i>nidulans</i> Pal homologues. External pH signaling may involve the PAL-8, PAL-9, PAL-6 and PAL-3 complex. PAL-1 may act together with PAL-2 and may recruit the PAC-3 protein. PAC-3 undergoes only one proteolytic processing and is likely involved in the activation of the <i>pal-1</i>, <i>pal-2</i>, <i>pal-9</i>, and <i>pac-3</i> genes and the repression of the <i>pal-6</i> and <i>pal-8</i> genes at alkaline conditions. aa, amino acid; ZF, C₂H₂ zinc finger; NLS, nuclear localization signal.</p

PAC-3 proteolytic processing at alkaline pH.

<p>PAC-3 protein levels were detected by western blot using the polyclonal anti-mCherry antibody and cell extracts from the wild-type and complemented (Δ<i>pac-3 pac-3</i>⁺) strains cultivated at pH 5.8 at 30°C for 24 h and then shifted to pH 4.2 and 7.8 at 30°C for 1 h. <b>(A)</b> Aliquots of 50 μg of total protein were loaded onto the gel. <b>(B)</b> Aliquots of 70 μg of total protein were loaded onto the gel. The arrows indicate the processed mCh-PAC-3 form at pH 5.8 and 4.2. <b>(C)</b> The protein α-tubulin was used as the loading control. The plots represent one of the three independent experiments. The numbers on the left represent the molecular weight in kD.</p